University of Southern California Dissertations and Theses

Utilization of bisphosphonate drugs in fluorescent imaging and targeted drug delivery

(USC Thesis Other)

Utilization of bisphosphonate drugs in fluorescent imaging and targeted drug delivery

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content i

Utilization of Bisphosphonate Drugs in
Fluorescent Imaging and Targeted Drug Delivery

by

Kim L. T. Nguyen

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)

December 2016

ii

DEDICATION

To my parents, and Dr. James Martin DeJovine,
who encouraged me to pursue my graduate studies in chemistry

And to those who have given me their unconditional love and support along this journey.
iii

ACKNOWLEDGEMENTS

When I was young, I had never thought of pursuing my career in chemistry. It was one of
my most feared subjects during my high school in Vietnam. However, my perception changed
when I moved to the U.S. Dr. James M. DeJovine, my AP chemistry teacher, was the person who
encouraged me to pursue my undergraduate major in chemistry, and showed me the possibility of
becoming a scientist. With his encouragement, I completed my undergraduate degree with a double
major in chemistry and biochemistry at California State University, Long Beach, and continued
my graduate study at University of Southern California with an emphasis in medicinal chemistry
in Dr. Charles McKenna’s lab. For all of his contributions to me being who I am today, I would
like to express my deepest gratitude to Dr. James M. DeJovine.
During my undergraduate studies, I was very fortunate to meet so many good friends and
great mentors through my journey. I would like to thank my undergraduate advisor, Brian L.
McClain, and my math professor, Dr. Will Murray, for their advices and encouragement over the
years.
Without the support and advice from the McKenna group, I would not be where I am today.
I would like to express my gratitude to my graduate advisor, Dr. Charles McKenna, for accepting
me to his research group, providing me the chance to pursue my research, and to collaborate with
great coinvestigators (Drs. David Jung and Judith Kempfle from Massachusetts Eye and Ear
Infirmary). I also would like to thank to Dr. Boris Kashemirov not only for his help in chemistry
on a daily basis, but also for the great conversations throughout the years.
iv

My journey through graduate school would definitely not be same without great support
from former and present McKenna lab members. A sincere acknowledgement to Kimberly Hui,
Michelle Zhao, Drs. Candy Hwang (and Quinn, our official lab dog), Jorge Osuna, Dana Mustafa,
Melissa Williams, Elena Ferri, Corinne Minard, and Eric Richard for the great times in OCW 201
and stimulating scientific discussions. Particularly, I would like to thank Drs. Candy Hwang, Dana
Mustafa, Elena Ferri, and Eric Richard for proofreading my writing.
Graduate school is both physically and mentally challenging, feeling at times like a five-
and-a-half-year marathon. I couldn’t have reached the finish line without support from my family
and friends. I would like to especially thank my high school best friends, Catherine Dang and
Caitlin Lang, as well as Caitlin’s family (Steve and Mary Lang, and aunt Teresa Trout) for their
help with English and their emotional support. Also, I would like to express my gratitude to my
high school teacher, Mrs. Carlberg and her husband, Dave Carlberg, for their love and support
throughout the years. I would like to also thank Dr. Lily Zhang and Ben Ducato for their great
friendship and warm memories during the past five and a half years. Finally, I would like to thank
my boyfriend, Dr. David D. Liu for his support during the toughest times and for being by my side
during the past 4 years.

v

TABLE OF CONTENTS

DEDICATION ii

ACKNOWLEDGEMENTS iii

List of Tables viii

List of Figures ix

List of Schemes xi

Abstract xii

CHAPTER 1 1
Bisphosphonates: Background, Mechanism of Action, Clinical Use and
Adverse Effects 1
1.1 A Brief History on the Development of Bisphosphonates 1
1.2 Chemical Synthesis of Bisphosphonates 6
1.3 Structural Activity Relationship Studies of Bisphosphonates 6
1.4 Cellular Effect of Bisphosphonates 8
1.5 Molecular Mechanisms of Action of Bisphosphonates 10
1.5.1 Mechanism of actions of Non-Nitrogen BPs 11
1.5.2 Mechanism of actions of N-BPs 12
1.6 Clinical Use of Bisphosphonates 17
1.7 Side Effects and Concerns 19
1.8 References 22

CHAPTER 2 40
Targeted Drug Delivery of using Functionalized Bisphosphonate Gold
Nanoparticles 40
2.1 Background 40
2.2 Results and Discussion 43
2.3 Conclusion 50
2.4 Experimental section 50
2.5 References 53
CHAPTER 3 55
Structure Relationship Activity Studies between Linker Lengths and the
Anti-Resorptivity of Dye-N-BP Conjugates 55
3.1 Introduction 55
3.2 Molecular docking using Autodock Vina 59
3.3 Chemical synthesis of fluorescent dye-BP conjugates with various linker lengths 60
3.4 Preliminary studies on the anti-resorptive activities of NIR-BP conjugates 63
vi

3.5 Conclusion 66
3.6 Experimental Section 67
3.6.1 Chemical Synthesis 67
3.6.2 Biological Experiments 87
3.7 References 87

CHAPTER 4 90
Determination of the Bone Binding Affinity of Novel Fluorescently Labeled
Bisphosphonates 90
4.1 The role of bone binding affinity in the pharmacology of bisphosphonates 90
4.2 Methods used to determine the bone binding affinity of bisphosphonates 91
4.3 Results and discussion 98
4.4 Conclusion 104
4.5 Experimental Section 105
4.5.1 Chemical Synthesis 105
4.5.2 HAP Binding Experiments 105
4.6 References 106

CHAPTER 5 109
Overview of the Anatomical Structure of the Human Ear and Drug Delivery
to the Ear 109
5.1 Anatomical structure of the human ear 109
5.2 Clinical considerations for drug delivery to the human inner ear 111
5.3 Hydrogels as a drug delivery vehicle to the human inner ear 112
5.3.1 Hyaluronic acid (HA) 113
5.3.2 Chitosan (CS) 113
5.3.3 Poloxamer 407 (P407) 114
5.4 Device-based delivery technologies to the human inner ear 115
5.5 Conclusion 118
5.6 References 119

CHAPTER 6 123
Local Cochlear Distribution of N-BP drugs in Animal and Cadaveric Human
Models Visualized using 6-FAM-ZOL for the Treatment of Otosclerosis 123
6.1 Otosclerosis and the use of N-BP drugs in treating the disorder 123
6.2 Synthesis of 6-FAM-ZOL 126
6.3 Local delivery of 6-FAM-ZOL to the cochlea 127
6.4 Ototoxic evaluation of the local delivery of ZOL visualized using 6-FAM-ZOL 134
6.5 Intracochlear delivery of 6-FAM-ZOL through the oval window in fresh cadaveric
human temporal bones 136
6.6 In vitro sustained delivery of N-BP drugs using Poloxamer 407 142
vii

6.7 Conclusion 148
6.8 Experimental Section 149
6.8.1 Chemical Synthesis of 6-FAM-ZOL 150
6.8.2 Animal Experiments 152
6.8.3 Cadaveric Human Temporal Bone Experiments 157
6.8.4 In Vitro Experiments of the Release of N-BP drugs using P407 159
6.9 References 161

CHAPTER 7 166
Bisphosphonates Prodrugs for Targeted Drug Delivery of Neurotrophic Agents
for the Treatment of Hearing Loss 166
7.1 Hearing Loss 166
7.2 Neurotrophic factors and their receptors 167
7.3 Molecular docking using Autodock Vina 168
7.4 Synthesis of a DHF derivative 170
7.5 Synthesis of DHF-linker 172
7.6 Synthesis of a BP-DHF conjugate 173
7.7 Neurotrophic activities of a DHF derivative and RIS-DHF conjugate 178
7.8 Conclusion 179
7.9 Experimental section 181
7.10 References 192

BIBLIOGRAPHY 194

APPENDICES
APPENDIX A: Chapter 2 Supporting Data 224
APPENDIX B: Chapter 3 Supporting Data 237
APPENDIX C: Chapter 4 Supporting Data 302
APPENDIX D: Chapter 6 Supporting Data 318
APPENDIX E: Chapter 7 Supporting Data 333

viii

List of Tables
Table 2.1. Various coupling conditions for the activation reaction of 2.6. 47
Table 2.2. Optimization of BPs and NHS-activated adipic acid 48
Table 3.1. HPLC methods for the purification of extended Rislinker and dye-BP
conjugates 68
Table 4.1. Ranking of clinically used BPs using various methods. 94
Table 4.2. Relative ranking of the HAP binding affinity of previously published
fluorescently labeled BPs and PCs. 101
Table 5.1. Summary of related applications, disorders and patent references associated
with different polymer systems. 115

ix

List of Figures
Figure 1.1. Chemical structure of pyrophosphate and bisphosphonate 2
Figure 1.2. Timeline on the development of bisphosphonates since 1960s.
6
3
Figure 1.3. Structure of clinical used bisphosphonates 7
Figure 1.4. Cellular mechanism of BPs.
6
9
Figure 1.5. Molecular mechanism of BPs.
6
10
Figure 1.6. Molecular mechanism of non-nitrogen BPs.
37
12
Figure 1.7. Effects of N-BPs on the mevalonate pathway and potential mechanism
of their effects on osteoclasts.
6, 37
16
Figure 1.8. Bone imaging using 99mTc-BP complex.
6
18
Figure 2.1. General structure of the proposed AuNP platform. 43
Figure 2.2. Chemical structure of 2.8 45
Figure 2.3. Possible conjugated products of 2.8 with lysine 46
Figure 2.4. Structure of the hydrolyzed product of 2.13 48
Figure 2.5. Comparison of 1H NMR spectra of the product after 3 and 5 times
of DOWEX exchanges. 49
Figure 3.1. The crystal structure of human FPPS co-crystallized with RIS.
14
56
Figure 3.2. Chemical structure of 5-FAM, 6-FAM, AF647 and Sulfo-Cy5 58
Figure 3.3. In Silico Molecular Docking Illustrating the Interaction of AF647RIS,
5-FAM-RIS with FPPS
14
using AutoDock Vina 1.1.2.
24
59
Figure 3.4. Effect of dye-BPs on OC formation. 64
Figure 3.5. Inhibitory effect of BPs on bone resorption effect of bovine bone slices. 65
Figure 3.6. OC cultures on bone slices. 66
Figure 4.1. Relationship between the bone binding affinity of bisphosphonate drugs
and their bone uptake. 91
Figure 4.2. Tissue distribution of
99m
Tc BP complexes.
12
92
Figure 4.3. Determination of the HAP binding affinity using FPLC. 95
Figure 4.4. HAP binding of ROX-RIS, ROX-RISPC, AF647-RIS, and AF647-RISPC
determined using two different methods.
1
96
Figure 4.5. Chemical structure of 5-FAM-RIS, 5-FAM-ZOL, ROX-RISPC,
ROX-RIS, AF647-RIS, AND AF647-RISPC 99
Figure 4.6. HAP binding of previously published fluorescently labeled RIS using the
newly established method. 101
Figure 4.7. HAP binding of fluorescently labeled RIS, ZOL, and RISPC determined
using HAP column and HAP disc methods. 103
Figure 5.1. Anatomic Structure of the Human Ear. 109
Figure 5.2. Design of New Medical Devices for Drug Delivery to the Ear. 116
Figure 6.1. A schematic diagram of the guinea pig cochlea viewed at a mid-modiolar
section. 127
Figure 6.2. Systemic delivery of 6-FAM-ZOL and quantification of fluorescent signal. 128
Figure 6.3. Local delivery of 6-FAM-ZOL via diffusion through the RWM and
quantification of fluorescent signal. 129
Figure 6.4. Intracochlear infusion of 6-FAM-ZOL. 131
x

Figure 6.5. Fluorescent photomicrographs of the cochlear taken at mid-modiolar
sections at various time points after direct intracochlear infusion of artificial
perilymph, 0.04X solution, and 0.08X solution. 134
Figure 6.6. DOAPEs and CAP measurements for the control, 0.04X and 0.08X systemic
doses at various frequencies. 136
Figure 6.7. CAP measurements after infusion of 0.08X systemic dose at the frequencies
of 24 (A), 16 (B), 12 (C), and 8kHz (D) with the average values generated from
independent experiments of either acute (1 day) or chronic experiments (4 weeks). 137
Figure 6.8. Human temporal bones after administration of 6-FAM-ZOL. 140
Figure 6.9. Quantification of fluorescently signal after intracochlear administration. 140
Figure 6.10. (A) Standard curve of the absorbance of ZOL in 10 mM PBS buffer
(pH 7.4) and (B) UV-Vis Spectra of Bisphosphonate in solution with P407 143
Figure 6.11. Release profiles of RIS (5 mM) and ZOL (5 mM) at the presence of
17% w/w P407 before (A) and after (B) normalization. 144
Figure 6.12. In vitro Release profile of various salts form of RIS with 17% w/w P407 145
Figure 6.13. Experimental (A, B) and Literature (C) Kinetic Release Profile of 2% w/w
MPS with P407. 146
Figure 6.14. Kinetic release profiles of RIS and ZOL under various experimental
conditions 147
Figure 6.15. (A) Release Profiles of 5-FAMZOL and 6-FAMZOL from 17 % w/w
P407 using 12 mm membrane. (B) Release Profiles of 5-FAMZOL and RIS from
17% and 25% w/w P407 using 6.5 mm membrane. 148
Figure 6.16. Experimental Setups for In vitro Release Studies of Bisphosphonate in
Formulation with P407 160
Figure 7.1. Illustration of the organ of Corti before and after exposure to cDNA BDNF. 168
Figure 7.2. Molecular docking of 7,8-DHF in trkB crystal structure (PDB code: 4AT3). 169
Figure 7.3. Structure of the DHF-derivative 7.1 170
Figure 7.4. pKa calculation of RIS-DHF (7.19) using MarvinSketch. 178
Figure 7.5. In vitro neurite outgrowth. 179
Figure 7.6. In vitro neurite outgrowth. 180

xi

List of Schemes
Scheme 1.1. General synthesis of N-BPs using microwave.
34
6
Scheme 2.1. Synthesis of the Extended RIS-linker 44
Scheme 2.2. Synthetic Scheme of the Target Compound 45
Scheme 2.3. Synthesis of NHS-activated pamidronate 47
Scheme 3.1. Synthesis of Rislinker 3.6 using the ‘magic linker’ 57
Scheme 3.2. Synthesis of Novel Risedronate Analogues with Various Linker Lengths 61
Scheme 3.3. Synthesis of Fluorescently-labeled Risedronate Analogues with Various
Linker Lengths 63
Scheme 6.1. Synthesis of 5,6-FAM-ZOL 126
Scheme 7.1. Synthesis of 7.1 171
Scheme 7.2. Synthesis of 7,8-DHF-Linker (7.11) 172
Scheme 7.3. Synthesis of RIS-DHF using Route 1 174
Scheme 7.4. Synthesis of RIS-DHF conjugate using Route 2 175
Scheme 7.5. Synthesis of RIS-DHF conjugate using Route 3 175
Scheme 7.6. Synthesis of 7.25 176
Scheme 7.7. Synthesis of RIS-DHF using route 4 177

xii

Abstract
Bisphosphonates are a commonly prescribed class of medications currently used to treat
postmenopausal osteoporosis, Paget’s disease, bone metastatic cancers, and other bone-related
diseases. They are stable analogues of pyrophosphates and belong to a class of compounds that
contains a P-C-P backbone. The presence of the two phosphonate groups enables the compounds
to chelate calcium, which is found in bone mineral. This chelating ability of bisphosphonates to
calcium is responsible for their specific uptake by the bone, where the drugs inhibit osteoclast
mediated bone resorption.
The unique high bone binding affinity of bisphosphonates is an attractive feature that could
be exploited for targeted delivery of other drugs to bone. This idea was further explored in the
work presented in this thesis for various applications, including targeted delivery of therapeutics
to the bone and bone imaging. Two categories of projects are covered in this thesis: 1) non-ear
related studies and 2) ear-related studies.
The first chapter briefly summarizes history of the development of bisphosphonate drugs,
their mechanism of action, clinical applications, and side effects. The three subsequent chapters
focus on work related to the synthesis and characterization of various nitrogen-containing
bisphosphonate analogues used in targeted drug delivery and bone imaging. Topics covered in
these chapters are 1) the synthesis of a novel N-containing bisphosphonate drug possessing an
activated carboxyl moiety for targeted delivery of peptide-coated gold nanoparticles to bone
metastatic niches; 2) the development of a novel series of near infrared dye-bisphosphonate
conjugates to explore the effect of different linker lengths on their anti-resorptive activities on
osteoclasts; and 3) the establishment of a new analytical method to determine the bone binding
affinity of these newly synthesized conjugates.
xiii

The latter half of this thesis centers on the utilization of bisphosphonates for targeted drug
delivery to the ear. This part of the thesis starts with a brief overview of the anatomy of the human
ear and describes the challenges of drug delivery to the ear. This is followed by discussion of
studies on drug delivery to the cochlea using bisphosphonates and imaging of cochlear drug
distribution in murine models using fluorescently labeled bisphosphonates in search for effective
treatments of ear disorders, such as otosclerosis and hearing loss.

1

CHAPTER 1
Bisphosphonates: Background, Mechanism of Action,
Clinical Use and Adverse Effects
1.1 A Brief History on the Development of Bisphosphonates
Human extracellular fluid is saturated with calcium phosphate. In vitro experiments have
shown that in the presence of calcium phosphate, collagen can be the nucleating agent for the
deposition of hydroxyapatite (HAP), the polymerized form of calcium phosphate.
1
The absence of
calcified soft tissues in the human body indicates the existence of a natural, endogenous factor that
helps prevent unwanted calcification.
2

Hypophosphatasia is a rare genetic disorder characterized by the absence of functional
alkaline phosphatase, which results in mineralization defects of the skeleton in affected patients.
3

Children affected by the disease have high levels of pyrophosphate (PPi) in their urine.
4
Studies
have demonstrated the importance of alkaline phosphatase in the in vivo metabolism of
pyrophosphate.
Extracellular pyrophosphates (PPi) are mainly generated by nucleoside triphosphate
pyrophosphohydrolases (NTP-PPases) located at the cell surface. These phosphatases, especially
tissue non-specific alkaline phosphatase (TNAP), are found mainly in the liver, cartilage, and
bone.
5
At its optimal concentration in the body fluid and under physiological conditions,
circulating alkaline phosphate plays the role of a pyrophosphatase.
4
The enzyme, therefore, plays
a key role in maintaining the levels of pyrophosphates below the critical concentration. At a level
higher than the critical concentration, pyrophosphates chelate calcium ions and subsequently
prevent the normal deposition of hydroxyapatite in the skeleton.
4, 6

2

Together, these results suggest that pyrophosphates are endogenous water softeners in
human body fluids. Their main role is to inhibit soft tissue calcification and to monitor bone
mineralization.
7
As a byproduct of the hydrolysis of nucleotide triphosphates (NTPs), only a small
fraction of intracellular PPi reaches the extracellular fluid.
8
Intracellular PPi is hypothesized to be
monitored by ANK (a protein that is expressed by the progressive ankylosis gene) , a trans-
membrane transporter.
9, 10
In fact, mutations of ANK are linked to an abnormality in the
metabolism of PPi and to the calcification of tissues.
11, 12, 13

Bisphosphonates (BPs) (diphosphonates) are analogues of pyrophosphates
5
that contain a
P-C-P backbone rather than the P-O-P backbone present in pyrophosphates (Fig. 1). While
polyphosphates are more susceptible to hydrolysis and are therefore of limited use in chelating
metals, the P-C-P backbone renders bisphosphonates chemically stable and resistant to hydrolysis.

Figure 1.1. Chemical structures of pyrophosphate and bisphosphonate
Despite their discovery and reported synthesis in the late 1800s,
14
BPs, specifically
germinal BPs, were not clinically used to treat diseases associated with calcium metabolism until
the 1960s.
5
Prior to clinical applications, BPs were mainly used in laundry detergents, where their
ability to chelate calcium and magnesium in water prevented the deposition of soil in the wash.
15

BPs were first investigated for clinical use in dental applications. Studies led by Proctor & Gamble
(P&G) indicated that addition of polyphosphates (e.g., pyrophosphates) inhibited crystal growth
on the surface of CaF
2
.
16
In addition, BPs are effective calcium chelating agents, preventing the
formation of dental calculus.
17
Performed in the 1960s, these key studies on the role of
3

pyrophosphate in preventing calcification, especially in vivo, eventually lead to the clinical use of
BPs in treating bone diseases.

Figure 1.2. Timeline for the development of bisphosphonates beginning in the 1960s.
5

Early interactions between Dr. Francis at P&G and Dr. Fleisch at the Davos Research
Institute in Switzerland were crucial to the development of clinical applications for BPs.
5, 15
Their
collaborative work resulted in pivotal findings on the in vivo effect of two geminal BPs, methylene
BP and methyl-hydroxy-BP (i.e. etidronate) in blocking the crystallization of calcium phosphate
in vitro and preventing aortic calcification in rats that consumed a high amount of vitamin D3.
18

In addition, BPs were also found to block the dissolution of HAP, suggesting their potential for
retarding bone resorption.
18

The opportunity to further explore this property of BPs arose in 1967 when Dr. Andrew
Bassett presented to Dr. Fleisch the case of a 16-month old patient, who was diagnosed with
myositis ossificans progressiva (MOP). This disease is characterized by the swelling of muscles
and connective tissues. These symptoms are followed by the ossification of soft tissue, which
4

develops in the later stages of the disease.
15, 19
Calcification of the patient’s chest muscle led to
severe respiratory problems and the young patient was in dire need for an effective treatment for
the disease.
Previous studies using PPi indicate that polyphosphates and PPi are ineffective in inhibiting
calcification in vivo when orally administered because they were hydrolyzed in the GI tract. The
compounds are only effective when intravenously administered.
20
For these reasons, Dr. Fleisch
recommended that Dr. Bassett contact and discuss with Dr. Francis (P&G) the use of BPs
developed by P&G for the treatment of MOP.
20
Due to the patient’s critical need, authorization by
the US Food and Drug Administration (FDA) was quickly obtained by P&G. The treatment
regimen for MOP consisted of etidronate administered daily at a dose of 10 mg/kg in a suitable
oral formulation.
21
After calcification of her tissues was under control, the patient continued to
receive etidronate to monitor her condition.
15

A great deal of effort was devoted in the 1980s to the development of more potent BPs that
selectively inhibited bone resorption rather than normal skeletal mineralization. The dosage of
etidronate to inhibit bone mineralization is only 10-fold larger than the dosage for bone resorption.
5

Development of new highly potent BPs, particularly N-heterocyclic BPs such as zoledronate and
risedronate (commercially available as Zometa and Actonel, respectively) has addressed this
concern.
22
Both compounds demonstrate a high efficacy and good safety profile in preventing
vertebral and non-vertebral osteoporotic fractures.
23, 24
In fact, post hoc analyses on the data
collected in phase III clinical trials suggest that a single dose of intravenous zoledronate would be
as effective as three annual doses that total the same amount as a single dose.
24

When administered to biological systems, BPs are absorbed mainly by the bones, teeth,
and soft tissue calcifications, where they chelate with calcium in the bone mineral and form a thin
5

layer (~2.5 nm) of BP on the bone surface.
15
A concentration gradient is present at the site of
absorption, with the highest concentration of BPs being found in the extracellular fluid and their
concentration decreasing to almost zero at the inorganic surface of the bone, with a difference in
absorption rate of about 40:1.
15
The absorption of BPs by the bone matrix results in the deceleration
of the dissolution rate of the HAP crystal as well as the inhibition of the deposition of HAP, as
observed in both in vitro and in vivo experiments.
25, 26, 27, 28
One possible explanation for this effect
in vivo is that the dissolution of the HAP crystal is thermodynamically favored over its deposition
onto bone surface.
5

The high specific binding of BPs to bone mineral helps avoid unwanted side effects and
improve their safety.
29
The drugs are highly stable in biological systems and are secreted from the
kidney in their original form. BPs have low oral bioavailability, which is rarely above 5% for many
of them. Despite this, the oral uptake of BPs is preferred over other routes of administration. The
absorption of BPs in the gastrointestinal (GI) tract is facilitated by paracellular transport and their
absorption can be improved by using EDTA. Chelation of EDTA to calcium in the GI tract induces
the opening of gap junctions between intestinal mucosa, therefore enhancing the absorption of
BPs.
30

Instead of decelerating over time, the inhibition of bone resorption induced by BPs
generally reaches a steady state level, even when BPs are administered continuously.
31
As
observed in animal models, this steady state level solely depends on the total administered dose,
delivered in either a small dose on a daily basis or a single large dose.
32
This notion is consistent
in human studies.
33
BPs bound to bone are inactive as long as the drugs are retained in the bone
matrix.
5
Some BPs, such as zoledronate and alendronate, have a longer lasting effect in comparison
to others (e.g., etidronate, risedronate). This could be correlated to their binding affinity to
6

hydroxyapatite (HAP).
5
This long lasting effect may also be attributed to the continuous recycling
of BPs off and onto the bone surface. This is confirmed by the existence of BPs in urine and plasma
many months after they were last administered.
5

1.2 Chemical Synthesis of Bisphosphonates
The synthesis of clinical N-BPs involves heating an appropriate carboxylic acid with
phosphorous acid and phosphorus trichloride, followed by hydrolysis, to obtain the final N-BPs of
interest. Conventional synthetic approach requires long reaction times with reflux of the reaction
mixture up to 3 days. A small scale synthesis of BPs has recently been reported using microwave
irradiation The method requires much shorter reaction times, less than 1 hour, and affords similar
yields when compared to the conventional approach.
34

Scheme 1.1. General synthesis of N-BPs using microwave irradiation.
34

1.3 Structural Activity Relationship Studies of Bisphosphonates
Currently used clinical BPs can be divided into two categories: simple BPs and N-BPs.
Simple BPs, the first generation of BPs, include etidronate (EHDP), clodronate (CLO), and
tiludronate (TLN) (Fig. 1.3). N-BPs, the newer generation of BP drugs, contain alendronate (ALN),
7

pamidronate (APD), risedronate (RIS), zoledronate (ZOL), and ibandronate (IBN) (Fig. 1.3).
Therapeutic effects of BPs, such as high bone binding affinity and anti-resorption, are highly
correlated to three structural factors. These include the nature of the R
1
and R
2
groups and the P-
C-P backbone motif.

Figure 1.3. Structure of clinically used bisphosphonates
The presence of a hydroxyl group at the R
1
position has a substantial effect on the binding
affinity of BPs to bone mineral. With R
1
being either a hydrogen or halogen atom, BPs form a
bidentate interaction with the metal atom, created by the two phosphonate groups. The α-OH group
at R
1
position in BPs increases their chelating ability by creating a third interaction with the metal
atom, in addition to the regular bidentate bond observed in simple BPs.
15

The second factor is the R
2
group, which mainly influences the anti-resorptive activity, i.e.
the inhibition of bone resorption, observed with BPs.
35, 36
R
2
groups containing a primary amine
that is connected to BP backbone via an alkyl chain, such as in APD and ALN, help increase
potency by 10-100 fold in comparison to simple BPs.
5, 37
Thus, N-BPs emerged as a new class of
8

BPs. This effect is not limited to primary amine N-BPs, but is also observed in tertiary N-BPs,
such as IBN.
38
The most potent BPs, including RIS, ZOL, and minodronate (MIN), share a
common structural feature: they all contain nitrogen-containing heterocyclic rings.
39
In addition to
its presence in the R
2
group, the nitrogen atom needs to have a critical distance from the
bisphosphonate groups.
40

Last but not least, the P-C-P backbone is also crucial in conferring to BPs with their
therapeutic activity. Many studies on the stability against hydrolysis have been performed for
various classes of phosphorus compounds, including monophosphonates, P-N-P and P-C-C-P, and
P-C-P compounds.
41
Susceptibility to hydrolysis of monophosphonate, P-C-C-P and P-N-P results
in their lack of inhibition against bone resorption. Only compounds with a P-C-P motif are stable
and both phosphonate groups are also required for the pharmacological activity of BPs.
41

1.4 Cellular Effect of Bisphosphonates
The high affinity of BPs to bone mineral promotes their localization at close proximity to
osteoclasts, which are bone resorbing cells. This facilitates their internalization into osteoclasts via
endocytosis to form intracellular vacuoles, confirmed by studies using either radiolabeled
42
or
fluorescently labeled ALN.
43
This has an extensive effect on the survival and function of
osteoclasts, including recruitment, differentiation, resorptive activity, apoptosis, and disruption of
the cytoskeleton.
44, 45, 46, 47, 48, 49
The phenomenon is later described as the disruption of prenylation-
dependent intracellular signaling within osteoclasts.
5

BPs mainly act on mature osteoclasts, possibly affecting their formation.
50, 51, 52
Bone
resorption is promoted by vacuolar-type proton pumps located at the ruffled border of the
membrane of osteoclasts. This, in turn, induces the acidification of the subcellular space beneath
the osteoclasts. The resulting acidic environment causes the dissolution of HAP mineral while the
9

degradation of the extracellular bone matrix, induced by proteolytic enzymes, occurs. Previously
absorbed BPs at the site of bone resorption are released from the bone mineral due to acidic pH.
53,
54
This results in a high local concentration of BPs at the site of bone resorption which is much
higher than the effective dose required to affect osteoclast morphology, leading to osteoclast
apoptosis in vitro.
55

Figure 1.4. Cellular mechanism of BPs.
5

Another therapeutic agent that is currently used to treat osteoporosis and bone associated
diseases is denosumab, a humanized antibody whose main effect is observed through its interaction
with RANK-ligand (RANKL).
56
The antibody mainly acts on the differentiation of osteoclasts by
interacting with RANK/RANKL (a key pathway that regulates the differentiation of precursors
into osteoclasts), resulting in the disappearance of osteoclasts during the course of treatment. In
contrast, the main cellular effect exerted by BPs is to disable the activity of osteoclasts. This
observation is confirmed by the presence of multinucleated osteoclasts found in bone biopsies of
patients treated with oral ALN.
57

Aside from their apoptotic effect on osteoclasts, BPs are found to promote the survival of
osteoblasts and osteocytes in vitro and in vivo when exposed to glucocorticoids.
58
This
phenomenon occurs as a result of the opening of connexin Cx43 hemichannels. This leads to the
activation of Src kinases and extracellular signal-regulated kinases (ERKs), creating an anti-
10

apoptotic effect in vitro.
59
However, the anti-apoptotic effect of BPs on osteocytes and osteoblasts
that is observed at low dose is not well understood.
5

The proximity of osteoclasts at the site of bone resorption makes them the only cell types
able to internalize BPs from bone mineral surface. However, more up-to-date studies suggest that
other cells, such as monocytes and macrophages, also have the ability to uptake BPs that are briefly
available in circulation.
60, 61
Studies on the uptake of BPs by osteoclasts using fluorescently labeled
BPs confirm that their cellular internalization is facilitated via endocytosis. BPs are possibly
internalized via transcytosis, a pathway used by osteoclasts to remove the products of bone matrix
degradation.
62
After entering the cells, the contents of these vesicles are released into the cytosol
via their acidification,
43
followed by their distribution to other organelles , where they exert their
effects.
1.5 Molecular Mechanisms of Action of Bisphosphonates

Figure 1.5. Molecular mechanism of BPs.
5

During the past four decades, many experiments have been conducted to elucidate the
molecular mechanism of action of BPs, particularly the inhibitory effect of BPs on different
11

enzymes.
63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74
Current literature divides the mechanism of action of BPs
into two categories based on their chemical structure: 1) non-nitrogen containing BPs and 2) N-
BPs.
1.5.1 Mechanism of actions of Non-Nitrogen BPs
Pioneering studies conducted by Klein et al. in 1989 measured the intracellular pH in
Dictyostelium slime mould amoebae. The results demonstrated that simple BPs, such as methylene
bisphosphonates, are incorporated into ATP to form non-hydrolysable ATP analogs, as confirmed
by
31
P NMR.
75
Subsequent studies conducted by others using various models, including human
cells and murine J774 macrophages, also indicated that other simple BPs, such as CLO and EHDP,
are metabolized in a similar manner to that of methylene bisphosphonates. This is confirmed using
either
31
P NMR or LC-MS to analyze the cell lysates.
76, 77, 78, 79, 80, 81, 82
Key enzymes involved in
this process include the type II class of aminoacyl-tRNA synthases
79, 80, 81, 82
and most recently, T4
RNA ligase.
83
This specific mechanism only works with simple BPs, which are sufficiently similar
to PPi, such that they can be incorporated into the active site of type II class of aminoacyl-tRNA
synthase and react with AMP to form AppCp analogues. On the other hand, N-BPs, such as ALN,
IBN, and APD, are too sterically-hindered to be accommodated into the active site of these
enzymes. The formation of their non-hydrolysable ATP analogs is not detected using the
previously mentioned detecting methods.
83
The accumulation of these non-hydrolysable ATP
(AppCp-type) analogs exerts a detrimental effect on the survival of osteoclasts, ultimately
promoting their apoptosis and thus inhibiting bone resorption.
47, 48, 84, 85, 86, 87

In particular, the apoptotic effect of non-nitrogen containing BPs is mainly induced through
their specific interaction with adenine nucleotide translocase (ANT), located at the mitochondrial
membrane.
88
The primary function of ANT is to maintain the mitochondrial potential via the
12

transmembrane transport of ATP. This is inhibited by AppCp type analogs, ultimately leading to
the activation of caspase-3, followed by caspase-3 mediated apoptosis-promoting kinase, Mts1.
88,
89, 90, 91
Studies investigating the survival of osteoclasts, in which the cells are first treated with
simple BPs, followed by introduction of caspase-3 inhibitors, further confirm this mechanism.
92

Other proteins, such as TNF-α and RANKL, are also found to promote survival of osteoclasts
against the cytotoxic effect induced by simple BPs.
93, 94
Simple BPs, such as CLO, EHDP, and
TLN, are selectively absorbed by bone and taken up by osteoclasts. This demonstrates their
specific apoptotic effect on osteoclasts, not affecting other bone cells in vivo. Thus, simples BPs
generally act as prodrugs. While they are dormant in their original BP form, their therapeutic effect
is activated once they are incorporated into the nucleoside triphosphate analogs.

Figure 1.6. Molecular mechanism of non-nitrogen BPs.
37

1.5.2 Mechanism of actions of N-BPs
In comparison to non-nitrogen containing BPs, N-BPs are significantly more potent than
simple BPs, by several orders of magnitudes. Their sterically hindered structure suggests that a
mechanism of action on osteoclasts that is distinct from that of simple BPs.
78, 83
Previous studies
demonstrate that while simple BPs do not affect the synthesis of cholesterol,
95, 96
N-BPs, such as
IBN, exhibit an inhibitory effect on squalene synthase and possibly on other enzymes in the
mevalonate pathway, which controls the synthesis of cholesterol in J774 macrophages.
95

13

Subsequent experiments further show a correlation between the apoptotic effect of N-BPs on J774
macrophages and their bone anti-resorptive activities.
97
Consequently, J774 macrophages are a
prime model for studying the anti-resorptivity of N-BPs on osteoclasts via their apoptotic effect.
Though found to be inhibitors of other enzymes in the mevalonate pathway, including
isopentenyl diphosphate (IPP) isomerase, squalene synthase, and geranylgeranyl diphosphate
(GGPP) synthase,
95, 98, 99
N-BPs mainly exert their effect on farnesyl pyrophosphate synthase
(FPPS) at nanomolar concentrations in in vitro experiments. N-BPs high inhibitory effect
corresponds well to their anti-resorptivity in vivo.
100, 101
Hence, FPPS is considered to be the main
enzyme affected by N-BPs in the mevalonate pathway.
101, 102, 103, 104
Farnesyl pyrophosphate (FPP)
plays an important role in protein post-translation modification, also known as prenylation, of
GTPases, such as Ras, Rab, Rho, and Rac.
105, 106
Ras, Rab, Rho, and Rac are important for the
normal function of osteoclasts, and their malfunction is detrimental to the survival of osteoclasts.
107,
108, 109, 110

Incorporation of FPP into proteins, or prenylation, has been observed using radiolabeled
mevalonate, FPP, and GGPP in in vitro and in vivo experiments. This is a valuable tool that could
be used to investigate the effect of N-BPs on the prenylation of proteins versus that of simple
BPs.
78, 105, 111
While several studies further validate the effect of N-BPs on the mevalonate pathway
and the role of the mevalonate pathway on the survival and function of osteoclasts,
101, 103, 111, 112,
113, 114
the mechanism by which N-BPs reach intracellular targets remains unclear. Additional
studies also indicate that the farnesylated proteins were less important than the geranylgeranylated
proteins in inhibiting bone resorption and inducing an apoptotic effect in osteoclasts.
115
This notion
was further validated in subsequent studies where the effects of N-BPs on osteoclast formation,
14

apoptosis, and bone resorption were counteracted by the addition of geranylgeraniol, an isoprenoid
lipid substrate of geranylgeranylation.
91, 113, 116

Studies further confirm the effect of N-BPs on FPPS by inhibiting the growth of
Dictyostelium slime mould amoebae. Mutant forms of FPPS found in Dictyostelium
117
and in
tumor cells incubated in low concentration of N-BPs indicate a lack of activity of N-BPs in
preventing growth and inducing cytotoxic effects.
118, 119
In addition, the activity levels of FPPS
were found to be increased. However, the in vivo effect of mutant FPPS resistance to N-BPs in
humans has yet to be investigated.
37
The structure of FPPS is well conserved among eukaryotes.
This results in the inhibitory effect of N-BPs observed not only in Dictyostelium,
117
but also in
Leishmania and trypanosome parasites.
120, 121, 122, 123
X-ray crystallographic studies of FPPS and
other enzymatic studies of FPPS with N-BPs show that N-BPs competitively bind to FPPS in a
similar manner at the active binding site to that of natural substrates, including isoprenoid
pyrophosphate (IPP) substrates, geranyl pyrophosphate (GPP) and dimethylallyl pyrophosphate
(DMAP).
114
The BP moiety stabilizes the binding of N-BPs to FPPS by interacting with Mg
2+
ions
available at the GPP/DMAPP binding site.
114, 124

Since most N-BPs accumulate at the bone surface, their activities as anti-tumor agents
mainly rely on their bioavailability to other cells, such as osteoclasts and monocytes. Structural-
relationship activity (SAR) studies performed on various derivatives of N-BPs show that
attenuation of mineral binding affinity by removal of a phosphonate group results in the loss of
key interactions with Mg
2+
ions located at the GPP/DMAPP binding site of FPPS, thus resulting
in the absence of an anti-prenylation effect.
112
Replacement of a phosphonate group with a
carboxylate group in N-BPs substantially reduces their high mineral binding affinity while
retaining their activities against FPPS, their inhibition on the prenylation of Rab GTPases in
15

vitro,
125, 126, 127, 128
as well as their in vitro anti-tumor effect.
129
Though the in vivo anti-tumor effect
of the phosphonocarboxylate compounds was previously reported,
130
their availability to tissues
other than bones may still be low. This could be due to their relatively strong bone binding affinity,
which can be attributed to the remaining phosphonate group, resulting in their accumulation in the
bone.
37

Besides their anti-prenylation effect on key proteins that control the function and survival
of osteoclasts, N-BPs also induce the accumulation of isopentenyl pyrophosphate (IPP). This
results in the acute phase reaction in patients after IV administration of N-BPs. This phenomenon
is attributed to the rapid accumulation of IPP in monocytes after IV administration of N-BPs.
131,
132
Exposure of IPP to γ,δ-T cells, specifically Vγ9Vδ2 T cells, activates these T-cells, leading to
the release of TNFα. This subsequently initiates the pro-inflammatory acute phase response.
133
In
vitro experiments demonstrate that this effect can be avoided through exposure to statins, which
prohibit the accumulation of IPP.
131

The accumulated IPP and DMAPP in cells that internalize N-BPs react with AMP, and
produce ApppI
134, 135, 136, 137
and ApppD.
138
Similar to the AppCp formed by the reaction of non-
nitrogen BPs with AMP, ApppI and ApppD inhibit the transport of ATP by interacting with
mitochondrial ANT, and ultimately lead to osteoclast apoptosis.
134
Thus, the molecular mechanism
of N-BPs on osteoclasts comprises of two separate effects: 1) the anti-prenylation effect and 2) the
apoptotic effect promoted by the accumulation of ApppI.
139
Among the two processes,
morphological changes observed in osteoclasts suggest that the anti-prenylation effect is the best
explanation for therapeutic effects of N-BPs.
37
Notably, the anti-resorption effect observed in N-
BPs is not due to their promotion of apoptosis of osteoclasts in a similar manner to that of simple
BPs.
92
In fact, cell toxicity and a decrease in the number of osteoclasts are not always the
16

justification of the therapeutic effect of N-BPs,
46, 140
as osteoclasts may still be present. Rather,
they exist in their fused, dormant form as ‘hypernucleated’ cells that are detached from the bone
surface or associated with superficial resorption lacunae.
57, 141

Figure 1.7. Effects of N-BPs on the mevalonate pathway and potential mechanism of their effects
on osteoclasts.
5, 37

Reported in the literature in the late 1970s, statins, a now commonly prescribed class of
drugs for the treatment of high cholesterol, also target the mevalonate pathway. The drugs interact
with a different target enzyme of the pathway, namely HMG-CoA reductase.
142
Since N-BPs and
statins are inhibitors of different enzymes of the mevalonate pathway, statins are believed to also
exhibit an anti-resorptive effect. This is proven by in vitro experiments, where statins inhibited
both the formation of osteoclasts and bone resorption.
143
In fact, both statins and N-BPs exhibit
similar apoptotic effects on J774 macrophages. Their apoptotic effect depends on protein synthesis
and is delayed for a period of 15-24 h.
144
Addition of geranylgeraniol without farnesol can
counteract the effect of statins on osteoclasts, indicating the importance of gerannylgeranylated
proteins in the survival and function of osteoclasts, such as Rho, Rac, and Rab. Among them, Rab
is of great importance.
145
There is no evidence of the effect of N-BPs on the level of cholesterol.
17

This is mainly attributed to selective absorption of each drug by a specific organ: statins are
primarily retained by the liver, while BPs are specifically absorbed by the bone.
1.6 Clinical Use of Bisphosphonates
The first human clinical trial in the late 1960s is the hallmark event that launched the
clinical use of BPs in treating diseases associated with calcification and metabolic bone disorders.
Besides myositis ossificans progressiva (MOP), BPs are also utilized to treat Paget’s disease, a
disorder characterized by abnormally high bone turnover rates, resulting in weak and misshaped
bones. A small clinical study performed at Oxford University using EHDP showed that BPs were
highly effective in the treatment of Paget’s disease
146
. Due to their long lasting effect in delaying
the onset of Paget’s disease, APD and ZOL are the main N-BPs that are used in the treatment of
this disease. However, the mechanism of their observed long lasting effect remains unclear. In
addition, BPs are also used in treating patients on glucocorticoids and in children with inherited
brittle bone disorder, i.e. osteogenesis imperfecta.
147, 148

Early key studies previously described further expand the use of BPs in treating other bone
diseases, including heterotopic ossification associated with total hip replacement or spinal cord
injury,
149
and hypercalcemia in malignancy.
150
Despite early investigations on the effect of BPs
(e.g., EHDP and CLO) in treating hypercalcemia of malignancy associated myeloma and bone
metastases,
151
their use did not become the standard of care in these conditions until the late
1990s.
152
Co-administration of BPs with common anti-cancer agents, including doxorubicin,
paclitaxel, and cisplatin, is reported to exhibit a synergistic antitumor effect in preclinical
experiments.
153
The observed effects include promotion of apoptosis of tumor cells, induction of
cytotoxicity, and inhibition of tumor proliferation.
153
Thus, BPs can potentially be used in
combination with chemotherapeutic agents to treat cancer.
18

Starting in the mid-1980s, the efficacy of BPs in increasing bone mineral density and
decreasing the rate of vertebrate fractures in high risk patients, especially in patients who were
regularly administered with EHDP, was also investigated for preventing and treating osteoporosis
in post-menopausal women.
154
Currently, BPs are used to treat osteoporosis in both men and
women. Commonly prescribed BPs to treat osteoporosis include EHDP, ALN, RIS, IBN, ZOL and
MIN, which is officially approved for use only in Japan. Their efficacy demonstrates a reduction
of up to 40% in hip fractures.
5
Intravenous ZOL is often preferred due to its consistent dosage and
lack of gastrointestinal side effects.
155, 156

Figure 1.8. Bone imaging using 99mTc-BP complex.
5

The strong chelating property of BPs to bone and metals can be further employed in other
bone-related applications, such as bone imaging. Chelation of BPs, using either tridentate or
bidentate compounds, to radioactive
99m
Tc, allows for the detection of abnormal bone metabolism
in the human body (see Figure 1.8).
19

In addition to their effect on the human mevalonate pathway, N-BPs are also found to be
effective in inhibiting the methylerythritol phosphate (MEP) pathway in protozoa parasites,
particularly Apicomplexan parasites.
157, 158, 159, 160
This creates a new exciting avenue to further
expand the use of BPs in treating parasitic diseases. Studies in the early 2000s revealed a wide
range of Apicomplexan parasites, including Toxoplasma,
161
Plasmodium spp.,
122

Cryptosporidia,
162
and Leishmanial spp.
163
While BPs inhibit GGPP synthase in Plasmodium spp.
and exert an anti-prenylation effect on the blood form parasites,
164
their lipophilic forms are active
in preventing the parasite from infecting the liver in vitro and in mice.
165
Similarly, BPs are also
found to be inhibitors of FPP synthase in Toxoplasma gondii. They affect the parasite growth in
vitro by preventing protein prenylation.
122
Utilization of an N-BPs, such as RIS, is shown to
improve the survival rate in mice infected with T. gondii.
166
Unlike their effects in Plasmodium
spp. and T. gondii, BPs are highly potent inhibitors of non-specific polyprenyl diphosphonate
(NPPP) synthase in C. parvum. Thus, they significantly halt the parasite growth.
159

1.7 Side Effects and Concerns
Common side effects of BPs include upper gastrointestinal irritation, acute phase response,
eye inflammation, and renal impairment. Upper gastrointestinal irritation affects about 20-30% of
BP users.
167
Eye inflammation, particularly uveitis, is another reported side effect of BPs and is
commonly observed in about 1% of patients who are first time BP users.
168
BPs are removed from
the human body via secretion in urine, mainly via glomerular filtration with the possible
involvement of tubular secretion. Thus, significant renal impairment is another potentially
dangerous side effect.
167
Reports of renal impairment are extremely rare and its underlying cause
remains unknown.
167
Other reported rare but serious adverse effects are hypocalcaemia, atrial
20

fibrillation, and esophageal cancer. Currently, they are not considered to be significant risks for
patients.
167

Acute phase response, a commonly observed adverse effect, occurs in patients who are BP-
naïve, particularly in intravenously administered patients. Symptoms are not limited to flu-like
symptoms, such as fever and muscle pain, but include inflammation of tissues in various organs,
such as the gastrointestinal system, upper respiratory tract and the eye.
169
This acute phase usually
appears within 15 days after the first administration of BPs and dissipates within few days. It can
be addressed using common nonsteroidal anti-inflammatory drugs.
170
This inflammatory reaction
becomes less frequent upon administration of subsequent doses. The observed side effect is
attributed to the involvement of BPs in the release of IPP from monocytes and their selective
receptor-mediated activation of γ,δ-T cells, resulting in the proliferation and activation of pro-
inflammatory cytokines.
131, 171
The full mechanism of this phenomenon is discussed in detail in
Section 1.5.2.
One of the most concerning side effects of BPs is osteonecrosis of the jaw (ONJ).
167

According to the American Society for bone and Mineral Research (ASBMR), ONJ is defined as
the failure of an area of exposed bone in the maxillofacial region to heal within 8 weeks.
172
ONJ
is first commonly detected in cancer patients who receive high doses of N-BPs administered
intravenously. The condition is not observed during clinical trials for both ALN and RIS. The rate
of occurrence of ONJ is estimated to be less than 2 per 100,000 patients per year. In clinical trials
for ZOL, ONJ is reported to occur in one patient (out of 7765 patients) in both the placebo and the
ZOL groups.
173, 174
Though observed in osteoporotic patients, ONJ in these patients is less severe
and the lesions are likely to heal.
175, 176
The mechanism of the manifestation of ONJ associated
with N-BP use is yet to be elucidated; nevertheless, the lack of distinction between different
21

dosages prescribed for cancer and osteoporotic patients creates difficulty in convincing patients to
use BPs.
Though BPs conserve the bone architecture and strength, it is possible that long-term use
of BPs would impair bone turnover, resulting in decreasing overall bone strength.
177, 178
Studies on
the effect of high dosage in animals indicate an association between the high dose and increased
micro-damages as well as fractures.
179, 180, 181

Fractures occurring at the upper femoral shaft were first reported in 2008 in patients who
underwent BP treatment in Singapore, followed by the U.S.
182
It is noteworthy that the rate of
femoral shaft fractures after the introduction of BPs does not change in comparison to the rate
prior to the use of BPs in the treatment of osteoporosis.
167
The notion of femoral shaft fractures is
not remarked in clinical trials of BPs.
183
Radiographs of fractures demonstrate two types of fracture
patterns, including classic fractures and atypical fractures, which are commonly observed in
patients treated with BPs. Studies performed in Swedish women in 2008 indicate an increasing
risk of atypical femoral fracture associated with those who receive treatment using BPs for more
than 2 years. Furthermore, a drug holiday for a year or more would reduce the risk of acquiring
such fractures.
184
Subsequent studies conducted by others on the association between the long term
use of BPs and atypical femoral fracture demonstrate that while there is an increasing risk of
atypical femoral fractures, the numbers reported for these fractures are well below that for classic
fractures.
167
Thus, treatment of osteoporosis using BPs is recommended to continue for 5-6 years
unless T-scores of patients are still below -2.5.
185, 186
Association of BPs with atypical femoral
fractures is widely accepted, however, it is inconclusive to state that BPs are the cause of fractures
that do not otherwise occur. Furthermore, BPs may just change the nature of femoral fractures
through their effect on the bone.
22

ONJ is observed not only in patients who are taking BPs, such as APD and ZOL, but also
occurs in patients who receive denosumab, where the rate of occurrence is similar to that of
ZOL.
187
Denosumab is an antibody that works by mainly inhibiting osteoclastogenesis. It is also
reported to be effective in increasing bone density in both the spine and hip bone, along with
reducing vertebral fracture risk as long as patients are compliant with the treatment.
188, 189
The
antibody is usually administered subcutaneously every 6 months. Treatment using denosumab in
combination with teriparatide
190
is as effective as treatment using ZOL and teriparatide.
191
As
reported in phase 3 clinical trials, a noticeable side effect of denosumab is the risk of cellulitis.
ONJ associated with the use of denosumab for osteoporotic patients does not pose a significant
risk, similar to what was observed in patients treated with BPs. Atypical femoral fractures
associated with the use of denosumab have been reported, though insufficient data has been
collected.
192

In general, patients are recommended to postpone treatment with BPs before undergoing
an invasive dental procedure. A popular approach to address the long-term use of BPs is to stop
the treatment for a period of time, also known as drug holidays. They are typically about two years
for ALN and one year for RIS.
193
ONJ and atypical femoral fractures are more commonly observed
in patients who received long-term treatment with BPs, typically 5 years or more.
167
Thus,
determination of the optimal time for the course of treatment is recommended based on the patients’
T-score.
1.8 References

1. Fleish, H.; Neuman, W. F., Mechanisms of calcification: role of collagen, polyphosphates,
and phosphatase. Am. J. Physiol. 1961, 200, 1296-1300.
2. Fleisch, H.; Bisaz, S., Isolation from urine of pyrophosphate, a calcification inhibitor. Am.
J. Physiol. 1962, 203, 671-675.
23

3. Whyte, M. P., Hypophosphatasia - aetiology, nosology, pathogenesis, diagnosis and
treatment. Nat. Rev. Endocrinol. 2016, 12, 233-246.
4. Russell, R. G., Excretion of Inorganic Pyrophosphate in Hypophosphatasia. Lancet 1965,
2, 461-464.
5. Russell, R. G., Bisphosphonates: the first 40 years. Bone 2011, 49, 2-19.
6. Russell, R. G.; Bisaz, S.; Donath, A.; Morgan, D. B.; Fleisch, H., Inorganic pyrophosphate
in plasma in normal persons and in patients with hypophosphatasia, osteogenesis imperfecta, and
other disorders of bone. J. Clin. Invest. 1971, 50, 961-969.
7. Fleisch, H.; Russell, R. G.; Straumann, F., Effect of pyrophosphate on hydroxyapatite and
its implications in calcium homeostasis. Nature 1966, 212, 901-903.
8. Jung, A.; Bisaz, S.; Gebauer, U.; Russell, R. G.; Morgan, D. B.; Fleisch, H., The uptake
and metabolism of (32-P)pyrophosphate by mouse calvaria in vitro. Biochem. J. 1974, 140, 175-
183.
9. Abhishek, A.; Doherty, M., Pathophysiology of articular chondrocalcinosis--role of ANKH.
Nat. Rev. Rheumatol. 2011, 7, 96-104.
10. Collins, M. T.; Boehm, M., It ANKH necessarily so. J. Clin. Endocrinol. Metab. 2011, 96,
72-74.
11. Terkeltaub, R. A., Inorganic pyrophosphate generation and disposition in pathophysiology.
Am. J. Physiol. Cell Physiol. 2001, 281, C1-C11.
12. Anderson, H. C.; Harmey, D.; Camacho, N. P.; Garimella, R.; Sipe, J. B.; Tague, S.; Bi,
X.; Johnson, K.; Terkeltaub, R.; Millan, J. L., Sustained osteomalacia of long bones despite major
improvement in other hypophosphatasia-related mineral deficits in tissue nonspecific alkaline
phosphatase/nucleotide pyrophosphatase phosphodiesterase 1 double-deficient mice. Am. J.
Pathol. 2005, 166, 1711-1720.
13. Timms, A. E.; Zhang, Y.; Russell, R. G.; Brown, M. A., Genetic studies of disorders of
calcium crystal deposition. Rheumatology (Oxford) 2002, 41, 725-729.
14. Von Baeyer, H.; Hofmann, K. A., Acetodiphosphoriga sauer. Ber 1897, 30, 1973.
15. Francis, M. D.; Valent, D. J., Historical perspectives on the clinical development of
bisphosphonates in the treatment of bone diseases. J. Musculoskelet. Neuronal. Interact. 2007, 7,
2-8.
16. Francis, M. D.; Gray, J. A.; Griebstein, W. J., The formation and influence of surface
phases on calcium phosphate solids. Adv. Oral Biol. 1968, 3, 83-120.
17. Francis, M. D.; Briner, W. W., The effect of phosphonates on dental enamel in vitro and
calculus formation in vivo. Calcif. Tissue Res. 1973, 11, 1-9.
24

18. Francis, M. D.; Russell, R. G.; Fleisch, H., Diphosphonates inhibit formation of calcium
phosphate crystals in vitro and pathological calcification in vivo. Science 1969, 165, 1264-1266.
19. Hashemi, J.; Shahfarhat, A.; Beheshtian, A., Fibrodysplasia ossificans progressiva: report
of a case and review of articles. Iran J. Radiol. 2011, 8, 113-117.
20. Schibler, D.; Russell, R. G.; Fleisch, H., Inhibition by pyrophosphate and polyphosphate
of aortic calcification induced by vitamin D3 in rats. Clin. Sci. 1968, 35, 363-372.
21. Bassett, C. A.; Donath, A.; Macagno, F.; Preisig, R.; Fleisch, H.; Francis, M. D.,
Diphosphonates in the treatment of myositis ossificans. Lancet 1969, 2, 845.
22. Widler, L.; Jaeggi, K. A.; Glatt, M.; Muller, K.; Bachmann, R.; Bisping, M.; Born, A. R.;
Cortesi, R.; Guiglia, G.; Jeker, H.; Klein, R.; Ramseier, U.; Schmid, J.; Schreiber, G.; Seltenmeyer,
Y.; Green, J. R., Highly potent geminal bisphosphonates. From pamidronate disodium (Aredia) to
zoledronic acid (Zometa). J. Med. Chem. 2002, 45, 3721-3738.
23. Harris, S. T.; Watts, N. B.; Genant, H. K.; McKeever, C. D.; Hangartner, T.; Keller, M.;
Chesnut, C. H., 3rd; Brown, J.; Eriksen, E. F.; Hoseyni, M. S.; Axelrod, D. W.; Miller, P. D.,
Effects of risedronate treatment on vertebral and nonvertebral fractures in women with
postmenopausal osteoporosis: a randomized controlled trial. Vertebral Efficacy With Risedronate
Therapy (VERT) Study Group. JAMA 1999, 282, 1344-1352.
24. Reid, I. R.; Black, D. M.; Eastell, R.; Bucci-Rechtweg, C.; Su, G.; Hue, T. F.; Mesenbrink,
P.; Lyles, K. W.; Boonen, S.; Trial, H. P. F.; Committees, H. R. F. T. S., Reduction in the risk of
clinical fractures after a single dose of zoledronic Acid 5 milligrams. J. Clin. Endocrinol. Metab.
2013, 98, 557-563.
25. Fleisch, H.; Russell, R. G.; Francis, M. D., Diphosphonates inhibit hydroxyapatite
dissolution in vitro and bone resorption in tissue culture and in vivo. Science 1969, 165, 1262-
1264.
26. Francis, M. D.; Flora, L.; King, W. R., The effects of disodium ethane-1-hydroxy-1, 1-
diphosphonate on adjuvant induced arthritis in rats. Calcif. Tissue Res. 1972, 9, 109-121.
27. Francis, M. D., The inhibition of calcium hydroxypatite crystal growth by
polyphosphonates and polyphosphates. Calcif. Tissue Res. 1969, 3, 151-162.
28. Briner, W. W.; Francis, M. D., In vitro and in vivo evaluation of anti-calculus agents. Calcif.
Tissue Res. 1973, 11, 10-22.
29. Cremers, S.; Papapoulos, S., Pharmacology of bisphosphonates. Bone 2011, 49, 42-49.
30. Janner, M.; Muhlbauer, R. C.; Fleisch, H., Sodium EDTA enhances intestinal absorption
of two bisphosphonates. Calcif. Tissue Int. 1991, 49, 280-283.
25

31. Reitsma, P. H.; Bijvoet, O. L.; Verlinden-Ooms, H.; van der Wee-Pals, L. J., Kinetic
studies of bone and mineral metabolism during treatment with (3-amino-1-hydroxypropylidene)-
1,1-bisphosphonate (APD) in rats. Calcif. Tissue Int. 1980, 32, 145-157.
32. Gasser, J. A.; Ingold, P.; Venturiere, A.; Shen, V.; Green, J. R., Long-term protective
effects of zoledronic acid on cancellous and cortical bone in the ovariectomized rat. J. Bone Miner.
Res. 2008, 23, 544-551.
33. Garnero, P.; Shih, W. J.; Gineyts, E.; Karpf, D. B.; Delmas, P. D., Comparison of new
biochemical markers of bone turnover in late postmenopausal osteoporotic women in response to
alendronate treatment. J. Clin. Endocrinol. Metab. 1994, 79, 1693-1700.
34. Mustafa, D. A.; Kashemirov, B. A.; McKenna, C. E., Microwave-assisted synthesis of
nitrogen-containing 1-hydroxymethylenebisphosphonate drugs. Tetrahedron Lett. 2011, 52, 2285-
2287.
35. Lawson, M. A.; Xia, Z.; Barnett, B. L.; Triffitt, J. T.; Phipps, R. J.; Dunford, J. E.; Locklin,
R. M.; Ebetino, F. H.; Russell, R. G., Differences between bisphosphonates in binding affinities
for hydroxyapatite. J. Biomed. Mater. Res. B Appl. Biomater. 2010, 92, 149-155.
36. Nancollas, G. H.; Tang, R.; Phipps, R. J.; Henneman, Z.; Gulde, S.; Wu, W.; Mangood, A.;
Russell, R. G.; Ebetino, F. H., Novel insights into actions of bisphosphonates on bone: differences
in interactions with hydroxyapatite. Bone 2006, 38, 617-627.
37. Rogers, M. J.; Crockett, J. C.; Coxon, F. P.; Monkkonen, J., Biochemical and molecular
mechanisms of action of bisphosphonates. Bone 2011, 49, 34-41.
38. Papapoulos, S. E.; Hoekman, K.; Lowik, C. W. G. M.; Vermeij, P.; Bijvoet, O. L. M.,
Application of an Invitro Model and a Clinical Protocol in the Assessment of the Potency of a New
Bisphosphonate. J. Bone Miner. Res. 1989, 4, 775-781.
39. Green, J. R.; Muller, K.; Jaeggi, K. A., Preclinical pharmacology of CGP 42'446, a new,
potent, heterocyclic bisphosphonate compound. J. Bone Miner. Res. 1994, 9, 745-751.
40. Russell, R. G.; Xia, Z.; Dunford, J. E.; Oppermann, U.; Kwaasi, A.; Hulley, P. A.;
Kavanagh, K. L.; Triffitt, J. T.; Lundy, M. W.; Phipps, R. J.; Barnett, B. L.; Coxon, F. P.; Rogers,
M. J.; Watts, N. B.; Ebetino, F. H., Bisphosphonates: an update on mechanisms of action and how
these relate to clinical efficacy. Ann. N.Y. Acad. Sci. 2007, 1117, 209-257.
41. Ebetino, F. H.; Hogan, A. M.; Sun, S.; Tsoumpra, M. K.; Duan, X.; Triffitt, J. T.; Kwaasi,
A. A.; Dunford, J. E.; Barnett, B. L.; Oppermann, U.; Lundy, M. W.; Boyde, A.; Kashemirov, B.
A.; McKenna, C. E.; Russell, R. G., The relationship between the chemistry and biological activity
of the bisphosphonates. Bone 2011, 49, 20-33.
42. Sato, M.; Grasser, W.; Endo, N.; Akins, R.; Simmons, H.; Thompson, D. D.; Golub, E.;
Rodan, G. A., Bisphosphonate action. Alendronate localization in rat bone and effects on
osteoclast ultrastructure. J. Clin. Invest. 1991, 88, 2095-2105.
26

43. Thompson, K.; Rogers, M. J.; Coxon, F. P.; Crockett, J. C., Cytosolic entry of
bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis. Mol.
Pharmacol. 2006, 69, 1624-1632.
44. Flanagan, A. M.; Chambers, T. J., Dichloromethylenebisphosphonate (Cl2mbp) Inhibits
Bone-Resorption through Injury to Osteoclasts That Resorb Cl2mbp-Coated Bone. Bone Miner.
1989, 6, 33-43.
45. Ito, M.; Chokki, M.; Ogino, Y.; Satomi, Y.; Azuma, Y.; Ohta, T.; Kiyoki, M., Comparison
of cytotoxic effects of bisphosphonates in vitro and in vivo. Calcif. Tissue Int. 1998, 63, 143-147.
46. Breuil, V.; Cosman, F.; Stein, L.; Horbert, W.; Nieves, J.; Shen, V.; Lindsay, R.; Dempster,
D. W., Human osteoclast formation and activity in vitro: effects of alendronate. J. Bone Miner.
Res. 1998, 13, 1721-1729.
47. Hughes, D. E.; Wright, K. R.; Uy, H. L.; Sasaki, A.; Yoneda, T.; Roodman, G. D.; Mundy,
G. R.; Boyce, B. F., Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo.
J. Bone Miner. Res. 1995, 10, 1478-1487.
48. Selander, K. S.; Monkkonen, J.; Karhukorpi, E. K.; Harkonen, P.; Hannuniemi, R.;
Vaananen, H. K., Characteristics of clodronate-induced apoptosis in osteoclasts and macrophages.
Mol. Pharmacol. 1996, 50, 1127-1138.
49. Murakami, H.; Takahashi, N.; Sasaki, T.; Udagawa, N.; Tanaka, S.; Nakamura, I.; Zhang,
D.; Barbier, A.; Suda, T., A Possible Mechanism of the Specific Action of Bisphosphonates on
Osteoclasts - Tiludronate Preferentially Affects Polarized Osteoclasts Having Ruffled Borders.
Bone 1995, 17, 137-144.
50. Hughes, D. E.; MacDonald, B. R.; Russell, R. G.; Gowen, M., Inhibition of osteoclast-like
cell formation by bisphosphonates in long-term cultures of human bone marrow. J. Clin. Invest.
1989, 83, 1930-1935.
51. Boonekamp, P. M.; van der Wee-Pals, L. J.; van Wijk-van Lennep, M. M.; Thesing, C. W.;
Bijvoet, O. L., Two modes of action of bisphosphonates on osteoclastic resorption of mineralized
matrix. Bone Miner. 1986, 1, 27-39.
52. Lowik, C. W. G. M.; Vanderpluijm, G.; Vanderweepals, L. J. A.; Vantreslongdegroot, H.
B.; Bijvoet, O. L. M., Migration and Phenotypic Transformation of Osteoclast Precursors into
Mature Osteoclasts - the Effect of a Bisphosphonate. J. Bone Miner. Res. 1988, 3, 185-192.
53. Masarachia, P.; Weinreb, M.; Balena, R.; Rodan, G. A., Comparison of the distribution of
3H-alendronate and 3H-etidronate in rat and mouse bones. Bone 1996, 19, 281-290.
54. Azuma, Y.; Sato, H.; Oue, Y.; Okabe, K.; Ohta, T.; Tsuchimoto, M.; Kiyoki, M.,
Alendronate distributed on bone surfaces inhibits osteoclastic bone resorption in vitro and in
experimental hypercalcemia models. Bone 1995, 16, 235-245.
27

55. Sato, M.; Grasser, W., Effects of Bisphosphonates on Isolated Rat Osteoclasts as Examined
by Reflected Light-Microscopy. J. Bone Miner. Res. 1990, 5, 31-40.
56. Baron, R.; Ferrari, S.; Russell, R. G. G., Denosumab and bisphosphonates: Different
mechanisms of action and effects. Bone 2011, 48, 677-692.
57. Weinstein, R. S.; Roberson, P. K.; Manolagas, S. C., Giant osteoclast formation and long-
term oral bisphosphonate therapy. N. Engl. J. Med. 2009, 360, 53-62.
58. Plotkin, L. I.; Weinstein, R. S.; Parfitt, A. M.; Roberson, P. K.; Manolagas, S. C.; Bellido,
T., Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J. Clin.
Invest. 1999, 104, 1363-1374.
59. Plotkin, L. I.; Bivi, N.; Bellido, T., A bisphosphonate that does not affect osteoclasts
prevents osteoblast and osteocyte apoptosis and the loss of bone strength induced by
glucocorticoids in mice. Bone 2011, 49, 122-127.
60. Roelofs, A. J.; Jauhiainen, M.; Monkkonen, H.; Rogers, M. J.; Monkkonen, J.; Thompson,
K., Peripheral blood monocytes are responsible for gammadelta T cell activation induced by
zoledronic acid through accumulation of IPP/DMAPP. Br. J. Haematol. 2009, 144, 245-250.
61. Roelofs, A. J.; Thompson, K.; Ebetino, F. H.; Rogers, M. J.; Coxon, F. P., Bisphosphonates:
molecular mechanisms of action and effects on bone cells, monocytes and macrophages. Curr.
Pharm. Des. 2010, 16, 2950-2960.
62. Coxon, F. P.; Thompson, K.; Roelofs, A. J.; Ebetino, F. H.; Rogers, M. J., Visualizing
mineral binding and uptake of bisphosphonate by osteoclasts and non-resorbing cells. Bone 2008,
42, 848-860.
63. Fast, D. K.; Felix, R.; Dowse, C.; Neuman, W. F.; Fleisch, H., The effects of
diphosphonates on the growth and glycolysis of connective-tissue cells in culture. Biochem. J.
1978, 172, 97-107.
64. David, P.; Nguyen, H.; Barbier, A.; Baron, R., The bisphosphonate tiludronate is a potent
inhibitor of the osteoclast vacuolar H(+)-ATPase. J. Bone Miner. Res. 1996, 11, 1498-1507.
65. Carano, A.; Teitelbaum, S. L.; Konsek, J. D.; Schlesinger, P. H.; Blair, H. C.,
Bisphosphonates directly inhibit the bone resorption activity of isolated avian osteoclasts in vitro.
J. Clin. Invest. 1990, 85, 456-461.
66. Zimolo, Z.; Wesolowski, G.; Rodan, G. A., Acid extrusion is induced by osteoclast
attachment to bone. Inhibition by alendronate and calcitonin. J. Clin. Invest. 1995, 96, 2277-2283.
67. Opas, E. E.; Rutledge, S. J.; Golub, E.; Stern, A.; Zimolo, Z.; Rodan, G. A.; Schmidt, A.,
Alendronate inhibition of protein-tyrosine-phosphatase-meg1. Biochem. Pharmacol. 1997, 54,
721-727.
28

68. Murakami, H.; Takahashi, N.; Tanaka, S.; Nakamura, I.; Udagawa, N.; Nakajo, S.; Nakaya,
K.; Abe, M.; Yuda, Y.; Konno, F.; Barbier, A.; Suda, T., Tiludronate inhibits protein tyrosine
phosphatase activity in osteoclasts. Bone 1997, 20, 399-404.
69. Felix, R.; Graham, R.; Russell, G.; Fleisch, H., The effect of several diphosphonates on
acid phosphohydrolases and other lysosomal enzymes. Biochim. Biophys. Acta 1976, 429, 429-
438.
70. Lerner, U. H.; Larsson, A., Effects of four bisphosphonates on bone resorption, lysosomal
enzyme release, protein synthesis and mitotic activities in mouse calvarial bones in vitro. Bone
1987, 8, 179-189.
71. Nishikawa, M.; Akatsu, T.; Katayama, Y.; Yasutomo, Y.; Kado, S.; Kugal, N.; Yamamoto,
M.; Nagata, N., Bisphosphonates act on osteoblastic cells and inhibit osteoclast formation in mouse
marrow cultures. Bone 1996, 18, 9-14.
72. Yu, X.; Scholler, J.; Foged, N. T., Interaction between effects of parathyroid hormone and
bisphosphonate on regulation of osteoclast activity by the osteoblast-like cell line UMR-106. Bone
1996, 19, 339-345.
73. Sahni, M.; Guenther, H. L.; Fleisch, H.; Collin, P.; Martin, T. J., Bisphosphonates act on
rat bone resorption through the mediation of osteoblasts. J. Clin. Invest. 1993, 91, 2004-2011.
74. Vitte, C.; Fleisch, H.; Guenther, H. L., Bisphosphonates induce osteoblasts to secrete an
inhibitor of osteoclast-mediated resorption. Endocrinology 1996, 137, 2324-2333.
75. Klein, G.; Martin, J. B.; Satre, M., Methylenediphosphonate, a Metabolic Poison in
Dictyostelium-Discoideum - P-31 Nmr Evidence for Accumulation of Adenosine 5'-
(Beta,Gamma-Methylenetriphosphate) and Diadenosine 5',5'''-P1,P4-(P2,P3-
Methylenetetraphosphate). Biochemistry-Us 1988, 27, 1897-1901.
76. Rogers, M. J.; Russell, R. G.; Blackburn, G. M.; Williamson, M. P.; Watts, D. J.,
Metabolism of halogenated bisphosphonates by the cellular slime mould Dictyostelium
discoideum. Biochem. Biophys. Res. Commun. 1992, 189, 414-423.
77. Frith, J. C.; Monkkonen, J.; Blackburn, G. M.; Russell, R. G.; Rogers, M. J., Clodronate
and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5'-(beta,
gamma-dichloromethylene) triphosphate, by mammalian cells in vitro. J. Bone Miner. Res. 1997,
12, 1358-1367.
78. Benford, H. L.; Frith, J. C.; Auriola, S.; Monkkonen, J.; Rogers, M. J., Farnesol and
geranylgeraniol prevent activation of caspases by aminobisphosphonates: biochemical evidence
for two distinct pharmacological classes of bisphosphonate drugs. Mol. Pharmacol. 1999, 56, 131-
140.
79. Rogers, M. J.; Ji, X.; Russell, R. G.; Blackburn, G. M.; Williamson, M. P.; Bayless, A. V.;
Ebetino, F. H.; Watts, D. J., Incorporation of bisphosphonates into adenine nucleotides by amoebae
of the cellular slime mould Dictyostelium discoideum. Biochem. J. 1994, 303 ( Pt 1), 303-311.
29

80. Pelorgeas, S.; Martin, J. B.; Satre, M., Cytotoxicity of dichloromethane diphosphonate and
of 1-hydroxyethane-1,1-diphosphonate in the amoebae of the slime mould Dictyostelium
discoideum. A 31P NMR study. Biochem. Pharmacol. 1992, 44, 2157-2163.
81. Rogers, M. J.; Xiong, X.; Brown, R. J.; Watts, D. J.; Russell, R. G.; Bayless, A. V.; Ebetino,
F. H., Structure-activity relationships of new heterocycle-containing bisphosphonates as inhibitors
of bone resorption and as inhibitors of growth of Dictyostelium discoideum amoebae. Mol.
Pharmacol. 1995, 47, 398-402.
82. Rogers, M. J.; Brown, R. J.; Hodkin, V.; Blackburn, G. M.; Russell, R. G.; Watts, D. J.,
Bisphosphonates are incorporated into adenine nucleotides by human aminoacyl-tRNA synthetase
enzymes. Biochem. Biophys. Res. Commun. 1996, 224, 863-869.
83. Sillero, M. A.; de Diego, A.; Silles, E.; Perez-Zuniga, F.; Sillero, A., Synthesis of
bisphosphonate derivatives of ATP by T4 RNA ligase. FEBS Lett. 2006, 580, 5723-5727.
84. Flanagan, A. M.; Chambers, T. J., Dichloromethylenebisphosphonate (Cl2MBP) inhibits
bone resorption through injury to osteoclasts that resorb Cl2MBP-coated bone. Bone Miner. 1989,
6, 33-43.
85. Schenk, R.; Merz, W. A.; Muhlbauer, R.; Russell, R. G.; Fleisch, H., Effect of ethane-1-
hydroxy-1,1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl 2 MDP) on the
calcification and resorption of cartilage and bone in the tibial epiphysis and metaphysis of rats.
Calcif. Tissue Res. 1973, 11, 196-214.
86. Selander, K.; Lehenkari, P.; Vaananen, H. K., The effects of bisphosphonates on the
resorption cycle of isolated osteoclasts. Calcif. Tissue Int. 1994, 55, 368-375.
87. Hiroi-Furuya, E.; Kameda, T.; Hiura, K.; Mano, H.; Miyazawa, K.; Nakamaru, Y.;
Watanabe-Mano, M.; Okuda, N.; Shimada, J.; Yamamoto, Y.; Hakeda, Y.; Kumegawa, M.,
Etidronate (EHDP) inhibits osteoclastic-bone resorption, promotes apoptosis and disrupts actin
rings in isolate-mature osteoclasts. Calcif. Tissue Int. 1999, 64, 219-223.
88. Lehenkari, P. P.; Kellinsalmi, M.; Napankangas, J. P.; Ylitalo, K. V.; Monkkonen, J.;
Rogers, M. J.; Azhayev, A.; Vaananen, H. K.; Hassinen, I. E., Further insight into mechanism of
action of clodronate: inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable,
adenine-containing metabolite. Mol. Pharmacol. 2002, 61, 1255-1262.
89. Benford, H. L.; McGowan, N. W.; Helfrich, M. H.; Nuttall, M. E.; Rogers, M. J.,
Visualization of bisphosphonate-induced caspase-3 activity in apoptotic osteoclasts in vitro. Bone
2001, 28, 465-473.
90. Kroemer, G.; Reed, J. C., Mitochondrial control of cell death. Nat. Med. 2000, 6, 513-519.
91. Reszka, A. A.; Halasy-Nagy, J. M.; Masarachia, P. J.; Rodan, G. A., Bisphosphonates act
directly on the osteoclast to induce caspase cleavage of mst1 kinase during apoptosis. A link
between inhibition of the mevalonate pathway and regulation of an apoptosis-promoting kinase. J.
Biol. Chem. 1999, 274, 34967-34973.
30

92. Halasy-Nagy, J. M.; Rodan, G. A.; Reszka, A. A., Inhibition of bone resorption by
alendronate and risedronate does not require osteoclast apoptosis. Bone 2001, 29, 553-559.
93. Sutherland, K. A.; Rogers, H. L.; Tosh, D.; Rogers, M. J., RANKL increases the level of
Mcl-1 in osteoclasts and reduces bisphosphonate-induced osteoclast apoptosis in vitro. Arthritis
Res. Ther. 2009, 11, R58.
94. Zhang, Q.; Badell, I. R.; Schwarz, E. M.; Boulukos, K. E.; Yao, Z.; Boyce, B. F.; Xing, L.,
Tumor necrosis factor prevents alendronate-induced osteoclast apoptosis in vivo by stimulating
Bcl-xL expression through Ets-2. Arthritis Rheum. 2005, 52, 2708-2718.
95. Amin, D.; Cornell, S. A.; Gustafson, S. K.; Needle, S. J.; Ullrich, J. W.; Bilder, G. E.;
Perrone, M. H., Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase
and cholesterol biosynthesis. J. Lipid Res. 1992, 33, 1657-1663.
96. Amin, D.; Cornell, S. A.; Perrone, M. H.; Bilder, G. E., 1-Hydroxy-3-
(methylpentylamino)-propylidene-1,1-bisphosphonic acid as a potent inhibitor of squalene
synthase. Arzneimittelforschung 1996, 46, 759-762.
97. Rogers, M. J.; Chilton, K. M.; Coxon, F. P.; Lawry, J.; Smith, M. O.; Suri, S.; Russell, R.
G., Bisphosphonates induce apoptosis in mouse macrophage-like cells in vitro by a nitric oxide-
independent mechanism. J. Bone Miner. Res. 1996, 11, 1482-1491.
98. Thompson, K.; Dunford, J. E.; Ebetino, F. H.; Rogers, M. J., Identification of a
bisphosphonate that inhibits isopentenyl diphosphate isomerase and farnesyl diphosphate synthase.
Biochem. Biophys. Res. Commun. 2002, 290, 869-873.
99. Kavanagh, K. L.; Dunford, J. E.; Bunkoczi, G.; Russell, R. G.; Oppermann, U., The crystal
structure of human geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement
and inhibitory product binding. J. Biol. Chem. 2006, 281, 22004-22012.
100. Dunford, J. E.; Kwaasi, A. A.; Rogers, M. J.; Barnett, B. L.; Ebetino, F. H.; Russell, R. G.;
Oppermann, U.; Kavanagh, K. L., Structure-activity relationships among the nitrogen containing
bisphosphonates in clinical use and other analogues: time-dependent inhibition of human farnesyl
pyrophosphate synthase. J. Med. Chem. 2008, 51, 2187-2195.
101. Dunford, J. E.; Thompson, K.; Coxon, F. P.; Luckman, S. P.; Hahn, F. M.; Poulter, C. D.;
Ebetino, F. H.; Rogers, M. J., Structure-activity relationships for inhibition of farnesyl diphosphate
synthase in vitro and inhibition of bone resorption in vivo by nitrogen-containing bisphosphonates.
J. Pharmacol. Exp. Ther. 2001, 296, 235-242.
102. Keller, R. K.; Fliesler, S. J., Mechanism of aminobisphosphonate action: characterization
of alendronate inhibition of the isoprenoid pathway. Biochem. Biophys. Res. Commun. 1999, 266,
560-563.
103. van Beek, E.; Pieterman, E.; Cohen, L.; Lowik, C.; Papapoulos, S., Nitrogen-containing
bisphosphonates inhibit isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase
31

activity with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo.
Biochem. Biophys. Res. Commun. 1999, 255, 491-494.
104. Bergstrom, J. D.; Bostedor, R. G.; Masarachia, P. J.; Reszka, A. A.; Rodan, G., Alendronate
is a specific, nanomolar inhibitor of farnesyl diphosphate synthase. Arch. Biochem. Biophys. 2000,
373, 231-241.
105. Zhang, F. L.; Casey, P. J., Protein prenylation: molecular mechanisms and functional
consequences. Annu. Rev. Biochem. 1996, 65, 241-269.
106. Konstantinopoulos, P. A.; Karamouzis, M. V.; Papavassiliou, A. G., Post-translational
modifications and regulation of the RAS superfamily of GTPases as anticancer targets. Nat. Rev.
Drug Discov. 2007, 6, 541-555.
107. Ridley, A. J.; Hall, A., The small GTP-binding protein rho regulates the assembly of focal
adhesions and actin stress fibers in response to growth factors. Cell 1992, 70, 389-399.
108. Ridley, A. J.; Paterson, H. F.; Johnston, C. L.; Diekmann, D.; Hall, A., The small GTP-
binding protein rac regulates growth factor-induced membrane ruffling. Cell 1992, 70, 401-410.
109. Zerial, M.; Stenmark, H., Rab GTPases in vesicular transport. Curr. Opin. Cell Biol. 1993,
5, 613-620.
110. Zhang, D.; Udagawa, N.; Nakamura, I.; Murakami, H.; Saito, S.; Yamasaki, K.; Shibasaki,
Y.; Morii, N.; Narumiya, S.; Takahashi, N.; et al., The small GTP-binding protein, rho p21, is
involved in bone resorption by regulating cytoskeletal organization in osteoclasts. J. Cell Sci. 1995,
108 ( Pt 6), 2285-2292.
111. Luckman, S. P.; Hughes, D. E.; Coxon, F. P.; Graham, R.; Russell, G.; Rogers, M. J.,
Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-
translational prenylation of GTP-binding proteins, including Ras. J. Bone Miner. Res. 1998, 13,
581-589.
112. Luckman, S. P.; Coxon, F. P.; Ebetino, F. H.; Russell, R. G.; Rogers, M. J., Heterocycle-
containing bisphosphonates cause apoptosis and inhibit bone resorption by preventing protein
prenylation: evidence from structure-activity relationships in J774 macrophages. J. Bone Miner.
Res. 1998, 13, 1668-1678.
113. Fisher, J. E.; Rogers, M. J.; Halasy, J. M.; Luckman, S. P.; Hughes, D. E.; Masarachia, P.
J.; Wesolowski, G.; Russell, R. G.; Rodan, G. A.; Reszka, A. A., Alendronate mechanism of action:
geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast
formation, bone resorption, and kinase activation in vitro. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
133-138.
114. Kavanagh, K. L.; Guo, K.; Dunford, J. E.; Wu, X.; Knapp, S.; Ebetino, F. H.; Rogers, M.
J.; Russell, R. G.; Oppermann, U., The molecular mechanism of nitrogen-containing
bisphosphonates as antiosteoporosis drugs. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 7829-7834.
32

115. Coxon, F. P.; Helfrich, M. H.; Van't Hof, R.; Sebti, S.; Ralston, S. H.; Hamilton, A.; Rogers,
M. J., Protein geranylgeranylation is required for osteoclast formation, function, and survival:
inhibition by bisphosphonates and GGTI-298. J. Bone Miner. Res. 2000, 15, 1467-1476.
116. van beek, E.; Lowik, C.; van der Pluijm, G.; Papapoulos, S., The role of
geranylgeranylation in bone resorption and its suppression by bisphosphonates in fetal bone
explants in vitro: A clue to the mechanism of action of nitrogen-containing bisphosphonates. J.
Bone Miner. Res. 1999, 14, 722-729.
117. Grove, J. E.; Brown, R. J.; Watts, D. J., The intracellular target for the antiresorptive
aminobisphosphonate drugs in Dictyostelium discoideum is the enzyme farnesyl diphosphate
synthase. J. Bone Miner. Res. 2000, 15, 971-981.
118. Ory, B.; Moriceau, G.; Trichet, V.; Blanchard, F.; Berreur, M.; Redini, F.; Rogers, M.;
Heymann, D., Farnesyl diphosphate synthase is involved in the resistance to zoledronic acid of
osteosarcoma cells. J. Cell Mol. Med. 2008, 12, 928-941.
119. Salomo, M.; Jurlander, J.; Nielsen, L. B.; Gimsing, P., How myeloma cells escape
bisphosphonate-mediated killing: development of specific resistance with preserved sensitivity to
conventional chemotherapeutics. Br. J. Haematol. 2003, 122, 202-210.
120. Montalvetti, A.; Bailey, B. N.; Martin, M. B.; Severin, G. W.; Oldfield, E.; Docampo, R.,
Bisphosphonates are potent inhibitors of Trypanosoma cruzi farnesyl pyrophosphate synthase. J.
Biol. Chem. 2001, 276, 33930-33937.
121. Martin, M. B.; Sanders, J. M.; Kendrick, H.; de Luca-Fradley, K.; Lewis, J. C.; Grimley, J.
S.; Van Brussel, E. M.; Olsen, J. R.; Meints, G. A.; Burzynska, A.; Kafarski, P.; Croft, S. L.;
Oldfield, E., Activity of bisphosphonates against Trypanosoma brucei rhodesiense. J. Med. Chem.
2002, 45, 2904-2914.
122. Martin, M. B.; Grimley, J. S.; Lewis, J. C.; Heath, H. T., 3rd; Bailey, B. N.; Kendrick, H.;
Yardley, V.; Caldera, A.; Lira, R.; Urbina, J. A.; Moreno, S. N.; Docampo, R.; Croft, S. L.; Oldfield,
E., Bisphosphonates inhibit the growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania
donovani, Toxoplasma gondii, and Plasmodium falciparum: a potential route to chemotherapy. J.
Med. Chem. 2001, 44, 909-916.
123. Montalvetti, A.; Fernandez, A.; Sanders, J. M.; Ghosh, S.; Van Brussel, E.; Oldfield, E.;
Docampo, R., Farnesyl pyrophosphate synthase is an essential enzyme in Trypanosoma brucei. In
vitro RNA interference and in vivo inhibition studies. J. Biol. Chem. 2003, 278, 17075-17083.
124. Rondeau, J. M.; Bitsch, F.; Bourgier, E.; Geiser, M.; Hemmig, R.; Kroemer, M.; Lehmann,
S.; Ramage, P.; Rieffel, S.; Strauss, A.; Green, J. R.; Jahnke, W., Structural basis for the
exceptional in vivo efficacy of bisphosphonate drugs. ChemMedChem 2006, 1, 267-273.
125. Coxon, F. P.; Ebetino, F. H.; Mules, E. H.; Seabra, M. C.; McKenna, C. E.; Rogers, M. J.,
Phosphonocarboxylate inhibitors of Rab geranylgeranyl transferase disrupt the prenylation and
membrane localization of Rab proteins in osteoclasts in vitro and in vivo. Bone 2005, 37, 349-358.
33

126. Baron, R. A.; Tavare, R.; Figueiredo, A. C.; Blazewska, K. M.; Kashemirov, B. A.;
McKenna, C. E.; Ebetino, F. H.; Taylor, A.; Rogers, M. J.; Coxon, F. P.; Seabra, M. C.,
Phosphonocarboxylates inhibit the second geranylgeranyl addition by Rab geranylgeranyl
transferase. J. Biol. Chem. 2009, 284, 6861-6868.
127. McKenna, C. E.; Kashemirov, B. A.; Blazewska, K. M.; Mallard-Favier, I.; Stewart, C. A.;
Rojas, J.; Lundy, M. W.; Ebetino, F. H.; Baron, R. A.; Dunford, J. E.; Kirsten, M. L.; Seabra, M.
C.; Bala, J. L.; Marma, M. S.; Rogers, M. J.; Coxon, F. P., Synthesis, chiral high performance
liquid chromatographic resolution and enantiospecific activity of a potent new geranylgeranyl
transferase inhibitor, 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid. J. Med.
Chem. 2010, 53, 3454-3464.
128. Coxon, F. P.; Helfrich, M. H.; Larijani, B.; Muzylak, M.; Dunford, J. E.; Marshall, D.;
McKinnon, A. D.; Nesbitt, S. A.; Horton, M. A.; Seabra, M. C.; Ebetino, F. H.; Rogers, M. J.,
Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl transferase that
specifically prevents Rab prenylation in osteoclasts and macrophages. J. Biol. Chem. 2001, 276,
48213-48222.
129. Varela, I.; Pereira, S.; Ugalde, A. P.; Navarro, C. L.; Suarez, M. F.; Cau, P.; Cadinanos, J.;
Osorio, F. G.; Foray, N.; Cobo, J.; de Carlos, F.; Levy, N.; Freije, J. M.; Lopez-Otin, C., Combined
treatment with statins and aminobisphosphonates extends longevity in a mouse model of human
premature aging. Nat. Med. 2008, 14, 767-772.
130. Fournier, P. G.; Daubine, F.; Lundy, M. W.; Rogers, M. J.; Ebetino, F. H.; Clezardin, P.,
Lowering bone mineral affinity of bisphosphonates as a therapeutic strategy to optimize skeletal
tumor growth inhibition in vivo. Cancer Res. 2008, 68, 8945-8953.
131. Thompson, K.; Rogers, M. J., Statins prevent bisphosphonate-induced gamma,delta-T-cell
proliferation and activation in vitro. J. Bone Miner. Res. 2004, 19, 278-288.
132. Gober, H. J.; Kistowska, M.; Angman, L.; Jeno, P.; Mori, L.; De Libero, G., Human T cell
receptor gammadelta cells recognize endogenous mevalonate metabolites in tumor cells. J. Exp.
Med. 2003, 197, 163-168.
133. Thompson, K.; Roelofs, A. J.; Jauhiainen, M.; Monkkonen, H.; Monkkonen, J.; Rogers, M.
J., Activation of gammadelta T cells by bisphosphonates. Adv. Exp. Med. Biol. 2010, 658, 11-20.
134. Monkkonen, H.; Auriola, S.; Lehenkari, P.; Kellinsalmi, M.; Hassinen, I. E.; Vepsalainen,
J.; Monkkonen, J., A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine
nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing
bisphosphonates. Br. J. Haematol. 2006, 147, 437-445.
135. Monkkonen, H.; Kuokkanen, J.; Holen, I.; Evans, A.; Lefley, D. V.; Jauhiainen, M.;
Auriola, S.; Monkkonen, J., Bisphosphonate-induced ATP analog formation and its effect on
inhibition of cancer cell growth. Anti-Cancer Drugs 2008, 19, 391-399.
136. Raikkonen, J.; Crockett, J. C.; Rogers, M. J.; Monkkonen, H.; Auriola, S.; Monkkonen, J.,
Zoledronic acid induces formation of a pro-apoptotic ATP analogue and isopentenyl
34

pyrophosphate in osteoclasts in vivo and in MCF-7 cells in vitro. Br. J. Haematol. 2009, 157, 427-
435.
137. Coscia, M.; Quaglino, E.; Iezzi, M.; Curcio, C.; Pantaleoni, F.; Riganti, C.; Holen, I.;
Monkkonen, H.; Boccadoro, M.; Forni, G.; Musiani, P.; Bosia, A.; Cavallo, F.; Massaia, M.,
Zoledronic acid repolarizes tumour-associated macrophages and inhibits mammary carcinogenesis
by targeting the mevalonate pathway. J. Cell Mol. Med. 2010, 14, 2803-2815.
138. Jauhiainen, M.; Monkkonen, H.; Raikkonen, J.; Monkkonen, J.; Auriola, S., Analysis of
endogenous ATP analogs and mevalonate pathway metabolites in cancer cell cultures using liquid
chromatography-electrospray ionization mass spectrometry. J. Chromatogr. B Analyt. Technol.
Biomed. Life Sci. 2009, 877, 2967-2975.
139. Mitrofan, L. M.; Pelkonen, J.; Monkkonen, J., The level of ATP analog and isopentenyl
pyrophosphate correlates with zoledronic acid-induced apoptosis in cancer cells in vitro. Bone
2009, 45, 1153-1160.
140. Sato, M.; Grasser, W., Effects of bisphosphonates on isolated rat osteoclasts as examined
by reflected light microscopy. J. Bone Miner. Res. 1990, 5, 31-40.
141. Jain, N.; Weinstein, R. S., Giant osteoclasts after long-term bisphosphonate therapy:
diagnostic challenges. Nat. Rev. Rheumatol. 2009, 5, 341-346.
142. Stancu, C.; Sima, A., Statins: mechanism of action and effects. J. Cell Mol. Med. 2001, 5,
378-387.
143. Staal, A.; Frith, J. C.; French, M. H.; Swartz, J.; Gungor, T.; Harrity, T. W.; Tamasi, J.;
Rogers, M. J.; Feyen, J. H., The ability of statins to inhibit bone resorption is directly related to
their inhibitory effect on HMG-CoA reductase activity. J. Bone Miner. Res. 2003, 18, 88-96.
144. Coxon, F. P.; Benford, H. L.; Russell, R. G.; Rogers, M. J., Protein synthesis is required
for caspase activation and induction of apoptosis by bisphosphonate drugs. Mol. Pharmacol. 1998,
54, 631-638.
145. Hall, A., Rho GTPases and the actin cytoskeleton. Science 1998, 279, 509-514.
146. Canfield, R.; Rosner, W.; Skinner, J.; McWhorter, J.; Resnick, L.; Feldman, F.;
Kammerman, S.; Ryan, K.; Kunigonis, M.; Bohne, W., Diphosphonate therapy of paget's disease
of bone. J. Clin. Endocrinol. Metab. 1977, 44, 96-106.
147. Glorieux, F. H.; Bishop, N. J.; Plotkin, H.; Chabot, G.; Lanoue, G.; Travers, R., Cyclic
administration of pamidronate in children with severe osteogenesis imperfecta. N. Engl. J. Med.
1998, 339, 947-952.
148. Rauch, F.; Glorieux, F. H., Osteogenesis imperfecta, current and future medical treatment.
Am. J. Med. Genet. C Semin. Med. Genet. 2005, 139C, 31-37.
35

149. Finerman, G. A.; Stover, S. L., Heterotopic ossification following hip replacement or spinal
cord injury. Two clinical studies with EHDP. Metab. Bone Dis. Relat. Res. 1981, 3, 337-342.
150. Singer, F. R.; Ritch, P. S.; Lad, T. E.; Ringenberg, Q. S.; Schiller, J. H.; Recker, R. R.;
Ryzen, E., Treatment of hypercalcemia of malignancy with intravenous etidronate. A controlled,
multicenter study. The Hypercalcemia Study Group. Arch. Intern. Med. 1991, 151, 471-476.
151. Ibrahim, A.; Scher, N.; Williams, G.; Sridhara, R.; Li, N.; Chen, G.; Leighton, J.; Booth,
B.; Gobburu, J. V.; Rahman, A.; Hsieh, Y.; Wood, R.; Vause, D.; Pazdur, R., Approval summary
for zoledronic acid for treatment of multiple myeloma and cancer bone metastases. Clin. Cancer
Res. 2003, 9, 2394-2399.
152. Coleman, R. E.; McCloskey, E. V., Bisphosphonates in oncology. Bone 2011, 49, 71-76.
153. Santini, D.; Caraglia, M.; Vincenzi, B.; Holen, I.; Scarpa, S.; Budillon, A.; Tonini, G.,
Mechanisms of disease: Preclinical reports of antineoplastic synergistic action of bisphosphonates.
Nat. Clin. Pract. Oncol. 2006, 3, 325-338.
154. Watts, N. B.; Harris, S. T.; Genant, H. K.; Wasnich, R. D.; Miller, P. D.; Jackson, R. D.;
Licata, A. A.; Ross, P.; Woodson, G. C., 3rd; Yanover, M. J.; et al., Intermittent cyclical etidronate
treatment of postmenopausal osteoporosis. N. Engl. J. Med. 1990, 323, 73-79.
155. Black, D. M.; Delmas, P. D.; Eastell, R.; Reid, I. R.; Boonen, S.; Cauley, J. A.; Cosman,
F.; Lakatos, P.; Leung, P. C.; Man, Z.; Mautalen, C.; Mesenbrink, P.; Hu, H.; Caminis, J.; Tong,
K.; Rosario-Jansen, T.; Krasnow, J.; Hue, T. F.; Sellmeyer, D.; Eriksen, E. F.; Cummings, S. R.;
Trial, H. P. F., Once-yearly zoledronic acid for treatment of postmenopausal osteoporosis. N. Engl.
J. Med. 2007, 356, 1809-1822.
156. Reid, I. R.; Brown, J. P.; Burckhardt, P.; Horowitz, Z.; Richardson, P.; Trechsel, U.;
Widmer, A.; Devogelaer, J. P.; Kaufman, J. M.; Jaeger, P.; Body, J. J.; Brandi, M. L.; Broell, J.;
Di Micco, R.; Genazzani, A. R.; Felsenberg, D.; Happ, J.; Hooper, M. J.; Ittner, J.; Leb, G.;
Mallmin, H.; Murray, T.; Ortolani, S.; Rubinacci, A.; Saaf, M.; Samsioe, G.; Verbruggen, L.;
Meunier, P. J., Intravenous zoledronic acid in postmenopausal women with low bone mineral
density. N. Engl. J. Med. 2002, 346, 653-661.
157. Szabo, C. M.; Matsumura, Y.; Fukura, S.; Martin, M. B.; Sanders, J. M.; Sengupta, S.;
Cieslak, J. A.; Loftus, T. C.; Lea, C. R.; Lee, H. J.; Koohang, A.; Coates, R. M.; Sagami, H.;
Oldfield, E., Inhibition of geranylgeranyl diphosphate synthase by bisphosphonates and
diphosphates: a potential route to new bone antiresorption and antiparasitic agents. J. Med. Chem.
2002, 45, 2185-2196.
158. Artz, J. D.; Wernimont, A. K.; Dunford, J. E.; Schapira, M.; Dong, A.; Zhao, Y.; Lew, J.;
Russell, R. G.; Ebetino, F. H.; Oppermann, U.; Hui, R., Molecular characterization of a novel
geranylgeranyl pyrophosphate synthase from Plasmodium parasites. J. Biol. Chem. 2011, 286,
3315-3322.
159. Artz, J. D.; Dunford, J. E.; Arrowood, M. J.; Dong, A.; Chruszcz, M.; Kavanagh, K. L.;
Minor, W.; Russell, R. G.; Ebetino, F. H.; Oppermann, U.; Hui, R., Targeting a uniquely
36

nonspecific prenyl synthase with bisphosphonates to combat cryptosporidiosis. Chem. Biol. 2008,
15, 1296-1306.
160. Coppens, I., Targeting lipid biosynthesis and salvage in apicomplexan parasites for
improved chemotherapies. Nat. Rev. Microbiol. 2013, 11, 823-835.
161. Ling, Y.; Sahota, G.; Odeh, S.; Chan, J. M.; Araujo, F. G.; Moreno, S. N.; Oldfield, E.,
Bisphosphonate inhibitors of Toxoplasma gondi growth: in vitro, QSAR, and in vivo
investigations. J. Med. Chem. 2005, 48, 3130-3140.
162. Moreno, B.; Bailey, B. N.; Luo, S.; Martin, M. B.; Kuhlenschmidt, M.; Moreno, S. N.;
Docampo, R.; Oldfield, E., (31)P NMR of apicomplexans and the effects of risedronate on
Cryptosporidium parvum growth. Biochem. Biophys. Res. Commun. 2001, 284, 632-637.
163. Rodriguez, N.; Bailey, B. N.; Martin, M. B.; Oldfield, E.; Urbina, J. A.; Docampo, R.,
Radical cure of experimental cutaneous leishmaniasis by the bisphosphonate pamidronate. J. Infect.
Dis. 2002, 186, 138-140.
164. Mukkamala, D.; No, J. H.; Cass, L. M.; Chang, T. K.; Oldfield, E., Bisphosphonate
inhibition of a Plasmodium farnesyl diphosphate synthase and a general method for predicting
cell-based activity from enzyme data. J. Med. Chem. 2008, 51, 7827-7833.
165. Singh, A. P.; Zhang, Y.; No, J. H.; Docampo, R.; Nussenzweig, V.; Oldfield, E., Lipophilic
bisphosphonates are potent inhibitors of Plasmodium liver-stage growth. Antimicrob. Agents
Chemother. 2010, 54, 2987-2993.
166. Yardley, V.; Khan, A. A.; Martin, M. B.; Slifer, T. R.; Araujo, F. G.; Moreno, S. N.;
Docampo, R.; Croft, S. L.; Oldfield, E., In vivo activities of farnesyl pyrophosphate synthase
inhibitors against Leishmania donovani and Toxoplasma gondii. Antimicrob. Agents Chemother.
2002, 46, 929-931.
167. Reid, I. R., Efficacy, effectiveness and side effects of medications used to prevent fractures.
J. Intern. Med. 2015, 277, 690-706.
168. Reid, I. R., Short-term and long-term effects of osteoporosis therapies. Nat. Rev.
Endocrinol. 2015, 11, 418-428.
169. Reid, I. R.; Gamble, G. D.; Mesenbrink, P.; Lakatos, P.; Black, D. M., Characterization of
and risk factors for the acute-phase response after zoledronic acid. J. Clin. Endocrinol. Metab.
2010, 95, 4380-4387.
170. Wark, J. D.; Bensen, W.; Recknor, C.; Ryabitseva, O.; Chiodo, J., 3rd; Mesenbrink, P.; de
Villiers, T. J., Treatment with acetaminophen/paracetamol or ibuprofen alleviates post-dose
symptoms related to intravenous infusion with zoledronic acid 5 mg. Osteoporos. Int. 2012, 23,
503-512.
171. Sanders, J. M.; Ghosh, S.; Chan, J. M.; Meints, G.; Wang, H.; Raker, A. M.; Song, Y.;
Colantino, A.; Burzynska, A.; Kafarski, P.; Morita, C. T.; Oldfield, E., Quantitative structure-
37

activity relationships for gammadelta T cell activation by bisphosphonates. J. Med. Chem. 2004,
47, 375-384.
172. Khan, A. A.; Morrison, A.; Hanley, D. A.; Felsenberg, D.; McCauley, L. K.; O'Ryan, F.;
Reid, I. R.; Ruggiero, S. L.; Taguchi, A.; Tetradis, S.; Watts, N. B.; Brandi, M. L.; Peters, E.;
Guise, T.; Eastell, R.; Cheung, A. M.; Morin, S. N.; Masri, B.; Cooper, C.; Morgan, S. L.;
Obermayer-Pietsch, B.; Langdahl, B. L.; Al Dabagh, R.; Davison, K. S.; Kendler, D. L.; Sandor,
G. K.; Josse, R. G.; Bhandari, M.; El Rabbany, M.; Pierroz, D. D.; Sulimani, R.; Saunders, D. P.;
Brown, J. P.; Compston, J.; International Task Force on Osteonecrosis of the, J., Diagnosis and
management of osteonecrosis of the jaw: a systematic review and international consensus. J. Bone
Miner. Res. 2015, 30, 3-23.
173. Grbic, J. T.; Landesberg, R.; Lin, S. Q.; Mesenbrink, P.; Reid, I. R.; Leung, P. C.; Casas,
N.; Recknor, C. P.; Hua, Y.; Delmas, P. D.; Eriksen, E. F.; Health, O.; Reduced Incidence with
Zoledronic Acid Once Yearly Pivotal Fracture Trial Research, G., Incidence of osteonecrosis of
the jaw in women with postmenopausal osteoporosis in the health outcomes and reduced incidence
with zoledronic acid once yearly pivotal fracture trial. J. Am. Dent. Assoc. 2008, 139, 32-40.
174. Grbic, J. T.; Black, D. M.; Lyles, K. W.; Reid, D. M.; Orwoll, E.; McClung, M.; Bucci-
Rechtweg, C.; Su, G., The incidence of osteonecrosis of the jaw in patients receiving 5 milligrams
of zoledronic acid: data from the health outcomes and reduced incidence with zoledronic acid once
yearly clinical trials program. J. Am. Dent. Assoc. 2010, 141, 1365-1370.
175. Assael, L. A., Oral bisphosphonates as a cause of bisphosphonate-related osteonecrosis of
the jaws: clinical findings, assessment of risks, and preventive strategies. J. Oral Maxillofac. Surg.
2009, 67, 35-43.
176. Manfredi, M.; Merigo, E.; Guidotti, R.; Meleti, M.; Vescovi, P., Bisphosphonate-related
osteonecrosis of the jaws: a case series of 25 patients affected by osteoporosis. Int. J. Oral
Maxillofac. Surg. 2011, 40, 277-284.
177. Odvina, C. V.; Zerwekh, J. E.; Rao, D. S.; Maalouf, N.; Gottschalk, F. A.; Pak, C. Y.,
Severely suppressed bone turnover: a potential complication of alendronate therapy. J. Clin.
Endocrinol. Metab. 2005, 90, 1294-1301.
178. Ott, S. M., Long-term safety of bisphosphonates. J. Clin. Endocrinol. Metab. 2005, 90,
1897-1899.
179. Mashiba, T.; Hirano, T.; Turner, C. H.; Forwood, M. R.; Johnston, C. C.; Burr, D. B.,
Suppressed bone turnover by bisphosphonates increases microdamage accumulation and reduces
some biomechanical properties in dog rib. J. Bone Miner. Res. 2000, 15, 613-620.
180. Mashiba, T.; Mori, S.; Burr, D. B.; Komatsubara, S.; Cao, Y.; Manabe, T.; Norimatsu, H.,
The effects of suppressed bone remodeling by bisphosphonates on microdamage accumulation and
degree of mineralization in the cortical bone of dog rib. J. Bone Miner. Metab. 2005, 23 Suppl, 36-
42.
38

181. Flora, L.; Hassing, G. S.; Cloyd, G. G.; Bevan, J. A.; Parfitt, A. M.; Villanueva, A. R., The
long-term skeletal effects of EHDP in dogs. Metab. Bone Dis. Relat. Res. 1981, 3, 289-300.
182. Kwek, E. B.; Goh, S. K.; Koh, J. S.; Png, M. A.; Howe, T. S., An emerging pattern of
subtrochanteric stress fractures: a long-term complication of alendronate therapy? Injury 2008, 39,
224-231.
183. Black, D. M.; Kelly, M. P.; Genant, H. K.; Palermo, L.; Eastell, R.; Bucci-Rechtweg, C.;
Cauley, J.; Leung, P. C.; Boonen, S.; Santora, A.; de Papp, A.; Bauer, D. C.; Fracture Intervention
Trial Steering, C.; Committee, H. P. F. T. S., Bisphosphonates and fractures of the subtrochanteric
or diaphyseal femur. N. Engl. J. Med. 2010, 362, 1761-1771.
184. Nieves, J. W.; Bilezikian, J. P.; Lane, J. M.; Einhorn, T. A.; Wang, Y.; Steinbuch, M.;
Cosman, F., Fragility fractures of the hip and femur: incidence and patient characteristics.
Osteoporos. Int. 2010, 21, 399-408.
185. Black, D. M.; Schwartz, A. V.; Ensrud, K. E.; Cauley, J. A.; Levis, S.; Quandt, S. A.;
Satterfield, S.; Wallace, R. B.; Bauer, D. C.; Palermo, L.; Wehren, L. E.; Lombardi, A.; Santora,
A. C.; Cummings, S. R.; Group, F. R., Effects of continuing or stopping alendronate after 5 years
of treatment: the Fracture Intervention Trial Long-term Extension (FLEX): a randomized trial.
JAMA 2006, 296, 2927-2938.
186. Black, D. M.; Reid, I. R.; Boonen, S.; Bucci-Rechtweg, C.; Cauley, J. A.; Cosman, F.;
Cummings, S. R.; Hue, T. F.; Lippuner, K.; Lakatos, P.; Leung, P. C.; Man, Z.; Martinez, R. L.;
Tan, M.; Ruzycky, M. E.; Su, G.; Eastell, R., The effect of 3 versus 6 years of zoledronic acid
treatment of osteoporosis: a randomized extension to the HORIZON-Pivotal Fracture Trial (PFT).
J. Bone Miner. Res. 2012, 27, 243-254.
187. Fizazi, K.; Carducci, M.; Smith, M.; Damiao, R.; Brown, J.; Karsh, L.; Milecki, P.; Shore,
N.; Rader, M.; Wang, H.; Jiang, Q.; Tadros, S.; Dansey, R.; Goessl, C., Denosumab versus
zoledronic acid for treatment of bone metastases in men with castration-resistant prostate cancer:
a randomised, double-blind study. Lancet 2011, 377, 813-822.
188. Cummings, S. R.; San Martin, J.; McClung, M. R.; Siris, E. S.; Eastell, R.; Reid, I. R.;
Delmas, P.; Zoog, H. B.; Austin, M.; Wang, A.; Kutilek, S.; Adami, S.; Zanchetta, J.; Libanati, C.;
Siddhanti, S.; Christiansen, C.; Trial, F., Denosumab for prevention of fractures in postmenopausal
women with osteoporosis. N. Engl. J. Med. 2009, 361, 756-765.
189. Papapoulos, S.; Lippuner, K.; Roux, C.; Lin, C. J. F.; Kendler, D. L.; Lewiecki, E. M.;
Brandi, M. L.; Czerwinski, E.; Franek, E.; Lakatos, P.; Mautalen, C.; Minisola, S.; Reginster, J.
Y.; Jensen, S.; Daizadeh, N. S.; Wang, A.; Gavin, M.; Libanati, C.; Wagman, R. B.; Bone, H. G.,
The effect of 8 or 5 years of denosumab treatment in postmenopausal women with osteoporosis:
results from the FREEDOM Extension study. Osteoporos. Int. 2015, 26, 2773-2783.
190. Leder, B. Z.; Tsai, J. N.; Uihlein, A. V.; Burnett-Bowie, S. A.; Zhu, Y.; Foley, K.; Lee, H.;
Neer, R. M., Two years of Denosumab and teriparatide administration in postmenopausal women
with osteoporosis (The DATA Extension Study): a randomized controlled trial. J. Clin. Endocrinol.
Metab. 2014, 99, 1694-1700.
39

191. Cosman, F.; Eriksen, E. F.; Recknor, C.; Miller, P. D.; Guanabens, N.; Kasperk, C.;
Papanastasiou, P.; Readie, A.; Rao, H.; Gasser, J. A.; Bucci-Rechtweg, C.; Boonen, S., Effects of
intravenous zoledronic acid plus subcutaneous teriparatide [rhPTH(1-34)] in postmenopausal
osteoporosis. J. Bone Miner. Res. 2011, 26, 503-511.
192. Paparodis, R.; Buehring, B.; Pelley, E. M.; Binkley, N., A case of an unusual
subtrochanteric fracture in a patient receiving denosumab. Endocr. Pract. 2013, 19, e64-68.
193. Watts, N. B.; Chines, A.; Olszynski, W. P.; McKeever, C. D.; McClung, M. R.; Zhou, X.;
Grauer, A., Fracture risk remains reduced one year after discontinuation of risedronate. Osteoporos.
Int. 2008, 19, 365-372.

40

CHAPTER 2
Targeted Drug Delivery using Functionalized Bisphosphonate
Gold Nanoparticles
2.1 Background
Bone metastasis is a major concern in cancers involving solid tumors. Breast and prostate
cancers in particular have a high propensity of metastasizing to bone, which is associated with
high mortality rate.
1
For instance, relapses with axially-distributed bone metastasis, verified by
autopsy, are present in approximately 80% of patients diagnosed with advanced forms of prostate
cancer.
2, 3
Patients with bone metastasis commonly experience severe bone pain and fractures.
4

While local interventions, including surgery or external radiation, can mitigate these symptoms,
they are only palliative remedies and frequently employed in patients at late metastatic stages.
5

Current palliative care approaches for patients diagnosed with bone metastases include
chemotherapy or hormone therapy in combination with bone-targeting agents.
5
Chemotherapy and
hormone therapy relieve patients from tumor burden, while bone-targeting agents, such as
bisphosphonates (BPs) and denosumab, disrupt bone turnover via their specific absorption by the
bone.
5
This helps delay related skeletal events (e.g., fractures, bone damage, hypercalcemia) as
well as preventing and treating osteoporosis, a common phenomenon observed during hormonal
therapy.
6
In turn, the inhibition of bone turnover diminishes bone destruction induced by metastatic
cells and, consequently, tumor growth.
5, 6
Yet, theses treatment do not address the key problem,
bone metastasis, and the risk of relapse.
Metastatic cells are active at the bone surface where BPs localize. BPs exert their effect on
osteoclast-mediated bone resorption and impede the production of bone-drive growth factors.
41

These growth factors are one of the many elements that participate in sustaining tumor
progression.
5
However, BPs are poor anti-cancer agents and, thus, ineffective in completely halting
the activities of metastatic cells. The unique high affinity of BPs for bone metastatic niches could
potentially benefit targeted delivery of antineoplastic agents to malignant cells at the bone sites
and prevent further bone metastasis. However, this would require the development of a versatile
carrier containing different moieties that allow its conjugation to not only BPs but also therapeutic
agents.
Thanks to advances in synthesis and functionalization, nanomaterials, particularly gold
nanoparticles (AuNPs), have recently emerged as a promising candidate for in vivo targeted drug
delivery in cancer.
7, 8
A key advantage of AuNPs is their high surface to volume ratio, allowing
dense functionalization with other targeting moieties while still maintaining their nano sizes
(diameter 10 - 60 nm).
9
This helps retain the enhanced permeability retention (EPR) effect for
efficient in vivo biodistribution to targeted tissue and good body clearance. AuNPs are highly
efficient at transporting therapeutic agents, such as doxorubicin, to cancer tumors,
10, 11
suggesting
that they are excellent platforms for selective in vivo delivery of anticancer agents.
The use of AuNPs as carriers in vivo greatly depends on robust surface chemistry of coating
the NPs with highly biocompatible ligands. The Pinaud lab at USC recently developed a surface
coating chemistry of AuNPs using metal-chelating biomimetic peptides,
12
containing two key
components. First, a metal-chelating binding domain consisting of multi-cysteine residues
facilitates coordination and hydrophobic interactions of the peptide to the surface of AuNPs. The
other component, a more hydrophilic domain, enhances the stability of AuNPs in physiological
environments and allows precise control of their biochemical properties.
13, 14
This domain also
enables selective, facile multi-functionalization via coating with various ligands containing a wide
42

range of attachment groups.
15
Direct functionalization can be achieved within the peptide coat or
via post-coating bioconjugation chemistries (e.g., linkages formed using succinimidyl ester reacted
with terminal lysine or hydrazine/aldehyde biorthogonal click-chemistry
14
). To enhance
biocompatibility, polyethylene glycol (PEG) is commonly incorporated into the AuNPs coating.
This has also been found to greatly prolong circulation half-time and increase targeting efficiency
in vivo.
9
Furthermore, this technique reportedly improved renal clearance of peptide-coated
nanomaterials compared to other bulkier surface chemistries.
16
Results suggest AuNPs coating
using this method are suitable for in vivo targeted drug delivery. In addition to their use as drug
carriers, AuNPs are also being employed in various diagnostic and therapeutic applications, such
as photothermal therapies and imaging.
9

As a collaborative project with the Pinaud lab, this work focuses on designing a novel
AuNP carrier using polypeptide-coated surface chemistry to develop a multimodal platform that
not only targets bone metastasis, but also transports chemotherapeutics to a targeted site. The
proposed nanoplatform would have three key functions: 1) to target AuNPs to bone metastatic
niches, 2) to promote the internalization of the nanoparticles by metastatic cells from the bone
surface, and 3) to release chemotherapeutic agents, such as doxorubicin, from AuNP carriers using
acid-triggered cleavage (see Figure 2.1). The presence of the AuNPs would induce site-specific
elimination of metastases and therefore, halt bone metastatic tumor progression.
The proposed AuNPs were first coated with cathepsin K peptides. Targeted delivery of the
NPs would be achieved by having multiple bisphosphonate moieties linked to cathepsin K peptides
coated on the outside nanoparticles. The peptide and BPs are connected by an amide linkage
formed between the ε-amino group on the peptide with an activated carboxylic BP derivative. This
43

synthetic work, therefore, is centered on the development of a carboxyl activated bisphosphonate
derivative.

Figure 2.1. Overview of the proposed AuNP platform.
2.2 Results and Discussion
Previous work developed in the McKenna group presented a new approach in modifying
N-containing bisphosphonates—particularly risedronate (RIS), zoledronate (ZOL), and
minodronate (MIN)—allowing the introduction of an additional amino group using an epoxide
linker.
17, 18
This new BP derivative, shown as 2.3 in Scheme 2.1, still exhibits significant
therapeutic activities of the BP parent drug in inhibiting bone absorption.
17, 19
Hence, 2.2, or RIS
linker, was chosen as the starting point for this synthetic work. A carboxyl moiety was incorporated
into the BP linker via its reaction with N-hydroxysuccinimide-activated succinic acid (Scheme
2.1).
44

H P L C w a t e r , p H 8 . 3
O
O
O
N - H y d r o x y s u c ci n i m i d e
C o u p l i n g R e a g e n t s
( e . g . , H A T U , D C C , E D C )
A n h . D M F
N
P
O H
P
N H
2
O H
O H
O H
O
O H
O
O H
N
P
O H
P
H
N
O H
O H
O H
O
O H
O
O H
O
O H
O
2 . 1
2 . 2
2 . 3
N
P
O H
P
H
N
O H
O H
O H
O
O H
O
O H
O
O
O
2 . 4
N
O
O
1 / N - h y d r o x y s u c c i n i m i d e
A n h . D M F
2 /

Scheme 2.1. Synthesis of the extended RIS-linker
Mass analysis of the reaction mixture revealed that the synthesis of 2.3 was efficient with
no RIS linker detected. The newly synthesized extended RIS-linker was purified via reverse phase
(RP) high liquid performance chromatography (HPLC) using a strong anion exchange (SAX)
column. The product of interest was eluted after 15 minutes and well separated from other
compounds in the reaction mixture. Compound 2.3 was characterized using
1
H,
31
P NMR
spectroscopy, and ESI-MS, with a mass of 455 m/z detected in negative mode, confirming its
formation. An unexpected side product was detected in this mode with a mass of 555 m/z,
appearing as the third peak in the chromatograph. The compound was determined to be 2.3 with
one additional succinic acid attached via an ester bond with the secondary hydroxyl group of the
RIS linker. Product 2.3 could be quantitatively recovered via basic hydrolysis of the side product
with sodium hydroxide.
The final step of the synthesis was to activate 2.3 with N-hydroxysuccinimide using
coupling reagents. A wide range of coupling reagents, including N,N'-Dicyclohexylcarbodiimide
(DCC), N,N'-Diisopropylcarbodiimide (DIC), and 1-[Bis(dimethylamino)methylene]-1H-1,2,3-
triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), was employed to investigate this
45

reaction. In order to conserve 2.3, pamidronic acid (PAM) (2.5), a readily available and simpler
system of N-BP, was utilized as the model compound to study the reaction (Scheme 2.2). Using
an approach similar to that depicted in Scheme 2.1, the PAM acid linker (2.6) was isolated with
quantitative yield from starting material (Scheme 2.2). Previously published work conducted by
Nazimov et al. reported that selective activation of carboxyl moiety in the presence of phosphate
group was feasible using HATU.
20
This coupling reaction of 2.6 with N-hydroxysuccinimide
(SuOH) using HATU was performed to obtain the target compound 2.7.

Scheme 2.2. Synthetic scheme of the target compound 2.7
The newly formed compound was obtained in quantitative yield and readily precipitated in
isopropanol with great purity. Unfortunately, experimental
1
H and
31
P spectroscopic data indicated
that the product obtained was not 2.7. Instead, the data suggested that its structure would be 2.8,
an NHS-carboxyl activated pamidronate with O-phosphorylisourea adduct. Further
characterization of the compound was not possible since it was not detectable in mass spectrometry.

Figure 2.2. Chemical structure of 2.8
46

Nevertheless, a test reaction of 2.8 with lysine was conducted to determine whether it
contained an activated carboxyl group. Analysis of the reaction mixture using mass spectrometry
indicated that besides 2.6, two conjugated products were formed, including 2.8 coupled with one
lysine molecule (2.9, Figure 2.3) and 2.8 coupled with two lysine molecules (2.10, Figure 2.3).

Figure 2.3. Possible conjugated products of 2.8 with lysine
To further confirm that the activation occurred at both the phosphonate and carboxylate
sites, the reaction mixture was adjusted to acidic pH via Dowex exchange and left stirring
overnight. Under acidic conditions, the P-N bond would be cleaved. Consequently, 2.9 would be
the only compound detected in the solution besides unreacted starting material 2.6. Again, analysis
of the reaction mixture using mass spectrometry confirmed that 2.9 was still present in the solution.
Optimization of the NHS-activation reaction was attempted using various conditions to maximize
the formation of 2.7 while suppressing the formation of 2.8 (Table 2.1). Nonetheless, 2.8 formed
in most cases, as verified by
1
H and
31
P NMR spectroscopy. This indicated that the selective
activation at a carboxyl group in the presence of bisphosphonate moiety was not achievable.

47

Solvent
Coupling
Reagent
Eq. Base Eq. Product Formation
DMF DCC 1.2 TBA 4 Decomposition of starting materials
DMF DCC 1.2 TBA ~3 Trace amount of product detected via MS
DMF HATU 2 TBA ~2.5 Trace amount of product detected via MS
DMF HATU 1.5 TBA 3 Product formed with an additional peak observed in
31
P
NMR (tested in coupling reaction with Lys)
DMF HATU 1.0 TBA 3 Similar results as seen in previous experiment
DMF HATU 1.0 TBA 5 Similar results as seen in previous experiment
DMF DIC 5 TBA 3 Activation at phosphate group detected via
31
P NMR
Dioxane: DMF DCC 1 TBA 3 Activation at phosphate group detected via
31
P NMR
Table 2.1. Various coupling conditions for the activation reaction of 2.6. TBA: tributylamine;
DCC: N,N'-dicyclohexylcarbodiimide; DIC: N,N′-Diisopropylcarbodiimide.
Since activation of both the carboxyl and phosphonyl sites was unavoidable, a new
approach was required to obtain the target compound. Instead of relying on a coupling reagent to
activate the carboxyl group in the last step, a dicarboxylic compound, such as adipic acid, would
be double-activated with N-hydroxysuccinimide (Scheme 2.3). The double-activated acid was
isolated and subsequently, reacted with pamidronate to yield the desired compound 2.13.

Scheme 2.3. Synthesis of NHS-activated pamidronate
Double NHS-activated adipic acid 2.12 was synthesized from reaction of 2.11 in the
presence of at least 2 equivalents of a coupling agent, such as N-(3-Dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (EDC), and SuOH in dioxane with quantitative yield. The
compound was solely soluble in either DMF or DMSO. The coupling of 2.12 with pamidronate,
48

as tributylammonium salts, was conducted in anhydrous DMF to prevent the hydrolysis of NHS-
activated acid. Reaction progress, monitored using
31
P NMR, indicated that the reaction was
significantly slow, yielding about 5% of 2.13 after 24 hours at room temperature. In order to
increase the reaction rate various conditions, such as increasing the reaction temperature and
adding different equivalents of 2.12, were utilized to increase the formation of 2.13 (Table 2.2).
Among the tested conditions, heating at 80
o
C for 9 hours in the presence of 1.3 equivalents of
2.12 consumed most of the starting material with the minimal amount of side product formed; with
only 2 hours at the same temperature, the reaction yielded approximately 37.5% of 2.13
decomposed into 2.14, according to
1
H NMR. Thus, the final condition for synthesis of 2.13 was
3 equivalents of 2.12 and heated at to 80
o
C for 2 hours.
Temperature (
o
C) Time (h) % of side product monitored using
31
P
NMR spectroscopy
Equivalent of Di-NHS
adipic ester
60 18 17 3
80 2 26 3
80 5.5 10 1.3
80 9 5.2 1.3
Table 2.2. Optimization of BPs and NHS-activated adipic acid
Generally, purification of BP derivatives requires the use of RP HPLC. However, the target
product 2.13 is not detectable via UV-Vis spectroscopy and is unstable in aqueous conditions. A
use of RP HPLC was not an option, purification was problematic. In order to purify 2.13 from
pamidronic acid, after the solvent was removed, the reaction mixture was quickly processed 3-5
times through columns of Dowex resins to remove the N-BP starting material.

Figure 2.4. Structure of the hydrolyzed product of 2.14
49

It is important to note that the yield of 2.13 decreased as the number of Dowex exchanges
increased. It was because 2.13 was hydrolyzed during Dowex exchanges and became 2.14 (Figure
2.4). For instance, while 12.5% of the target product degraded after running through Dowex
columns 3 times, 65% of target product remained after 5 Dowex exchanges (Figure 2.5),
determined via
1
H NMR spectroscopy.

Figure 2.5. Comparison of
1
H NMR spectra of the product after 3 and 5 times of DOWEX
exchanges. The relative yield was estimated based on the ratio of the integration of the signals 4
protons of the succinimidyl ester (dashed blue boxes) versus the 2 protons in the alkyl chain next
to the amino group (solid red boxes) in pamidronate.
Isolating the target product from starting BP materials was difficult due to instability of
NHS-activated carboxyl moiety in aqueous solvents. Thus, in order to obtain the maximal amount
of 2.13, the purity of the target product was compromised. Two different samples with different
50

purities were prepared. The first sample was passed through Dowex resin 3 times and contained
27% of 2.5 and 60% of 2.13, in which 16% of 2.13 decomposed into 2.14. The second sample was
purified by passing the reaction mixture through Dowex resin 5 times and comprised of 10% and
82% of 2.5 and 2.13, respectively. In this sample, 35% of 2.13 decomposed into 2.14.
2.3 Conclusion
In conclusion, a derivative of N-BP containing an activated carboxyl moiety was
successfully synthesized. The target product was obtained via the coupling reaction of pamidronic
acid with double-activated adipic acid rather than by using conventional approaches to activate a
carboxyl moiety employing coupling reagents. Purification of the compound presented a unique
challenge due its instability in aqueous solvents, which required a fast and efficient purification
method. Rapid Dowex exchange of reaction mixture helped to address these concerns while
removing most of the starting material. A fraction of product still decomposed, and its degradation
depended on the number of times the reaction mixture was exchanged with Dowex resin.
2.4 Experimental section
Reagents and Spectral Measurements. All chemical compounds were purchased from either
Aldrich or Alfa Aesar. Triethylamine (TEA) was distilled from KOH, and dioxane was distilled
from sodium. Other compounds were used as supplied by the manufacturer. HPLC separations
were performed on a Rainan Dynamax Model SD-200 system with a Rainan Dynamax absorbance
detector Model UV-DII. NMR spectra were recorded on either 400 MHz or 500 MHz Varian
spectrometers. MS was conducted on a Thermo-Finnigan LCQ DECA XP MAX Ion Trap mass
spectrometer operated in ESI mode.
Synthesis of 1-(3-amino-2-hydroxypropyl)-3-(2-hydroxy-2,2-diphosphonoethyl) pyridin-1-ium
(2.2). The compound was synthesized using previously reported method.
17, 18

51

1
H NMR (500 MHz, Deuterium Oxide) δ 8.83 (s, 1H), 8.66 (d, J = 6.0 Hz, 1H), 8.56 (d, J = 8.0
Hz, 1H), 7.98 – 7.93 (m, 1H), 4.82 (s, 1H), 4.53 – 4.44 (m, 1H), 4.36 (t, J = 9.7 Hz, 1H), 3.56 (d,
J = 7.1 Hz, 1H), 3.48 (t, J = 12.3 Hz, 2H), 3.37 (d, J = 15.2 Hz, 1H), 3.13 – 3.03 (m, 1H).
31
P NMR
(202 MHz, Deuterium Oxide) δ 16.80 – 16.45 (m).
Synthesis of 1-(3-(3-carboxypropanamido)-2-hydroxypropyl)-3-(2-hydroxy-2,2-diphosphono-
ethyl)pyridin-1-ium (2.3). In a 10 mL flask, 100.1 mg of 2.1 (1.0 mmol, 1 equivalent) and 230 mg
of N-hydroxysuccinimide (SuOH) (2.0 mmol, 2 equivalents) were dissolved in 0.5 mL of
anhydrous DMF. The reaction was left stirring for 30 minutes. Compound 2.2 (50.68 mg, 0.09
mmol, 0.09 equivalent) was dissolved in 0.5 mL of water and pH was adjusted to 8.3 using solid
sodium carbonate. The solution was added into solution of 2.1, and the reaction mixture was stirred
overnight. Purification of 2.3 was performed via RP HPLC using preparative strong anion
exchange (SAX) column. Buffers A and B for the purification were water and 0.5 M
triethylammonium bicarbonate (TEAB) (pH 7.5), respectively. Method for the purification had the
following gradients: 0-30% of buffer B for 10 min, followed by staying at 30% of buffer B for 5
min, and 30-100% of buffer B for 25 min, with flow rate at 9.0 mL/min and detection set at 260
nm. The target product was eluted at 22 min and verified via MS. Solvent was removed and the
product was collected as triethylammonium salts. (66%)

1
H NMR (500 MHz, Deuterium Oxide) δ 8.68 (s, 1H), 8.52 (d, J = 6.1 Hz, 1H), 8.43 (d, J = 8.1
Hz, 1H), 7.82 (dd, J = 8.0, 6.2 Hz, 1H), 4.65 (d, J = 2.9 Hz, 1H), 4.33 – 4.24 (m, 1H), 4.09 (dq, J
= 9.2, 6.5, 4.6 Hz, 1H), 3.42 – 3.22 (m, 4H), 2.42 – 2.37 (m, 4H).
31
P NMR (202 MHz, Deuterium
Oxide) δ 17.0-15.1.
Synthesis of 4-((3-hydroxy-3,3-diphosphonopropyl)amino)-4-oxobutanoic acid (2.6). In a 10 mL
flask, 255.7 mg of 2.1 (2.56 mmol, 1 equivalent) and 368.1 mg of SuOH (3.2 mmol, 1.4 equivalents)
52

were dissolved in 0.5 mL of anhydrous DMF. The reaction was left stirring for 30 minutes.
Pamidronic acid (2.5) (111.6 mg, 0.47 mmol, 0.2 equivalent) was dissolved in 1.0 mL of water,
and pH was adjusted to 8.3 using solid sodium carbonate. The solution of 2.1 was added into the
solution of 2.5 in three portions and the reaction mixture was stirred overnight. Complete
consumption of pamidronate was monitored using
31
P NMR spectroscopy. Solvent was removed
and the reaction mixture was dissolved in water. Dowex exchange was performed and solvent was
removed from the collected eluent. The target product was precipitated using ether and dried under
vacuum, yielding 70.6 mg (44%).
1
H NMR (500 MHz, Deuterium Oxide) δ 3.57 – 3.38 (m, 2H), 2.65 (t, J = 6.3 Hz, 2H), 2.53 (t, J
= 6.2 Hz, 2H), 2.27 – 2.05 (m, 2H).
31
P NMR (202 MHz, Deuterium Oxide) δ 18.5 – 17.7 (m).
Synthesis of 1,1'-[(1,6-Dioxo-1,6-hexanediyl)bis(oxy)]di(2,5-pyrrolidinedione) (2.12). Adipic acid
(500.4 mg, 3.4 mmol, 1 equivalent), SuOH (985 mg, 8.6 mmol, 2.5 equivalent) and N-(3-
Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) (1.635 g, 8.6 mmol, 2.5
equivalent) were dissolved in 13 mL of dioxane, and the reaction mixture was stirred overnight.
Solvent was removed and 2.12 was precipitated in isopropanol. The white solid was washed with
ether and dried under vacuum, yielding 1.16 g. (95%)
1
H NMR (400 MHz, DMSO-d6) δ 2.79 (s, 8H), 2.75 – 2.68 (m, 4H), 1.74 – 1.63 (m, 4H).
Synthesis of (3-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexanamido)-1-hydroxypropane-1,1-
diyl)bis(phosphonic acid) (2.13). In a 25 mL flask, 50.2 mg of 2.5 (0.214 mmol, 1 equivalent) was
dissolved in 1.0 mL of water. The solution was adjusted to basic using 117.9 mg of tributylamine
(0.636 mmol, 3 equivalent) dissolved in 1.0 mL ethanol. The final pH of the solution was 7.9.
Solvent was removed and the residue was washed with anhydrous DMF twice. In the same flask,
219.39 mg of 2.12 (0.27 mmol, 3.0 equivalent), was added and the residue was dissolved in 2 mL
53

of anhydrous DMF. The reaction mixture was heated to 80
o
C and left stirring for 2 hours. Solvent
was removed. The reaction mixture was dissolved in 0.5 mL of water and passed through Dowex
columns for 3 times, yielding 9.5 mg of sample containing 60% 2.13, 27% starting material, and
13% side product determined using
31
P NMR spectroscopy.
1
H NMR (500 MHz, Deuterium Oxide) δ 3.42 – 3.35 (m, 2H), 3.25 (dt, J = 14.2, 6.9 Hz, 1H), 2.81
(s, 3H), 2.62 (t, J = 6.4 Hz, 2H), 2.16 (t, J = 24.6 Hz, 7H), 1.60 (dd, J = 23.8, 7.1 Hz, 4H), 1.53 –
1.39 (m, 1H).
31
P NMR (202 MHz, Deuterium Oxide) δ 18.7-18.5 (s, 2P).
2.5 References
1. Croucher, P. I.; McDonald, M. M.; Martin, T. J., Bone metastasis: the importance of the
neighbourhood. Nat. Rev. Cancer 2016, 16, 373-386.
2. Logothetis, C. J.; Navone, N. M.; Lin, S. H., Understanding the biology of bone metastases:
key to the effective treatment of prostate cancer. Clin. Cancer Res. 2008, 14, 1599-1602.
3. Shah, R. B.; Mehra, R.; Chinnaiyan, A. M.; Shen, R.; Ghosh, D.; Zhou, M.; Macvicar, G.
R.; Varambally, S.; Harwood, J.; Bismar, T. A.; Kim, R.; Rubin, M. A.; Pienta, K. J., Androgen-
independent prostate cancer is a heterogeneous group of diseases: lessons from a rapid autopsy
program. Cancer Res. 2004, 64, 9209-9216.
4. Suva, L. J.; Griffin, R. J.; Makhoul, I., Mechanisms of bone metastases of breast cancer.
Endocr. Relat. Cancer 2009, 16, 703-713.
5. Suva, L. J.; Washam, C.; Nicholas, R. W.; Griffin, R. J., Bone metastasis: mechanisms and
therapeutic opportunities. Nat. Rev. Endocrinol 2011, 7, 208-218.
6. Coleman, R. E.; McCloskey, E. V., Bisphosphonates in oncology. Bone 2011, 49, 71-76.
7. Lal, S.; Clare, S. E.; Halas, N. J., Nanoshell-enabled photothermal cancer therapy:
impending clinical impact. Acc. Chem. Res. 2008, 41, 1842-1851.
8. Loo, C.; Lin, A.; Hirsch, L.; Lee, M. H.; Barton, J.; Halas, N.; West, J.; Drezek, R.,
Nanoshell-enabled photonics-based imaging and therapy of cancer. Technol. Cancer Res. Treat.
2004, 3, 33-40.
9. Pedrosa, P.; Vinhas, R.; Fernandes, A.; Baptista, P. V., Gold Nanotheranostics: Proof-of-
Concept or Clinical Tool? Nanomaterials 2015, 5, 1853-1879.
54

10. Aryal, S.; Grailer, J. J.; Pilla, S.; Steeber, D. A.; Gong, S. Q., Doxorubicin conjugated gold
nanoparticles as water-soluble and pH-responsive anticancer drug nanocarriers. J. Mater. Chem.
2009, 19, 7879-7884.
11. Kim, B.; Han, G.; Toley, B. J.; Kim, C. K.; Rotello, V. M.; Forbes, N. S., Tuning payload
delivery in tumour cylindroids using gold nanoparticles. Nat. Nanotechnol. 2010, 5, 465-472.
12. Pinaud, F.; King, D.; Moore, H. P.; Weiss, S., Bioactivation and cell targeting of
semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides. J. Am. Chem. Soc.
2004, 126, 6115-6123.
13. Clarke, S.; Pinaud, F.; Beutel, O.; You, C.; Piehler, J.; Dahan, M., Covalent
monofunctionalization of peptide-coated quantum dots for single-molecule assays. Nano Lett.
2010, 10, 2147-2154.
14. Iyer, G.; Pinaud, F.; Xu, J.; Ebenstein, Y.; Li, J.; Chang, J.; Dahan, M.; Weiss, S., Aromatic
aldehyde and hydrazine activated peptide coated quantum dots for easy bioconjugation and live
cell imaging. Bioconjug Chem. 2011, 22, 1006-1011.
15. Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan,
G.; Wu, A. M.; Gambhir, S. S.; Weiss, S., Quantum dots for live cells, in vivo imaging, and
diagnostics. Science 2005, 307, 538-544.
16. Schipper, M. L.; Iyer, G.; Koh, A. L.; Cheng, Z.; Ebenstein, Y.; Aharoni, A.; Keren, S.;
Bentolila, L. A.; Li, J.; Rao, J.; Chen, X.; Banin, U.; Wu, A. M.; Sinclair, R.; Weiss, S.; Gambhir,
S. S., Particle size, surface coating, and PEGylation influence the biodistribution of quantum dots
in living mice. Small 2009, 5, 126-134.
17. Kashemirov, B. A.; Bala, J. L.; Chen, X.; Ebetino, F. H.; Xia, Z.; Russell, R. G.; Coxon, F.
P.; Roelofs, A. J.; Rogers, M. J.; McKenna, C. E., Fluorescently labeled risedronate and related
analogues: "magic linker" synthesis. Bioconjug Chem. 2008, 19, 2308-2310.
18. McKenna, C. E.; Kashemirov, B. A.; Bala, J. L. F. Synthesis of drug conjugates via reaction
with epoxide-containing linkers. US20080312440A1, 2008.
19. Sun, S.; Blazewska, K. M.; Kadina, A. P.; Kashemirov, B. A.; Duan, X.; Triffitt, J. T.;
Dunford, J. E.; Russell, R. G.; Ebetino, F. H.; Roelofs, A. J.; Coxon, F. P.; Lundy, M. W.;
McKenna, C. E., Fluorescent Bisphosphonate and Carboxyphosphonate Probes: A Versatile
Imaging Toolkit for Applications in Bone Biology and Biomedicine. Bioconjug Chem. 2016, 27,
329-340.
20. Nizamov, S.; Willig, K. I.; Sednev, M. V.; Belov, V. N.; Hell, S. W., Phosphorylated 3-
heteroarylcoumarins and their use in fluorescence microscopy and nanoscopy. Chemistry (Easton)
2012, 18, 16339-16348.

55

CHAPTER 3
Structure Relationship Activity Studies between Linker Lengths
and the Anti-Resorptivity of Dye-N-BP Conjugates
3.1 Introduction
Bisphosphonate drugs (BPs), especially the 3
rd
generation of nitrogen-containing BPs (N-
BPs) such as RIS and ZOL, are well known for their high bone affinity and clinical use in the
treatment osteoporosis and metastatic bone cancers.
1
However, the exact mechanism of their
cellular uptake and skeletal distribution remains unclear. In addition, the link between anti-
resorptive activity and their bone binding affinity has yet to be fully elucidated.
1
Thus, developing
suitable imaging probes via conjugation of N-BPs to fluorescent dyes
2
or labeling them with
radioactive tags,
3
would be valuable. Such tools could be used to map the skeletal distribution and
cellular uptake of N-BPs by osteoclasts and other cells, which may lead to a deeper understanding
of how N-BPs elicit their anti-tumor effect.
4

Studies performed in the 1990s using
3
H-labeled alendronate (ALN) and etidronate (EHDP)
revealed the localization of BP at the surfaces of both bone resorption and bone formation sites.
5,
6
Subsequently, Coxon et al. further elucidated the mechanism of the internalization of BPs and
quantified the uptake of BPs by osteoclasts and non-resorbing cells using ALN fluorescently
labeled with Alexa Fluor 488 (AF-ALN).
7
The AF-ALN was synthesized via conjugation of the γ-
amino group on ALN to NHS-activated Alexa Fluor 488 (AF488). Its purity was reported to be
approximately 7%.
8
Using a similar synthetic approach, other fluorescently labeled pamidronate
(PAM) conjugates were also prepared, including IRDye78-PAM (PAM78)
9
and IRDye800CW-
56

PAM (PAM800).
10
They were utilized for various imaging applications, including osteoclast
imaging, atherosclerosis imaging, and detection of microcalcification in breast cancer.
10, 11, 12, 13

Figure 3.1. The crystal structure of human FPPS co-crystallized with RIS.
14
Superimposed in gray
is the avian apo structure of FPPS.
Despite the facile conjugation between the existing amino group on N-BPs and NHS-
activated fluorescent dyes, this approach has two major drawbacks. First, as observed in the crystal
structure of human FPPS co-crystallized with RIS and ZOL, H-bonding of the nitrogen atom in
the pyridine ring of N-BPs with both the O
γ
of Thr-201 and the carbonyl oxygen of Lys-200 is are
key interactions of N-BPs with FPPS (Figure 3.1).
14
Therefore, using the nitrogen to form an amide
linkage to the fluorescent dye substantially decreases the basicity of nitrogen atom, and is
hypothesized to have a drastic effect on its interaction with FPPS and ultimately reduces their anti-
resorption activity. Second, this synthetic method cannot be applied to third generation N-BPs,
such RIS, ZOL and MIN, due to the absence of a terminal amino group. Therefore, a novel
synthetic route is required to obtain the fluorescently labeled RIS, ZOL, and MIN.
57

The McKenna group has developed a new synthetic approach described as a ‘magic linker’
to obtain novel fluorescently labeled derivatives of RIS and its analogs, including its
phosphonocarboxylate and deoxy forms.
15, 16
The fluorescently labeled BP analogues are prepared
by reacting the appropriate 'magic' epoxide linker with heterocyclic N-BPs under mild and aqueous
conditions (Scheme 3.1). Reaction progress is frequently monitored using
31
P NMR to reduce
formation of undesired dialkylated byproduct. This reaction introduces a primary amino group that
allows for facile conjugation of the N-BP linker with a succinimidyl ester-activated fluorescent
dye, yielding the desired dye-BP conjugate. The final conjugate can be easily purified using
reverse phase (RP) HPLC performed on a C18 column. Characterization and purity can be assessed
using UV-Vis, fluorescence, and NMR (
1
H and
31
P) spectroscopies. These probes enable
visualization of the uptake of heterocyclic N-BPs by bones, bone tissues, and cells. Their bone
binding affinity and anti-resorption/anti-prenylation activity of the parent drugs are still partially
retained with effective inhibitory concentration in the micromolar range.
15, 17

Scheme 3.1. Synthesis of RIS-linker 3.6 using the ‘magic linker’
Two of the major drawbacks of in vivo fluorescence imaging using visible dyes is the
limited penetration depth of visible light and tissue auto-fluorescence at these wavelengths. Near-
58

infrared (NIR) dyes, with emission ranging between 650 nm and 900 nm, offer many advantages
in observing in vitro and in vivo processes at the molecular level, including lower cellular
phototoxicity, higher penetration depth, and reduced scattering and background signal.
18
Thus,
they can potentially be a valuable clinical tool for noninvasive imaging, such as identification of
bone erosions in rheumatoid arthritis
19
or intraoperative imaging.
20
Despite many preliminary
preclinical and clinical studies, there are currently no other NIR dyes that are approved for use in
humans beside indocyanine green (ICG) and methylene blue (MB).
21

Figure 3.2. Chemical structure of dyes: 5-FAM, 6-FAM, AF647 and Sulfo-Cy5
While the 5,6-carboxyfluorescein (5,6-FAM) labeled BPs (e.g., 5,6-FAM-RIS and 5,6-
FAM-ZOL) partially retained anti-prenylation/anti-resorptive activity in J774 macrophages and in
osteoclasts. However, no significant anti-prenylation activity was observed with the NIR-BP
conjugates, including Alexa Fluor 647 (AF647), Cy5, and IRDye 800CW labeled compounds.
22

The synthetic method using the 'magic' epoxide linker has yet to yield biologically active NIR dye-
BP conjugates.
23

Here, a series of fluorescently labeled RIS conjugates with varying linker lengths was
prepared to attempt to reduce the fluorophore interference with cellular activity, and possibly
expand the utility of these NIR agents to address the unmet need for pharmacologically active
probes for noninvasive imaging. Linker lengths between the fluorescent dye and the pyridyl moiety
were extended to increase the distance of the dye from the GPP/DMAPP binding site, where N-
59

BPs mainly interact with the enzyme. The selected NIR dyes that were utilized for preparation of
the novel NIR-BP conjugates include AF647 and Sulfo-Cy5. Sulfo-Cy5 was used instead of its
original form Cy5, due to its higher solubility in aqueous solutions. In addition, 5,6-FAM dye-BP
conjugates with various linker lengths were also synthesized for reference. Investigations of their
in vitro anti-resorption and anti-prenylation activity were also performed on osteoclasts to further
elucidate the relationship between the biological activities of dye-BP conjugates and the length of
linkers.
3.2 Molecular docking using Autodock Vina

Figure 3.3. In Silico Molecular Docking Illustrating the Interaction of AF647RIS and 5-FAM-RIS
with FPPS
14
using AutoDock Vina 1.1.2.
24

In order to examine our hypothesis on the effect of the size of the chromophore on its
interference with the ability of dye-BP conjugates entering the active site of human farnesyl
pyrophosphate synthase (FPPS) enzyme, in silico molecular docking using AutoDock Vina 1.1.2
24

was performed for AF647-RIS and 5-FAM-RIS conjugates with the crystal structure of human
FPPS co-crystallized with RIS (PDB code 1YQ7).
14
Docking results were analyzed and their
60

interaction in the active site of the protein were compared to that of RIS in its co-crystallized
structure with human FPPS. Molecular docking calculations indicated that due to steric hindrance,
the size of AF647 prevented AF-RIS from entering the active site (Figure 3.2). Consequently, the
newly proposed AF-RIS conjugates containing an additional spacer between BP and the
fluorophore should enable the entrance of N-BP into the GPP/DMAPP binding site of human FPPS.
3.3 Chemical synthesis of fluorescent dye-BP conjugates with various linker lengths
The previously reported synthetic approach employing the ‘magic’ epoxide linker
functionalizes heterocyclic N-BPs (e.g., RIS).
15
This not only offers an active amino group that
readily reacts with NHS-activated fluorescent dyes but also provides a great spot for further
modifying the linker length between N-BPs and the fluorescent dye. Increasing the distance
between the fluorescent dye and the pyridyl moiety of RIS-linker can be achieved by reacting the
original RIS-linker with compounds containing a carboxylic acid and a NBoc-protected amino
group at the other end, which are commercially available.
The general scheme for the synthesis of these new RIS-linkers are shown in Scheme 3.2.
First, the carboxylic moiety was transformed into its activated ester form using N-
hydroxysuccinimide (NHS) in the presence of coupling reagents, such as EDC, with yield up to
80%. This was followed by reaction of the NHS-activated ester with RIS-linker under aqueous,
basic conditions to yield the extended RIS-linker. It is noteworthy that the previously reported
method for the synthesis of RIS-linker utilized the sodium salt of RIS, resulting in its high
hygroscopicity when precipitated in ether. Thus, monosodium salt of RIS was converted into its
acid form and subsequently converted into triethylammonium salts, which resulted in large crystal
formation when precipitated in ether with no significant hygroscopicity observed. Except for V2-
linker, the remaining unreacted carboxylic acid compound was easily removed via extraction of
61

the reaction mixture under acidic condition. The leftover RIS-linker was separated from the novel
RIS-linker via RP-HPLC using either preparative C18 column or strong anion exchange (SAX)
column. The pure extended NBoc protected RIS-linker was obtained, and NBoc deprotection was
performed using trifluoroacetic acid (TFA).
O G
O
N
O
O
H O G
O
E D C
A n h . D i o x a n e
N
P
O H
P
N H
2
O H
D M F/ H
2
O
p H 8 . 3
3 . 7 : G =
3 . 6
N H B o c
3 . 9 : G =
H
2
C C H
2
O
4
N H B o c
O H
O H
O
O H
O
O H
N
P
H O
P
H
N
O H
O H
O H
O
O H
O
O H
G
N H B o c
O
N
O
O
O H
3 . 8 : G =
6
3 . 1 0 : G =
3 . 1 2 : G =
H
2
C C H
2
O
4
3 . 1 1 : G =
6
3 . 1 3 : G =
3 . 1 5 : G =
H
2
C C H
2
O
4
3 . 1 4 : G =
6
T F A : H
2
O ( 1 : 1 v/ v )
N
P
H O
P
H
N
O H
O H
O H
O
O H
O
O H
G
N H
2
O
3 . 1 6 : G =
3 . 1 8 : G =
H
2
C C H
2
O
4
3 . 1 7 : G =
6

Scheme 3.2. Synthesis of Novel Risedronate Analogues with Various Linker Lengths
Without further purification, the new extended RIS-linkers were coupled with
commercially available NHS-activated fluorescent dyes under basic, aqueous conditions,
affording the desired dye-BP conjugates with various linker lengths. Detailed synthetic steps for
the new fluorescently-labeled risedronate analogues are depicted in Scheme 3.3. To examine the
effect of linkers on the bone binding affinity and anti-resorption/anti-prenylation of BP conjugates,
three different linkers were employed in this project. The chosen linkers contained an extension of
62

4, 8, and 14 atoms, totaling 12 novel fluorescently labeled BPs. Linker of length of 4, 8, and 14
atoms were noted as V1, M, and V2, respectively (Scheme 3.3).
The final fluorescently labeled BP conjugates were purified via RP-HPLC using a semi-
preparative C18 column. The previously reported method for the purification of AF647-BP
conjugates utilizes 0.1M triethylammonium acetate buffers containing methanol as the organic
solvent.
22
However, the new AF647-BPs with additional linkers formed a stable acetate salt,
resulting in a significant amount of residual acetate, obscuring the
1
H NMR spectrum of final
compound. Therefore, an alternative buffer system, 0.1M triethylammonium bicarbonate
containing acetonitrile as organic solvent was utilized to purify the dye-BPs conjugates from the
unreacted dye. The new purification method yielded similar separation of dye and dye-BP
conjugates as the previously reported method. Additionally, previous purification method for 5,6-
FAM-BPs used preparative TLC, where the conjugates were overloaded onto the plates and
developed in pure methanol to separate the dye from the desired product. Due to their negative
charge, BPs were trapped on the silica particles and they could only partially be removed via
extraction with water. This phenomenon was especially evident in the purification of conjugates
with linkers, such as V1 and M, resulting in their low yield. Moreover, silica was partially
dissolved in the water extract, causing additional complications in purifying the conjugates using
C18 RP-HPLC. Thus, extraction with ethyl acetate was employed instead to remove the leftover
dye. Since previous data suggested there is a significant difference between the anti-resorption of
5-FAM and 6-FAM conjugates, the 5- and 6-isomer of FAM conjugated analogues, obtained as a
mixture of 5,6-FAM after coupling reaction, were individually isolated at the final purification
step using preparative C18 RP HPLC.
63

Scheme 3.3. Synthesis of Fluorescently-labeled Risedronate Analogues with Various Linker
Lengths
Identity and purity of these compounds were verified using
1
H NMR,
31
P NMR, UV-Vis,
fluorescence spectroscopies, mass spectrometry, and LC-MS. All new dye conjugates were
lyophilized and stored at -20 ˚C. Among these conjugates, 5,6-FAM and AF647 labeled
compounds were stable in water as long as they were stored at -20 ˚C, S-Cy5-BP conjugates
decomposed even when stored in water at -20 ˚C, and therefore required immediate lyophilization
for long-term storage. Their decomposition was easily verified using fluorescence spectroscopy.
3.4 Preliminary studies on the anti-resorptive activities of NIR-BP conjugates
Preliminary biological assays on murine osteoclasts were conducted with various
concentrations of fluorescently labeled BP conjugates, ranging from 0.1 µM to 10 µM. In this
assay, the following compounds were analyzed: 3.19a - 3.19c (RIS-V1) and 3.21a - 3.21c (RIS-
64

V2). Quantitative analysis of osteoclast (OC) formation was evaluated based on two criteria: 1)
the number of osteoclast cells formed and 2) the area of the multinucleated osteoclast colonies.

Figure 3.4. Effect of dye-BPs on OC formation. (A) Visualization of OC colonies using tartrate-
resistant acid phosphatase (TRAP) activity staining. (B) Quantification of the number and area of
OCs. Note that the concentrations of RIS used for the experiments were 0.01, 0.1, and 1µM.
(Unpublished data)
The assays were also performed with 5-FAM-RIS and 6-FAM-RIS for comparison. Data
indicated that these novel dye-BP conjugates significantly affected OC formation at 10 μM but not
at or below 1 μM. In addition, while AF647-RIS was not effective in reducing the formation of
osteoclast even at 10 mM, AF647-RIS-V1 and AF647-RIS-V2 were both active, with the AF-RIS-
V1 exhibiting higher activity. Also, it is important to note that the concentrations of RIS (the parent
drug) used for this assay were 0.01 μM, 0.1 μM, and 1 μM, which were 10 fold less than those of
dye-BP conjugates.
65

Figure 3.5. Inhibitory effect of BPs on bone resorption effect on bovine bone slices. Visualization
of the resorption pit using either confocal fluorescence microscopy (A) or toluidine staining (B).
No fluorescence signal was observed in the vehicle sample. (Unpublished data)
Evaluation of on the anti-resorptive effect of BP conjugates was conducted on bovine bone
slices where OC precursors were co-cultured with BPs for ten days. Resorption pits were
visualized via two separate methods with 1) confocal fluorescence microscopy, followed by 2)
toluidine staining. Fluorescent imaging at 10 μM was not possible due to self-quenching of the
fluorophores, which commonly occurred when the concentration of fluorescent dye was too high.
Reduced resorption pits observed at 10 μM conjugate concentrations might be a result of reduced
OC formation caused by the probes as observed using toluidine staining (Figure 3.5B). As seen in
Figure 3.5A, the dye-BP-conjugates, particularly 6-FAM-RIS-V1 (3.19b) and 6-FAM-RIS-V2
(3.21b), exhibit strong binding to newly formed resorption lacunae. Furthermore, the area of the
66

resorption pits determined either via confocal fluorescence microscopy or toluidine staining
demonstrated similar measurements (Figure 3.6A).

Figure 3.6. OC cultures on bone slices. A- comparison between vehicle-treated and 5-FAM-RIS
treated bone slices visualized using either confocal fluorescence microscopy or toluidine staining.
B- Measurement of pit area determined by toluidine staining. (Unpublished data)
3.5 Conclusion
In conclusion, a new series of near-IR and visible fluorescent BP probes with extended
linker lengths was synthesized. Three different linkers were used: 1) RIS-V1 (4-atom extension);
2) RIS-M (8-atom extension) and 3) RIS-V2 (14-atom extension). These novel dye-BP-conjugates
significantly affected OC formation at 10 μM but not at or below 1 μM. The reduction in resorption
pits observed at 10 μM conjugate concentrations was caused by reduced OC formation induced by
the probes. The dye-BP-conjugates, particularly 6-FAM-RIS-V1 and V2, exhibit strong binding
to newly formed resorption lacunae. Six compounds in the series were evaluated for inhibition of
osteoclast formation and anti-resorption of bovine bone slices. Other compounds need to be tested
for a conclusive evaluation of the relationship between linker length and anti-resorption. In
addition, experiments for anti-prenylation using J774 macrophages should also be conducted to
67

compare the original dye-BP series with the new ones extended with various linker lengths to fully
assess the structure-activity relationship between dye-linker-BPs and anti-resorption.
3.6 Experimental Section
3.6.1. Chemical Synthesis
Reagents and Spectral Measurements. 5(6)-carboxyfluorescein succinimidyl ester (FAM, SE),
BOC-beta-alanine, 7-((tert-butoxycarbonyl) amino) heptanoic acid and BOC-tetraoxapenta-
decanoic acid were purchased from Sigma Aldrich. AF647 succinimidyl ester (SE, AF647) and
5,6-FAM succinimidyl ester were purchased from Life Technologies. Sulfo-Cy5 succinimidyl
ester (SE, S-Cy5) was purchased from Lumiprobe. Risedronate monosodium was a kind gift from
Warner Chilcott (formerly P&G Pharmaceuticals). All other chemical compounds were purchased
from either Aldrich or Alfa Aesar. Triethylamine (TEA) was distilled from KOH and dioxane was
distilled from sodium. Other compounds were used as supplied by the manufacturer. Thin layer
chromatography (TLC) was performed on Merck Silica Gel 60 F254 plates, and the developed
plates were visualized under a UV lamp at 354 nm. HPLC separations were performed on a Rainan
Dynamax Model SD-200 system with a Rainan Dynamax absorbance detector Model UV-DII.
NMR spectra were recorded on either 400 MHz or 500 MHz Varian spectrometers. UV spectra
were recorded on a DU 800 spectrometer, fluorescence emission spectra were taken on a Jobin
Yvon Horiba FluoroMax-3 fluorimeter, and samples were dissolved in 1X PBS (pH 7.4). Molar
extinction coefficient used for 5,6-FAM, AF647, and S-Cy5, are 73,000, 240,000 and 271,000 M
-
1
cm
-1
, respectively, as provided by the manufacturers. Mass spectra of several of the new dye-
risedronate conjugates were collected at UC Riverside High Resolution Mass Spectrometry
Facility for high resolution mass spectrometric analysis (HRMS) performed by Dr. Ron New on a
PE Biosystems DE-STR MALDI TOF spectrometer with a WinNT (2000) Data System. LC-MS
68

of several of the new dye-risedronate conjugates was obtained on a Thermo-Finnigan LCQ DECA
XP MAX Ion Trap LC/MS/MS spectrometer operated in ESI mode.
General Synthesis: The activation of NBoc-protected carboxylic acids was synthesized via
reacting the commercially available NBoc-protected acids with N-hydroxysuccinimide (SuOH)
and coupling reagents (e.g., EDC) in anhydrous solvents (e.g., DMF, or dioxane). The NHS-
activated acid was then reacted with RIS-linker, synthesis of which has been previously reported.
15

Deprotection of the newly synthesized extended RIS-linker (ExtRIS-linker) was performed using
a mixture of trifluoroacetic acid (TFA) and water (1:1 v/v) after RP-HPLC purification using either
preparative SAX or C18 columns. The newly synthesized ExtRIS-linker was dissolved in water
and pH was adjusted to 8.3 using sodium carbonate. Dissolved in anhydrous DMF, SE-5(6)-
carboxy fluorescein, SE-AF647 or SE-Sulfo-Cy5, was added dropwise into the ExtRIS-linker
solution in order to obtain fluorescently labeled RIS conjugates. Purification of the dye-BP
conjugates was performed using semi preparative C18 RP HPLC. Methods used for the
purification was listed in Table 3.1.
Table 3.1. HPLC methods for the purification of extended RIS-linker and dye-BP conjugates
Method Column Mobile Phase Gradient (%B) Detection
(nm)
A Phenomenex
Luna
®
C18 HPLC
Column (5µm,
250 mm x 10 mm)
A: 0.1 M triethylammonium
bicarbonate (TEAB), pH 7.0
B: 100% MeCN
Flow rate: 8.0 mL/min
0-10 min: 8%
10-15 min: 8-25%
15-40: 25%
260
B Macherey-Nagel
(21.4 mm × 150
mm) SP15/25
Nucleogel
A: water
B: 0.1 M TEAB, pH 7.8
Flow rate: 8 mL/min
0-20 min: 0-10%
20-25 min: 10%
25-40 min: 10-
100%
260
C Beckman
Ultrasphere ODS
C18 (250 x 10
A: 0.1 TEAB, 10% MeOH,
pH 7.0
B: 0.1 M TEAB, 75%
MeOH, pH 7.8
0-20 min: 0-10%
20-25 min: 10%
25-40 min: 10-
100%
0-10 min:
260 nm
10-100 min:
493 nm
69

mm, 5 μm, 80 Å
pore size)
Flow rate: 4 mL/min
D Phenomenex
Luna
®
C18 HPLC
Column (5µm,
250 mm x 10 mm)
A: 0.1 TEAB, 10% MeOH,
pH 7.0
B: 0.1 M TEAB, 75%
MeOH, pH 7.8
Flow rate: 4 mL/min
0-20 min: 0-10%
20-25 min: 10%
25-40 min: 10-
100%
0-10 min:
260 nm
10-100 min:
493 nm
E Phenomenex
Luna
®
C18 HPLC
Column (5µm,
100 mm x 10 mm)
A: 0.1 TEAB, 10% MeCN,
pH 7.0
B: 0.1 M TEAB, 75%
MeCN, pH 7.8
Flow rate: 4.5 mL/min
0-5 min: 15%
5-30 min: 15-30%
30-35 min: 30-
40%
35-60 min: 40%
0-7 min:
260 nm
7-30 min:
598 nm
F Phenomenex
Luna
®
C18 HPLC
Column (5µm,
100 mm x 10 mm)
A: 0.1 TEAB, 10% MeCN,
pH 7.0
B: 0.1 M TEAB, 75%
MeCN, pH 7.8
Flow rate: 4.5 mL/min
0-7 min: 10%
7-37 min: 10-30%
37-45 min: 30-
40%
45-60 min: 40%
0-7 min:
260 nm
7-30 min:
598 nm

Synthesis of tert-butyl allylcarbamate (3.2): In a round bottom flask, freshly distilled allyl-amine
(3.6 mL, 47 mmol, 1 equivalent) was dissolved in 25 mL DCM. Added to the solution was BOC 2O
(10.5 g, 48 mmol, 1 equivalent) and the mixture was left stirring overnight. Additional DCM was
added into the mixture (V = 15 mL) and the organic layer was washed with 5% citric acid (1X),
brine (1X), followed by drying over sodium sulfate. Solvent was removed and the solid was
precipitated using hexanes, yielding 6.41 g of 3.2 (85%) as white solid. The compound was used
for the next reaction without further purification.
1
H NMR (CDCl3) δ 5.94 – 5.66 (m, 1H), 5.29 – 4.94 (m, 2H), 4.57 (s, 1H), 3.74 (s, 2H), 1.45 (s,
9H).
Synthesis of tert-butyl (oxiran-2-ylmethyl) carbamate (3.3): In a round bottom flask, 0.562 g of 3.2
(3.6 mmol, 1.00 equivalent) was dissolved in 20 mL of DCM. Added to the flask was 1.73 g of
MCPBA (75% pure) (7.5 mmol, 2.1 equivalent) and the reaction mixture was chilled in an ice bath
for 30 minutes. The reaction mixture was then warmed up to room temperature and left stirring
70

overnight. Additional DCM was added into the mixture (V = 20mL) and the organic layer was
washed with 10% Na2SO3 (2X), saturated NaHCO3, and water (1X), followed by drying over
sodium sulfate. Solvent was removed, yielding 0.52 g of 3.3 (83%) as a clear oil. The compound
was used for the next reaction without further purification.
Synthesis of 1-(3-((tert-butoxycarbonyl) amino)-2-hydroxypropyl)-3-(2-hydroxy-2,2-diphospho-
noethyl) pyridin-1-ium (3.5): In a round bottom flask, 0.283 g of risedronate acid form (1.00 mmol,
1 equivalent) was dissolved in 4 mL of water and the pH of the solution was adjusted to 6.3 using
TEA. 0.173 g of 3.3 (1.00 mmol, 1 equivalent) was dissolved in minimal volume of methanol (~
300 µL) and added into the BP solution. The reaction mixture was stirred in a water bath at 40
o
C
for 18 hours and reaction progress was monitored using
31
P NMR. The reaction was left stirring
until most of the starting material was consumed and minimal amount of dialkylation product was
detected. Yield of the reaction was 85% verified via
31
P NMR. The reaction mixture was then
washed with ether (3X). The aqueous layer was collected and solvent was removed.
1
H NMR (500 MHz, Deuterium Oxide) δ 8.71 (s, 1H), 8.48 (d, J = 33.4 Hz, 2H), 8.33 (s, 0H), 8.12
(s, 0H), 7.84 (s, 1H), 7.47 (s, 0H), 4.66 (s, 1H), 4.29 (s, 1H), 4.05 (s, 1H), 3.33 (s, 2H), 3.23 (s,
2H), 3.16 (dd, J = 15.0, 6.7 Hz, 1H), 1.33 (s, 9H).
31
P{
1
H} NMR (D2O): 202 MHz δ 17.26 – 16.95
(m), 16.70, 16.45 – 15.97 (m), 15.50 – 15.18 (m).
Synthesis of 1-(3-amino-2-hydroxypropyl)-3-(2-hydroxy-2,2-diphosphonoethyl) pyridin-1-ium
(3.6): Compound 3.5 was dissolved in 4 mL of water and 4 mL of trifluoroacetic acid was
subsequently added. The reaction mixture was left stirring overnight. Solvent was removed and
product 3.6 was precipitated using ether. The precipitate was filtered and dried under vacuum.
Product 3.6 was collected as white solid in the form of triethyl ammonium acetate salt. Compound
was used for the next step without further purification.
71

1
H NMR (500 MHz, Deuterium Oxide) δ 8.83 (s, 1H), 8.66 (d, J = 6.0 Hz, 1H), 8.56 (d, J = 8.0
Hz, 1H), 7.98 – 7.93 (m, 1H), 4.82 (s, 1H), 4.53 – 4.44 (m, 1H), 4.36 (t, J = 9.7 Hz, 1H), 3.56 (d,
J = 7.1 Hz, 1H), 3.48 (t, J = 12.3 Hz, 2H), 3.37 (d, J = 15.2 Hz, 1H), 3.13 – 3.03 (m, 1H).
31
P NMR
(202 MHz, Deuterium Oxide) δ 16.80 – 16.45 (m).
Synthesis of 1-(3-(3-carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzamido)-2-hydroxypro-
pyl)-3-(2-hydroxy-2,2-diphosphonoethyl)pyridin-1-ium (3.7a) (5-FAM-RIS) and 1-(3-(4-carboxy-
3-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzamido)-2-hydroxypropyl)-3-(2-hydroxy-2,2-diphos-
phonoethyl)pyridin-1-ium (3.7b) (6-FAM-RIS): In 0.5 ml HPLC water, 75.9 mg of 3.6 (0.13 mmol,
4 equivalent) was dissolved and pH was adjusted to 8.30 using solid Na 2CO3. 5(6)-
Carboxyfluorescein, N-hydroxysuccinimide ester (a mixture of 5-{[(2,5-dioxopyrrolidin-1-
yl)oxy]carbonyl}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid and 4-{[(2,5-
dioxopyrrolidin-1-7yl)oxy]carbonyl}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid, 15.7
mg (0.033 mmol, 1 equivalent) was dissolved in anhydrous DMF (~50 mg per 200 μL) and added
dropwise into solution of 3.6. The solution became dark orange and some solid precipitate
appeared. The pH of the solution was adjusted again using solid Na 2CO3, dissolving all the
precipitate. The reaction was stirred overnight. The unreacted dye was removed via purification
by TLC with 100% MeOH as the eluent. The FAM-labeled compound stayed at the origin while
the dye moved upward in the TLC plates, giving a yellow upper band, while the phosphonate-
containing compounds remain at the origin. The desire product was extracted with HPLC water
from the silica, centrifuged, and concentrated under vacuum to yield a dark red-orange solid. The
compound was then dissolved in water and filtered through Nanosep 30K Omega filter.
Purification of 5,6-FAM-RIS was performed using method C. The final amount of labeled product
72

was calculated from the UV absorption spectrum taking ε = 73000 M
-1
cm
-1
in 1X PBS buffer at
pH 7.4 and the isolated 3.7a and 3.7b are lyophilized, yielding red-orange solid 15.3 mg (65%).
3.7a:
1
H NMR (500 MHz, Deuterium Oxide) δ 8.75 (s, 1H), 8.53 (d, J = 6.1 Hz, 1H), 8.44 (d, J =
8.1 Hz, 1H), 8.08 (s, 1H), 7.90 – 7.75 (m, 2H), 7.19 (d, J = 7.9 Hz, 1H), 6.98 (d, J = 9.2 Hz, 2H),
6.54 – 6.37 (m, 4H), 4.77 (d, J = 13.2 Hz, 1H), 4.39 (d, J = 13.3 Hz, 1H), 4.27 (s, 1H), 3.64 (d, J
= 18.6 Hz, 1H), 3.52 (dd, J = 14.0, 6.9 Hz, 1H), 3.31 (t, J = 12.1 Hz, 2H).
31
P NMR (202 MHz,
Deuterium Oxide) δ 17.07-15.55
ESI-MS (negative ion, M-): calcd 715.11 m/z, found [M-2H]
-
= 713.2 m/z.
3.7b:
1
H NMR (500 MHz, Deuterium Oxide) δ 8.71 (s, 1H), 8.49 (d, J = 6.1 Hz, 1H), 8.42 (d, J =
8.1 Hz, 1H), 7.89 (d, J = 9.6 Hz, 1H), 7.78 (t, J = 8.7 Hz, 2H), 7.53 (s, 1H), 7.02 (d, J = 9.2 Hz,
2H), 6.56 – 6.48 (m, 3H), 4.74 – 4.70 (m, 2H), 4.38 – 4.28 (m, 1H), 4.18 (s, 1H), 3.56 (dd, J =
14.2, 4.4 Hz, 1H), 3.43 (dd, J = 14.2, 7.0 Hz, 1H), 3.37 – 3.24 (m, 2H).
31
P NMR (202 MHz,
Deuterium Oxide) δ 16.77 – 15.84 (m).
ESI-MS (negative ion, M-): calcd 715.11 m/z, found [M-2H]
-
= 713.2 m/z.
Synthesis of 1-(6-((2-hydroxy-3-(3-(2-hydroxy-2,2-diphosphonoethyl) pyridin-1-ium-1-yl)propyl)
amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethyl-5-sulfonatoindolin-2-ylide-
ne)penta-1,3-dien-1-yl)-3H-indol-1-ium-5-sulfonate (3.7c) (S-Cy5-RIS): In 1.0 ml HPLC water,
22.4 mg of 3.6 as TEA and TFA salt (0.039 mmol) was dissolved and pH was adjusted to 8.30
using solid Na2CO3. For this reaction, 0.5 mL of solution of 13 was utilized. Sulfo-Cy5,
succinimidyl ester, 5.0 mg (6.6 µmol, 0.3 equivalent) was dissolved in 150µL anhydrous DMF
and added dropwise into solution of 13. The solution became dark blue. The reaction was stirred
overnight. Purification of Sulfo-Cy5-labeled compound 3.7c was performed using Method F. The
final amount of labeled product is calculated from the UV absorption spectrum taking ε = 271000
73

M
-1
cm
-1
at maximum absorption in 1X PBS buffer at pH 7.4 and the final product 3.7c is
lyophilized, yielding a dark blue solid (20%).
1
H NMR (400 MHz, Deuterium Oxide) δ 8.65 (s, 1H), 8.41 (dd, J = 14.1, 7.0 Hz, 2H), 7.86 (d, J
= 13.1 Hz, 2H), 7.80 – 7.72 (m, 1H), 7.72 – 7.61 (m, 4H), 7.18 (d, J = 8.4 Hz, 2H), 6.40 (t, J =
12.6 Hz, 1H), 6.09 (dd, J = 13.3, 10.9 Hz, 2H), 4.26 – 4.14 (m, 2H), 4.07 – 3.85 (m, 4H), 3.44 (s,
3H), 3.35 – 3.10 (m, 8H), 2.25 (t, J = 6.8 Hz, 2H), 2.05 (t, J = 7.1 Hz, 2H), 1.52 (s, 18H).
31
P NMR
(202 MHz, Deuterium Oxide): δ 16.9-15.6 (br, 2P).
LC-MS (negative ion, M-): tretention = 3.44 min, calcd 982.99 m/z, found [M-4H]
2-
= 489.6 m/z.
Synthesis of 2-(5-(3-(6-((2-hydroxy-3-(3-(2-hydroxy-2,2-diphosphonoethyl) pyridin-1-ium-1-yl)
propyl)amino)-6-oxohexyl)-3-methyl-5-sulfo-1-(3-sulfopropyl)indolin-2-ylidene)penta-1,3-dien-
1-yl)-3,3-dimethyl-5-sulfo-1-(3-sulfopropyl)-3H-indol-1-ium (3.7d) (AF647-RIS): In a 10 mL
round bottom flask, 1 mg of Alexa Fluor 647 (0.86 µmol, 1 equivalent), commercially available
as TEA salt form (3 equivalent), was dissolved in 200 µL of anhydrous DMF. Added to the solution
were 1 µL of freshly distilled TEA (9 equivalent, 7.74 µmol) and TsTu (1.1 equivalent). The
reaction was stirred for 1 hour. Formation of the NHS-activated AF647 was confirmed using ESI-
MS. Solvent was removed and the compound was used for the coupling reaction without further
purification. In 1.3 ml HPLC water, 13.1 mg of 3.6 as TEA and TFA salt (0.023 mmol) was
dissolved and pH was adjusted to 8.30 using solid Na2CO3. For this reaction, 100 µL of solution
of 3.6 was utilized and diluted with 150 µL. The NHS-activated AF647 was dissolved in 100 µL
anhydrous DMF and added dropwise into solution of 3.6. The solution became dark blue and the
reaction was stirred overnight. Purification of AF647-labeled compound 3.7d was performed using
Method E. The final amount of labeled product is calculated from the UV absorption spectrum
74

taking ε = 240000 M
-1
cm
-1
at maximum absorption in 1X PBS buffer at pH 7.4 and the final
product 3.7d is lyophilized, yielding a dark blue solid (76%).
1
H NMR (400 MHz, Deuterium Oxide) δ 8.66 (s, 1H), 8.44 (d, J = 6.2 Hz, 2H), 8.02 (t, J = 14.0
Hz, 2H), 7.82 – 7.67 (m, 4H), 7.38 – 7.18 (m, 2H), 6.58 (d, J = 27.2 Hz, 1H), 6.32 (t, J = 13.0 Hz,
2H), 4.28 – 3.90 (m, 6H), 3.27 (d, J = 12.9 Hz, 3H), 2.19 – 2.05 (m, 4H), 1.97 (t, J = 7.6 Hz, 2H),
1.59 (d, J = 5.0 Hz, 8H), 1.29 (d, J = 12.8 Hz, 3H), 0.83 (d, J = 3.2 Hz, 2H), 0.46 (s, 1H).
ESI-MS (negative ion, M-): calcd 1199.21 m/z, found [M-5H]
3-
= 398.2 m/z.
Synthesis of 2,5-dioxopyrrolidin-1-yl 3-((tert-butoxycarbonyl) amino) propanoate (3.11): In a dry
flask containing a magnetic stir bar, 0.50 g of 3.8 (2.64 mmol) and 0.31 g of N-hydroxysuccinimide
(SuOH) (2.72 mmol, 1.03 equivalent) were dissolved in 3mL of distilled dioxane. Subsequently,
1.25 equivalent of 3-(Ethyliminomethyleneamino)-N,N-dimethylpropan-1-amine in hydrogen
chloride form (EDC) (0.63 g, 3.3 mmol) was added into the flask. The solution was cloudy, and
2mL of distilled dioxane was added into the reaction mixture. The reaction was stirred under
nitrogen for 30 minutes and then left for stirring overnight at room temperature. Dioxane was
removed under vacuum. The reaction mixture was dissolved in chloroform and washed with HPLC
water 3x. The organic layer was dried of over Na2SO4 and concentrated under vacuum. The desire
product was precipitated in ethanol and dried in a desiccator, yielding 0.47g of product (62%). The
1
H NMR spectral data matched the previously reported values for the compound.
1
H NMR (CDCl3): δ 5.09 (brt, 1H), 3.52 (q, J = 9.5, 6.6 Hz, 2H), 2.91-2.78 (m, 6H), 1.44 (s, 9H).

Synthesis of 1-(3-(3-((tert-butoxycarbonyl)amino)propanamido)-2-hydroxypropyl)-3-(2-hydroxy-
2,2-diphos-phonoethyl)pyridin-1-ium (3.14): In a flask, 97.1 mg of 3.8 (0.197 mmol) was
dissolved in 1.0mL of HPLC water. The solution was adjusted to pH 8.3 using solid Na 2CO3. In
0.5mL of anhydrous DMF, 56.0mg of 3.6 (0.195 mmol, 1.0 equivalent) was dissolved, which was
75

then added dropwise into the solution of 3.8. The reaction was stirred overnight. Product 3.14 was
obtained after SAX HPLC purification, yielding 66% (75.93 mg).
1
H NMR (D2O): δ 8.82 (s, 1H), 8.64 – 8.59 (m, 1H), 8.55 (d, J = 8.1 Hz, 1H), 7.93 (dd, J = 8.0,
6.1 Hz, 1H), 4.40 (dd, J = 13.5, 9.5 Hz, 1H), 4.25 – 4.17 (m, 1H), 3.54 – 3.25 (m, 6H), 2.54 – 2.43
(m, 2H), 1.40 (s, 9H).
31
P NMR (202 MHz, Deuterium Oxide): δ 16.8-15.7 (br, 2P)
Synthesis of 1-(3-(3-aminopropanamido)-2-hydroxypropyl)-3-(2-hydroxy-2,2-diphosphonoethyl)
pyridin-1-ium (3.17): Product 3.14 was dissolved in HPLC water, and an equal volume of TFA
was slowly added. The solution was stirred overnight at rt, giving quantitative yield of 3.17. The
solvent was removed under vacuum, and the collected product was then used without further
purification.
1
H NMR (D2O): δ 8.60 (s, 1H), 8.48 (d, J = 6.1 Hz, 1H), 8.36 (d, J = 8.1 Hz, 1H), 7.80 – 7.73 (m,
1H), 4.59 (dd, J = 13.6, 2.5 Hz, 1H), 4.24 (dd, J = 13.7, 9.1 Hz, 1H), 4.00 (q, J = 7.6 Hz, 1H), 3.42
– 3.23 (m, 4H), 3.17 (dd, J = 14.1, 7.0 Hz, 1H), 3.07 (t, J = 6.5 Hz, 2H), 2.64 – 2.48 (m, 2H).
31
P
NMR (202 MHz, Deuterium Oxide): δ 18.3-15.8 (br, 2P)
Synthesis of 1-(3-(3-(3-carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzamido)propanamido)-
2-hydroxy-propyl)-3-(2-hydroxy-2,2-diphosphonoethyl)pyridin-1-ium) (3.20a) (5-FAM-RIS-V1)
and (1-(3-(3-(4-carboxy-3-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzamido)propanamido)-2-hy-
droxypropyl) -3-(2-hydroxy-2,2-diphosphonoethyl)pyridin-1-ium) (3.20b) (6-FAM-RIS-V1): In
0.5 ml HPLC water, 75.9 mg of 3.17 (0.11 mmol) was dissolved and pH was adjusted to 8.30 using
solid Na2CO3. 5(6)-Carboxyfluorescein, N-hydroxysuccinimide ester (a mixture of 5-{[(2,5-
dioxopyrrolidin-1-yl)oxy]carbonyl}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid and 4-
{[(2,5-dioxopyrrolidin-1-7yl)oxy]carbonyl}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid,
11.63 mg (0.025 mmol, 4 equivalent) was dissolved in anhydrous DMF (~50 mg per 200 μL) and
76

added dropwise into solution of 3.17. The solution became dark orange and some solid precipitate
appeared. The pH of the solution was adjusted again using solid Na 2CO3, dissolving all the
precipitate. The reaction was stirred overnight. The reaction mixture was adjusted to acid Ph (~4-
5) using acetic acid. The unreacted dye was removed via extraction with EtOAc: Acetone (5:1 v/v)
(3X). The aqueous was collected and solvent was removed. The purification of 5,6-FAM-RIS-V1
was performed using semi-preparative C18 reverse-phase HPLC and method D. The final amount
of labeled product is calculated from the UV absorption spectrum taking ε = 73000 M
-1
cm
-1
in 1X
PBS buffer at pH 7.4 and the isolated 3.20a and 3.20b are lyophilized, yielding red-orange solid
(30%).
3.20a (as triethylammonium salt):
1
H NMR (D2O): δ 8.69 (s, 1H), 8.44 (dd, J = 24.9, 7.1 Hz, 2H),
8.07 (s, 1H), 7.88 – 7.71 (m, 2H), 7.33 (d, J = 7.9 Hz, 1H), 7.08 (d, J = 9.2 Hz, 2H), 6.68 – 6.52
(m, 4H), 4.36 – 4.22 (m, 2H), 4.10 (s, 1H), 3.63 (t, J = 6.8 Hz, 2H), 3.46 – 3.24 (m, 5H), 2.57 (t, J
= 6.7 Hz, 2H).
31
P NMR (202 MHz, Deuterium Oxide): δ 17.8-14.5 (br, 2P)
LC-MS (negative ion, M-): tretention = 11.25 min, calcd 786.15 m/z, found [M-2H]
-
= 784.13 m/z.
3.20b (as triethylammonium salt):
1
H NMR (D2O): δ 8.62 (s, 1H), 8.37 (d, J = 5.4 Hz, 2H), 7.84
(d, J = 9.7 Hz, 1H), 7.78 (d, J = 8.2 Hz, 1H), 7.74 – 7.67 (m, 1H), 7.47 (s, 1H), 7.02 (d, J = 11.3
Hz, 2H), 6.57 (s, 2H), 6.53 (d, J = 10.6 Hz, 2H), 4.52 (d, J = 12.4 Hz, 3H), 4.19 – 4.10 (m, 1H),
4.02 (s, 1H), 3.55 (s, 3H), 3.26 (m, 6H), 2.48 (t, J = 5.8 Hz, 2H).
31
P NMR (202 MHz, Deuterium
Oxide): δ 18.7-13.9 (br, 2P)
HRMS (negative ion MALDI, [M-H]-): calcd 786.15 m/z; found [M-2H]
-
= 784.129 m/z.
1-(6-((3-((2-hydroxy-3-(3-(2-hydroxy-2,2-diphosphonoethyl)pyridin-1-ium-1-yl)propyl)amino)-
3-oxopropyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfo-2-((1E,3E)-5-((E)-1,3,3-trimethyl-5-sul-
foindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium (3.20c) (S-Cy5-RIS-V1): In 0.6 ml HPLC
77

water, 12.7 mg of 3.17 as TEA and TFA salt (0.022 mmol) was dissolved and pH was adjusted to
8.30 using solid Na2CO3. Sulfo-Cy5, succinimidyl ester, 5.0 mg (6.6 µmol, 0.3 equivalent) was
dissolved in 150 µL anhydrous DMF and added dropwise into solution of 3.17. The solution
became dark blue. The reaction was stirred overnight. Purification of Sulfo-Cy5-labeled compound
3.20c was performed using semi-preparative reverse-phase HPLC and method F. The final amount
of labeled product is calculated from the UV absorption spectrum taking ε = 271000 M
-1
cm
-1
at
maximum absorption in 1X PBS buffer at pH 7.4 and the final product 3.20c is lyophilized,
yielding a dark blue solid (41%).
1H NMR (400 MHz, Deuterium Oxide) δ 8.64 (s, 1H), 8.45 (d, J = 6.1 Hz, 1H), 8.39 (d, J = 8.1
Hz, 1H), 7.88 – 7.72 (m, 3H), 7.67 (s, 2H), 7.63 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 8.4 Hz, 2H), 6.32
(t, J = 12.4 Hz, 1H), 6.10 – 5.84 (m, 2H), 4.28 – 4.12 (m, 2H), 4.00 (s, 1H), 3.89 (t, J = 6.8 Hz,
2H), 3.42 (s, 3H), 3.31 (d, J = 11.5 Hz, 2H), 3.24 – 3.16 (m, 1H), 2.13 (t, J = 7.2 Hz, 2H), 1.64 (s,
2H), 1.47 (s, 15H), 1.22 (d, J = 6.7 Hz, 2H).
31
P NMR (202 MHz, Deuterium Oxide): δ 16.7-15.1
(br, 2P).
LC-MS (negative ion, M-): tretention = 3.42 min, calcd 1053.3 m/z, found [M-3H]
2-
= 525.38 m/z.
Synthesis of 1-(6-((3-((2-hydroxy-3-(3-(2-hydroxy-2,2-diphosphonoethyl) pyridin-1-ium-1-yl)pro-
pyl)amino)-3-oxopropyl)amino)-6-oxohexyl)-3,3-dimethyl-2-((1E,3E)-5-((E)-1,3,3-trimethyl-5-
sulfonatoindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium-5-sulfonate (3.20d) (AF647-RIS-
V1): In 0.5 ml HPLC water, 16.2 mg of 3.17 as triethylammonium salt (0.03 mmol) was dissolved
and pH was adjusted to 8.30 using solid Na2CO3. Alexa Fluo 647, succinimidyl ester, 1.0 mg
(1µmol, 0.1 equivalent) was dissolved in 150µL anhydrous DMF and added dropwise into solution
of 3.17. The solution became dark blue. The reaction was stirred overnight. Purification AF647-
labeled compound, AF647-RIS-V1, was performed using semi-preparative reverse-phase HPLC
78

and method E. The final amount of labeled product is calculated from the UV absorption spectrum
taking ε = 240000 M
-1
cm
-1
in 1X PBS buffer at pH 7.4 and the isolated 3.20d is lyophilized,
yielding a dark blue solid (47.9%).
1
H NMR (D2O): δ 8.66 (s, 1H), 8.44 (dd, J = 13.9, 7.1 Hz, 2H), 8.02 (t, J = 13.1 Hz, 2H), 7.86 –
7.62 (m, 5H), 7.28 (t, J = 8.1 Hz, 2H), 6.58 (t, J = 12.5 Hz, 1H), 6.32 (t, J = 13.7 Hz, 2H), 4.30 –
3.97 (m, 8H), 3.36 – 3.17 (m, 7H), 3.00 – 2.84 (m, 7H), 2.36 – 2.05 (m, 10H), 1.94 – 1.85 (m, 3H),
1.65 – 1.54 (m, 10H), 0.75 (s, 1H), 0.48 (s, 1H).
31
P NMR (202 MHz, Deuterium Oxide): δ 17.0-
15.9 (br, 2P)
HRMS (negative ion MALDI, [M-2H]
2-
): calcd 1269.28 m/z; found [M-3H]
2-
= 632.6217 m/z.
Synthesis of 2,5-dioxopyrrolidin-1-yl 7-((tert-butoxycarbonyl)amino)heptanoate (3.12): In a dry
flask containing a magnetic stir bar, 100.7 g of 1 (0.41 mmol) and 63.6 mg of N-
hydroxysuccinimide (SuOH) (0.55mmol, 1.25 equivalent) were dissolved in 3mL of distilled
dioxane. Subsequently, 1.25 equivalent of 3-(Ethyliminomethyleneamino)-N,N-dimethylpropan-
1-amine in HCl form (EDC) (106.5 mg, 0.55 mmol) was added into the flask. The solution was
cloudy, and 2mL of distilled dioxane was added into the reaction mixture. The reaction was stirred
under nitrogen for 30 minutes and then left for stirring overnight at room temperature. Dioxane
was removed under vacuum. The reaction mixture was dissolved in chloroform and washed with
HPLC water (1X), followed by a wash with brine (1X). The organic layer was dried of over
Na2SO4 and concentrated under vacuo, yielding 132 mg of crude product (62% yield based on
1
H
NMR spectrum). The crude product was used for the subsequent reaction without purification.
1
H NMR (600 MHz, Chloroform-d) δ 4.65 (s, 1H), 3.03 (s, 2H), 2.77 (s, 3H), 2.53 (d, J = 7.4 Hz,
1H), 1.68 (p, J = 7.4 Hz, 1H), 1.61 – 1.48 (m, 1H), 1.53 – 1.18 (m, 15H).
79

Synthesis of 1-(3-(7-((tert-butoxycarbonyl)amino)heptanamido)-2-hydroxypropyl)-3-(2-hydroxy-
2,2-diphos-phonoethyl) pyridin-1-ium (3.15): 124.5 mg (0.22 mmol, 0.9 equivalent) of Compound
3.6 in the form of TEA/TFA salt, was dissolved in 1.0 mL of HPLC water, and pH was adjusted
to 8.3 using solid Na2CO3. Compound 3.12 was dissolved in 300 µL anhydrous DMF and added
dropwise into solution of 3.6. The reaction was left stirred overnight at room temperature. Product
3.15 was purified using SAX HPLC, method B, and obtained as TEA salt, yielding 110 mg (73%).
1
H NMR (500 MHz, Deuterium Oxide) δ 8.68 (s, 1H), 8.46 (dd, J = 33.4, 7.0 Hz, 2H), 7.86 – 7.77
(m, 1H), 4.35 – 4.20 (m, 1H), 4.07 (s, 1H), 3.44 – 3.18 (m, 5H), 2.91 (t, J = 6.7 Hz, 3H), 2.17 (t, J
= 7.4 Hz, 2H), 1.53 – 1.38 (m, 2H), 1.29 (s, 12H).
31
P NMR (202 MHz, Deuterium Oxide): δ
16.34-16.01 (m, 2P).
Synthesis of 1-(3-(7-aminoheptanamido)-2-hydroxypropyl)-3-(2-hydroxy-2,2-diphosphono-
ethyl)pyridin-1-ium (3.18). Product 3.15 was dissolved in HPLC water, and an equal volume of
TFA was slowly added. The solution was stirred overnight at room temperature, giving
quantitative yield of 3.18. The solvent was removed in vacuo, and the collected product was then
used without further purification.
1
H NMR (500 MHz, Deuterium Oxide) δ 8.66 (s, 1H), 8.47 (dd, J = 49.3, 6.6 Hz, 2H), 7.97 – 7.72
(m, 1H), 4.41 – 4.20 (m, 1H), 4.06 (s, 1H), 3.29 (dd, J = 74.2, 12.4 Hz, 4H), 2.84 (t, J = 7.1 Hz,
2H), 2.16 (t, J = 7.3 Hz, 2H), 1.72 – 1.38 (m, 4H), 1.22 (d, J = 13.3 Hz, 4H).
31
P NMR (202 MHz,
Deuterium Oxide): δ 18.8-14.1 (br, 2P).
Synthesis of 1-(3-(7-(3-carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzamido)heptanamido)-
2-hydroxy-propyl)-3-(2-hydroxy-2,2-diphosphonoethyl)pyridin-1-ium (5-FAM-ExtRis-M) (3.21a)
and 1-(3-(7-(4-carboxy-3-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzamido)heptanamido)-2-hydro-
xypropyl) -3-(2-hydroxy-2,2-diphosphonoethyl)pyridin-1-ium (6-FAM-RIS-M) (3.21b): In 0.8 ml
80

HPLC water, 20 mg of 3.18 (0.029 mmol) was dissolved and pH was adjusted to 8.30 using solid
Na2CO3. 5(6)-Carboxyfluorescein, N-hydroxysuccinimide ester (a mixture of 5-{[(2,5-
dioxopyrrolidin-1-yl)oxy]carbonyl}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid and 4-
{[(2,5-dioxopyrrolidin-1-7yl)oxy]carbonyl}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid,
31.6 mg (0.067 mmol, 2 equivalent) was dissolved in 600 μL anhydrous DMF and added dropwise
into solution of 3.18. The solution became dark orange and some solid precipitate appeared. The
pH of the solution was adjusted again using solid Na 2CO3, dissolving all the precipitate. The
reaction was stirred overnight. The unreacted dye was removed via extraction with EtOAc:
Acetome (5:1 v/v). The aqueous layer was collected and solvent was removed. Purification and
isolation of compound 3.21a and 3.21b were performed using semi-preparative reverse-phase
HPLC and method D. The final amount of labeled product is calculated from the UV absorption
spectrum taking ε = 73000 M
-1
cm
-1
at maximum absorption in 1X PBS buffer at pH 7.4 and the
isolated final product 3.21a and 3.21b were lyophilized, yielding an orange solid (17%).
3.21a, as triethylammonium salt:
1
H NMR (400 MHz, Deuterium Oxide) δ 9.50 (s, 1H), 9.31 (d, J
= 5.9 Hz, 1H), 9.25 (d, J = 7.6 Hz, 1H), 8.94 (s, 1H), 8.73 – 8.56 (m, 2H), 8.05 (d, J = 7.8 Hz, 1H),
7.88 (d, J = 9.2 Hz, 2H), 7.55 – 7.38 (m, 4H), 5.10 (dd, J = 13.6, 9.5 Hz, 1H), 4.89 (s, 1H), 4.26 –
3.99 (m, 7H), 3.77 (d, J = 7.2 Hz, 2H), 3.02 (t, J = 7.3 Hz, 2H), 2.36 (s, 3H), 2.12 (s, 3H).
31
P NMR
(202 MHz, Deuterium Oxide): δ 16.8-15.4(br, 2P).
LC-MS (negative ion, M-): tretention = 3.67 min, calcd 842.21 m/z, found [M-2H]
-
= 840.43 m/z.
3.21b, as triethylammonium salt:
1
H NMR (400 MHz, Deuterium Oxide) δ 8.65 (s, 1H), 8.44 (d,
J = 6.1 Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H), 7.81 (s, 2H), 7.80 – 7.73 (m, 1H), 7.41 (s, 1H), 7.03 (d,
J = 9.2 Hz, 2H), 6.67 – 6.53 (m, 4H), 4.22 (dd, J = 13.6, 9.8 Hz, 1H), 4.01 (s, 1H), 3.37 – 3.25 (m,
81

3H), 3.25 – 3.12 (m, 5H), 2.08 (t, J = 7.4 Hz, 2H), 1.43 (q, J = 6.7 Hz, 4H), 1.20 (s, 5H).
31
P NMR
(202 MHz, Deuterium Oxide): δ 16.9-15.4 (br, 2P).
LC-MS (negative ion, M-): tretention = 3.67 min, calcd 842.21 m/z, found [M-2H]
-
= 840.33 m/z.
Synthesis of 1-(6-((7-((2-hydroxy-3-(3-(2-hydroxy-2,2-diphosphonoethyl)pyridin-1-ium-1-yl)pro-
pyl)amino)-7-oxoheptyl)amino)-6-oxohexyl)-3,3-dimethyl-5-sulfo-2-((1E,3E)-5-((E)-1,3,3-trime-
thyl-5-sul-foindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium (Sulfo-Cy5-RIS-M) (3.21c): In
0.5 ml HPLC water, 14.0 mg of 3.18 as TEA and TFA salt (0.020 mmol) was dissolved and pH
was adjusted to 8.30 using solid Na2CO3. Sulfo-Cy5, succinimidyl ester, 5.0 mg (6.6 µmol, 0.3
equivalent) was dissolved in 150 µL anhydrous DMF and added dropwise into solution of 15. The
solution became dark blue. The reaction was stirred overnight. Purification Sulfo-Cy5-labeled
compound 3.21c was performed using semi-preparative reverse-phase HPLC and method F. The
final amount of labeled product is calculated from the UV absorption spectrum taking ε = 271000
M
-1
cm
-1
at maximum absorption in 1X PBS buffer at pH 7.4 and the final product 3.21c is
lyophilized, yielding a dark blue solid (45%).
1
H NMR (400 MHz, Deuterium Oxide) δ 8.64 (s, 1H), 8.47 (d, J = 6.1 Hz, 1H), 8.41 (d, J = 8.1
Hz, 1H), 7.95 – 7.73 (m, 3H), 7.69 (s, 2H), 7.65 (dt, J = 8.4, 2.1 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H),
6.35 (t, J = 12.4 Hz, 1H), 6.11 – 5.85 (m, 2H), 4.22 (dd, J = 13.8, 9.6 Hz, 2H), 4.04 – 3.87 (m, 5H),
3.61 (t, J = 6.9 Hz, 2H), 3.43 (s, 4H), 3.21 (s, 8H), 2.86 (t, J = 7.0 Hz, 2H), 2.06 (d, J = 7.2 Hz,
4H), 1.77 – 1.72 (m, 1H), 1.72 – 1.58 (m, 2H), 1.50 (s, 15H), 1.38 – 1.26 (m, 3H), 1.09 – 1.02 (m,
4H).
31
P NMR (202 MHz, Deuterium Oxide): δ 16.8-14.9 (br, 2P).
LC-MS (negative ion, M-): tretention = 18.24 min, calcd 1109.36 m/z, found 552.91 [M-3H]/2z.
Synthesis of 2-(5-(3-(6-((7-((2-hydroxy-3-(3-(2-hydroxy-2,2-diphosphonoethyl) pyridin-1-ium-1-
yl)propyl)amino)-7-oxoheptyl)amino)-6-oxohexyl)-3-methyl-5-sulfo-1-(3-sulfopropyl)indolin-2-
82

ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-1-(3-sulfopropyl)-3H-indol-1-ium (AF647-RIS-
M) (3.21d). In 0.3 ml HPLC water, 3.0 mg of 3.18 as TEA and TFA salt (4.3 µmol) was dissolved
and pH was adjusted to 8.30 using solid Na2CO3. Sulfo-Cy5, succinimidyl ester, 5.0 mg (1.0 µmol,
0.3 equivalent) was dissolved in 50µL anhydrous DMF and added dropwise into solution of 3.18.
The solution became dark blue. The reaction was stirred overnight. Purification of compound 21
was performed using semi-preparative reverse-phase HPLC and method E. The final amount of
labeled product is calculated from the UV absorption spectrum taking ε = 240000 M
-1
cm
-1
at
maximum absorption in 1X PBS buffer at pH 7.4 and the final product 3.21d is lyophilized,
yielding a dark blue solid (48%).
1H NMR (400 MHz, Deuterium Oxide) δ 8.64 (s, 1H), 8.48 (d, J = 5.8 Hz, 1H), 8.40 (d, J = 8.8
Hz, 1H), 8.00 (td, J = 13.3, 4.0 Hz, 2H), 7.83 – 7.66 (m, 6H), 7.26 (dd, J = 8.5, 3.9 Hz, 2H), 6.55
(t, J = 12.3 Hz, 1H), 6.29 (t, J = 14.9 Hz, 2H), 4.29 – 3.99 (m, 8H), 3.30 (d, J = 12.0 Hz, 3H), 3.23
– 3.11 (m, 3H), 2.89 (dp, J = 13.4, 6.8 Hz, 9H), 2.60 (s, 1H), 2.29 – 1.99 (m, 9H), 1.87 (dd, J =
13.9, 6.9 Hz, 2H), 1.45 – 1.19 (m, 8H), 1.09 – 0.90 (m, 7H), 0.81 (t, J = 7.4 Hz, 1H), 0.47 (s, 1H).
31
P NMR (202 MHz, Deuterium Oxide): δ 16.6-15.2 (br, 2P).
LC-MS (negative ion, M-): tretention = 3.35 min, calcd 1325.34 m/z, found [M-3H]
2-
= 661.05 m/z.
Synthesis of 2,5-dioxopyrrolidin-1-yl 2,2-dimethyl-4-oxo-3,8,11,14,17-pentaoxa-5-azaicosan-20-
oate (3.13): In a dry flask containing a magnetic stir bar, 41.6 mg of 3.10 (0.11 mmol) and 16.4
mg of N-hydroxysuccinimide (SuOH) (0.14 mmol, 1.25 equivalent) were dissolved in 1.5 mL of
freshly distilled dioxane. Subsequently, 1.25 equivalent of 3-(Ethyliminomethyleneamino)-N,N-
dimethylpropan-1-amine (EDC) (27.35 mg, 0.14 mmol) was added into the flask. The reaction was
stirred under nitrogen for an hour and then left for stirring overnight at room temperature. Solvent
was removed and the reaction mixture was dissolved in chloroform, followed by washes with water
83

(2x). The organic layer was dried over Na2SO4. Solvent was removed and the final product, as a
colorless oil, was used without any additional purification for the next step.
Synthesis of 1-(23-hydroxy-2,2-dimethyl-4,20-dioxo-3,8,11,14,17-pentaoxa-5,21-diazatetraco-
san-24-yl)-3-(2-hydroxy-2,2-diphosphonoethyl) pyridin-1-ium (3.16): In a flask, 69.16 mg of 3.13
(0.14 mmol), as trifluoroacetate sodium salts, was dissolved in 1.0 mL of HPLC water. The
solution was adjusted to pH 8.3 using solid Na2CO3. Dissolved in 0.5mL of anhydrous DMF, 3.13
was added dropwise into solution of 3.6 and stirred overnight. Purification of ExtRis-V2 was
performed using preparative reverse-phase HPLC and method A. Product 3.16 was obtained as
triethylammonium salts, yielding 76% (69.6 mg).
1
H NMR (D2O): δ 8.70 (s, 1H), 8.50 (d, J = 6.1 Hz, 1H), 8.44 (d, J = 8.1 Hz, 1H), 7.82 (dd, J =
8.0, 6.1 Hz, 1H), 4.30 (dd, J = 13.5, 9.5 Hz, 1H), 4.15 – 4.06 (m, 1H), 3.69 (t, J = 6.1 Hz, 2H),
3.59 – 3.54 (m, 12H), 3.47 (t, J = 5.4 Hz, 2H), 3.45 – 3.34 (m, 1H), 3.33 (s, 2H), 3.27 (dd, J = 14.1,
6.9 Hz, 1H), 2.48 (t, J = 6.1 Hz, 2H), 1.31 (s, 9H).
31
P NMR (202 MHz, Deuterium Oxide): δ 17.4-
14.4 (br, 2P)
Synthesis of 1-(1-amino-18-hydroxy-15-oxo-3,6,9,12-tetraoxa-16-azanonadecan-19-yl)-3-(2-hy-
droxy-2,2-diphosphonoethyl) pyridin-1-ium (3.19): Product 3.16 was dissolved in HPLC water,
and an equal volume of TFA was slowly added. The solution was stirred overnight at rt, giving
quantitative yield of 3.19 (79.4 mg, as triethylammonium trifluoroacetate salts). The solvent was
removed in vacuo, and the collected product was then used without further purification.
1
H NMR (D2O): δ 8.68 (s, 1H), 8.52 (d, J = 6.0 Hz, 1H), 8.44 (d, J = 8.0 Hz, 1H), 7.83 (t, J = 7.0
Hz, 1H), 4.30 (dd, J = 13.4, 9.4 Hz, 1H), 4.10 (s, 1H), 3.72 – 3.53 (m, 18H), 3.44 – 3.20 (m, 4H),
2.47 (t, J = 6.0 Hz, 2H).
31
P NMR (202 MHz, Deuterium Oxide): δ 18.3-13.7 (br, 2P).
84

Synthesis of (1-(1-(3-carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)phenyl)-20-hydroxy-1,17-
dioxo-5,8,11,14-tetraoxa-2,18-diazahenicosan-21-yl)-3-(2-hydroxy-2,2-diphosphonoethyl)pyri-
din-1-ium) (3.22a) (5-FAM-RIS-V2) and 1-(1-(4-carboxy-3-(6-hydroxy-3-oxo-3H-xanthen-9-
yl)phe-nyl)-20-hydroxy-1,17-dioxo-5,8,11,14-tetraoxa-2,18-diazahenicosan-21-yl)-3-(2-hydroxy-
2,2 diphos-phonoethyl)pyridin-1-ium (3.22b) (6-FAM-RIS-V2): In 0.5 ml HPLC water, 12.1 mg of
3.19 (0.02 mmol) was dissolved and pH was adjusted to 8.30 using solid Na 2CO3. 5(6)-
Carboxyfluorescein, N-hydroxysuccinimide ester (a mixture of 5-{[(2,5-dioxopyrrolidin-1-
yl)oxy]carbonyl}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid and 4-{[(2,5-
dioxopyrrolidin-1-7yl)oxy]carbonyl}-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid, 9.85
mg (0.02 mmol, 1 equivalent) was dissolved in anhydrous DMF (~50 mg per 200 μL) and added
dropwise into solution of 3.19. The solution became dark orange and some solid precipitate
appeared. The pH of the solution was adjusted again using solid Na 2CO3, dissolving all the
precipitate. The reaction was stirred overnight. The unreacted dye was removed via purification
by TLC with 100% MeOH as the eluent. The FAM-labeled compound stayed at the origin while
the dye moved upward in the TLC plates, giving a yellow upper band, while the phosphonate-
containing compounds remain at the origin. The desire product was extracted with HPLC water
from the silica, centrifuged, and concentrated in vacuo to yield a dark red-orange solid. The
compound was then dissolved in water and filtered through Nanosep 30K Omega filter.
Purification of 5,6-FAM-RIS-V2 was performed using semi-preparative reverse-phase HPLC and
method C. The final amount of labeled product is calculated from the UV absorption spectrum
taking ε = 73000 M
-1
cm
-1
in 1X PBS buffer at pH 7.4 and the isolated 3.22a and 3.22b are
lyophilized, yielding red-orange solids (34%).
85

3.22a (as triethylammonium salt):
1
H NMR (D2O): δ 8.68 (s, 1H), 8.45 (s, 2H), 8.10 (s, 1H), 7.87
(d, J = 9.5 Hz, 1H), 7.84 – 7.71 (m, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.07 (d, J = 9.0 Hz, 2H), 6.59 –
6.51 (m, 4H), 4.30 – 4.15 (m, 1H), 4.04 (s, 1H), 3.73 – 3.41 (m, 19H), 3.34 (d, J = 14.4 Hz, 4H),
2.39 (t, J = 6.2 Hz, 2H.
31
P NMR (202 MHz, Deuterium Oxide): δ 17.8-14.5 (br, 2P)
HRMS (negative ion MALDI, [M-H]-): calcd 962.25 m/z; found 960.2387 m/z.
3.22b (as triethylammonium salt):
1
H NMR (D2O): δ 8.68 (s, 1H), 8.44 (d, J = 5.2 Hz, 2H), 7.77
(d, J = 7.2 Hz, 1H), 8.00 – 7.37 (m, 2H), 7.06 (d, J = 8.9 Hz, 2H), 6.51 (d, J = 9.0 Hz, 4H), 4.23
(dd, J = 13.4, 9.6 Hz, 1H), 4.06 (s, 1H), 3.60 – 3.43 (m, 11H), 3.39 – 3.23 (m, 11H), 2.34 (s, 2H).
31
P NMR (202 MHz, Deuterium Oxide): δ 17.1-15.8 (br, 2P).
HRMS (negative ion MALDI, [M-H]
-
): calcd 962.25 m/z; found [M-2H]
-
= 960.23 m/z.
Synthesis of 1-(2-hydroxy-1-(3-(2-hydroxy-2,2-diphosphonoethyl)-1l4-pyridin-2-ylium-1-yl)-5,21
-dioxo-8,11,14,17-tetraoxa-4,20-diazahexacosan-26-yl)-3,3-dimethyl-5-sulfo-2-((1E,3E)-5-((E)-
1,3,3-trimethyl-5-sulfoindolin-2-ylidene) penta-1,3-dien-1-yl)-3H-indol-1-ium (3.22c) (S-Cy5-
RIS-V2): In 0.5 ml HPLC water, 14.0 mg of 3.19 as TEA and TFA salt (0.017 mmol) was dissolved
and pH was adjusted to 8.30 using solid Na2CO3. Sulfo-Cy5, succinimidyl ester, 5.0 mg (6.6 µmol,
0.3 equivalent) was dissolved in 150µL anhydrous DMF and added dropwise into solution of 3.19.
The solution became dark blue. The reaction was stirred overnight. Purification of Sulfo-Cy5-
labeled compound 3.22c was performed using semi-preparative reverse-phase HPLC and method
F. The final amount of labeled product is calculated from the UV absorption spectrum taking ε =
271000 M
-1
cm
-1
at maximum absorption in 1X PBS buffer at pH 7.4 and the final product 3.22c is
lyophilized, yielding a dark blue solid (76%).
1
H NMR (400 MHz, Deuterium Oxide) δ 8.81 (s, 1H), 8.63 (d, J = 5.8 Hz, 1H), 8.56 (d, J = 7.8
Hz, 1H), 8.04 – 7.87 (m, 3H), 7.83 (s, 2H), 7.79 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 6.47
86

(t, J = 12.5 Hz, 1H), 6.23 – 6.03 (m, 2H), 4.49 – 4.31 (m, 1H), 4.21 (s, 1H), 4.05 (s, 2H), 3.74 (t, J
= 5.9 Hz, 2H), 3.67 – 3.54 (m, 15H), 3.54 – 3.41 (m, 4H), 3.36 (s, 2H), 3.26 (s, 1H), 2.55 (t, J =
5.9 Hz, 2H), 2.23 (t, J = 6.8 Hz, 2H), 1.80 (d, J = 8.2 Hz, 2H), 1.63 (s, 14H).
31
P NMR (202 MHz,
Deuterium Oxide): δ 17.0-15.2 (br, 2P).
LC-MS (negative ion, M-): tretention = 3.40 min, calcd 1228.40 m/z, found [M-2H]
-
= 1226.76 m/z.
Synthesis of 2-(5-(3-(2-hydroxy-1-(3-(2-hydroxy-2,2-diphosphonoethyl) pyridin-1-ium-1-yl)-5,21-
dioxo-8,11,14,17-tetraoxa-4,20-diazahexacosan-26-yl)-3-methyl-5-sulfo-1-(3-sulfopropyl)indolin
-2-ylidene) penta-1,3-dien-1-yl)-3,3-dimethyl-5-sulfo-1-(3-sulfopropyl)-3H-indol-1-ium (AF647-
RIS-V2) (3.22d): In 0.5 ml HPLC water, 7.35 mg of 3.19 as triethylammonium salt (0.01 mmol)
was dissolved and pH was adjusted to 8.30 using solid Na 2CO3. Alexa Fluor 647, succinimidyl
ester, 1.0 mg (1µmol, 0.1 equivalent) was dissolved in 200µL anhydrous DMF and added dropwise
into solution of 3.19. The solution became dark blue. The reaction was stirred overnight.
Purification AF647-labeled compound, AF647-RIS-V2, was performed using semi-preparative
reverse-phase HPLC and method E. The final amount of labeled product is calculated from the
UV absorption spectrum taking ε = 240000 M
-1
cm
-1
in 1X PBS buffer at pH 7.4 and the isolated
3.22d is lyophilized, yielding a dark blue solid (20.3%).
HRMS (negative ion MALDI, [M-2H]
2-
): calcd 1445.38 m/z; found found [M-5H]
3-
= 480.12 m/z.
1
H NMR (D2O): δ 8.67 (s, 1H), 8.48 (d, J = 6.2 Hz, 1H), 8.42 (d, J = 8.3 Hz, 1H), 8.03 (t, J = 13.1
Hz, 2H), 7.83 – 7.70 (m, 5H), 7.28 (t, J = 8.7 Hz, 2H), 6.32 (dd, J = 19.9, 13.5 Hz, 2H), 4.15 (s,
6H), 3.62 (t, J = 6.2 Hz, 2H), 3.57 – 3.42 (m, 15H), 3.40 – 3.27 (m, 5H), 2.92 (dq, J = 15.0, 7.4
Hz, 6H), 2.42 (t, J = 6.2 Hz, 2H), 2.18 – 2.05 (m, 5H), 1.93 (t, J = 7.4 Hz, 2H), 1.63 – 1.55 (m,
9H), 1.02 (d, J = 16.4 Hz, 3H), 0.74 (s, 1H), 0.48 (s, 1H).
31
P NMR (202 MHz, Deuterium Oxide)::
δ 16.6-15.6 (br, 2P)
87

3.6.2. Biological Experiments
Evaluation on the anti-resorption of novel fluorescently labeled BPs on osteoclast
formation was performed at Dr. Brandon F. Boyce’s lab, University of Rochester. The biological
assays were conducted by Drs. Shuting Sun, Xiaodong Hou, and Zhenqiang Yao. All assays were
triplicated and detailed procedure of these assays is shown below.
Effect of BPs on osteoclast formation
Wild type (WT) mouse bone marrow cells (~5×10
4
)

in 96-well plates were cultured with
recombinant macrophage colony-stimulating factor (M-CSF) (10 ng/ml) for 2 days to enrich for
osteoclast precursors (OCPs), which were then treated with RANKL (10 ng/ml) in the presence of
the indicated concentration (0.1–10 µM) of BP-conjugate for 3 days when multinucleated
osteoclasts (OCs) had formed. Staining of tartrate-resistant acid phosphatase (TRAP) activity was
performed to identify TRAP+ OCs containing at least three nuclei (Figure 3.4A) and to quantify
OC number and area (Figure 3.4B).
Effect of BPs on bone resorption on bovine bone slices
OC cultures on bone slices in 96-well plates in the presence of dye-BP conjugates for 10
days when a large number of resorption pits were present. The resorption pits were visualized by
using confocal fluorescence microscopy (Figure 3.5) and the pit area were measured. Subsequently,
the cells were brushed off the bone slices, followed by toluidine blue staining (Figure 3.#).
Measurements of the resorption pit areas were repeated. The measurement of the areas determined
by two different visualization techniques were compared.
3.7 References
1. Ebetino, F. H.; Hogan, A. M. L.; Sun, S. T.; Tsoumpra, M. K.; Duan, X. C.; Triffitt, J. T.;
Kwaasi, A. A.; Dunford, J. E.; Barnett, B. L.; Oppermann, U.; Lundy, M. W.; Boyde, A.;
Kashemirov, B. A.; McKenna, C. E.; Russell, R. G. G., The relationship between the chemistry
and biological activity of the bisphosphonates. Bone 2011, 49, 20-33.
88

2. Torres Martin de Rosales, R.; Finucane, C.; Mather, S. J.; Blower, P. J., Bifunctional
bisphosphonate complexes for the diagnosis and therapy of bone metastases. Chem. Commun.
(Camb) 2009, 4847-4849.
3. Maalouf, M. A.; Wiemer, A. J.; Kuder, C. H.; Hohl, R. J.; Wiemer, D. F., Synthesis of
fluorescently tagged isoprenoid bisphosphonates that inhibit protein geranylgeranylation. Bioorg.
Med. Chem. 2007, 15, 1959-1966.
4. Rogers, M. J.; Crockett, J. C.; Coxon, F. P.; Monkkonen, J., Biochemical and molecular
mechanisms of action of bisphosphonates. Bone 2011, 49, 34-41.
5. Sato, M.; Grasser, W.; Endo, N.; Akins, R.; Simmons, H.; Thompson, D. D.; Golub, E.;
Rodan, G. A., Bisphosphonate action. Alendronate localization in rat bone and effects on
osteoclast ultrastructure. J. Clin. Invest. 1991, 88, 2095-2105.
6. Masarachia, P.; Weinreb, M.; Balena, R.; Rodan, G. A., Comparison of the distribution of
3H-alendronate and 3H-etidronate in rat and mouse bones. Bone 1996, 19, 281-290.
7. Coxon, F. P.; Thompson, K.; Roelofs, A. J.; Ebetino, F. H.; Rogers, M. J., Visualizing
mineral binding and uptake of bisphosphonate by osteoclasts and non-resorbing cells. Bone 2008,
42, 848-860.
8. Thompson, K.; Rogers, M. J.; Coxon, F. P.; Crockett, J. C., Cytosolic entry of
bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis. Mol.
Pharmacol. 2006, 69, 1624-1632.
9. Zaheer, A.; Lenkinski, R. E.; Mahmood, A.; Jones, A. G.; Cantley, L. C.; Frangioni, J. V.,
In vivo near-infrared fluorescence imaging of osteoblastic activity. Nat. Biotechnol. 2001, 19,
1148-1154.
10. Bhushan, K. R.; Tanaka, E.; Frangioni, J. V., Synthesis of conjugatable bisphosphonates
for molecular imaging of large animals. Angew. Chem., Int. Ed. 2007, 46, 7969-7971.
11. Figueiredo, J. L.; Passerotti, C. C.; Sponholtz, T.; Nguyen, H. T.; Weissleder, R., A novel
method of imaging calcium urolithiasis using fluorescence. J. Urol. (Hagerstown, MD, U.S.) 2008,
179, 1610-1614.
12. Jaffer, F. A.; Libby, P.; Weissleder, R., Optical and multimodality molecular imaging:
insights into atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 2009, 29, 1017-1024.
13. Subramanian, S.; Jaffer, F. A.; Tawakol, A., Optical molecular imaging in atherosclerosis.
J. Nucl. Cardiol. 2010, 17, 135-144.
14. Kavanagh, K. L.; Guo, K.; Dunford, J. E.; Wu, X.; Knapp, S.; Ebetino, F. H.; Rogers, M.
J.; Russell, R. G.; Oppermann, U., The molecular mechanism of nitrogen-containing
bisphosphonates as antiosteoporosis drugs. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 7829-7834.
89

15. Kashemirov, B. A.; Bala, J. L.; Chen, X.; Ebetino, F. H.; Xia, Z.; Russell, R. G.; Coxon, F.
P.; Roelofs, A. J.; Rogers, M. J.; McKenna, C. E., Fluorescently labeled risedronate and related
analogues: "magic linker" synthesis. Bioconjug Chem. 2008, 19, 2308-2310.
16. McKenna, C. E.; Kashemirov, B. A.; Bala, J. L. F. Synthesis of drug conjugates via reaction
with epoxide-containing linkers. US20080312440A1, 2008.
17. Roelofs, A. J.; Coxon, F. P.; Ebetino, F. H.; Lundy, M. W.; Henneman, Z. J.; Nancollas,
G. H.; Sun, S. T.; Blazewska, K. M.; Bala, J. L. F.; Kashemirov, B. A.; Khalid, A. B.; McKenna,
C. E.; Rogers, M. J., Fluorescent Risedronate Analogues Reveal Bisphosphonate Uptake by Bone
Marrow Monocytes and Localization Around Osteocytes In Vivo. J. Bone Miner. Res. 2010, 25,
606-616.
18. Guo, Z.; Park, S.; Yoon, J.; Shin, I., Recent progress in the development of near-infrared
fluorescent probes for bioimaging applications. Chem. Soc. Rev. 2014, 43, 16-29.
19. Tanaka, S., Regulation of bone destruction in rheumatoid arthritis through RANKL-RANK
pathways. World J. Orthop. 2013, 4, 1-6.
20. Mondal, S. B.; Gao, S.; Zhu, N.; Liang, R.; Gruev, V.; Achilefu, S., Real-time fluorescence
image-guided oncologic surgery. Adv. Cancer Res. 2014, 124, 171-211.
21. Gibbs, S. L., Near infrared fluorescence for image-guided surgery. Quant. Imaging Med.
Surg. 2012, 2, 177-187.
22. Sun, S.; Blazewska, K. M.; Kadina, A. P.; Kashemirov, B. A.; Duan, X.; Triffitt, J. T.;
Dunford, J. E.; Russell, R. G.; Ebetino, F. H.; Roelofs, A. J.; Coxon, F. P.; Lundy, M. W.;
McKenna, C. E., Fluorescent Bisphosphonate and Carboxyphosphonate Probes: A Versatile
Imaging Toolkit for Applications in Bone Biology and Biomedicine. Bioconjug Chem. 2016, 27,
329-340.
23. Roelofs, A. J.; Stewart, C. A.; Sun, S. T.; Blazewska, K. M.; Kashemirov, B. A.; McKenna,
C. E.; Russell, R. G. G.; Rogers, M. J.; Lundy, M. W.; Ebetino, F. H.; Coxon, F. P., Influence of
Bone Affinity on the Skeletal Distribution of Fluorescently Labeled Bisphosphonates In Vivo. J.
Bone Miner. Res. 2012, 27, 835-847.
24. Trott, O.; Olson, A. J., AutoDock Vina: improving the speed and accuracy of docking with
a new scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010, 31,
455-461.

90

CHAPTER 4
Determination of the Bone Binding Affinity of
Novel Fluorescently Labeled Bisphosphonates
4.1 The role of bone binding affinity in the pharmacology of bisphosphonates
As previously described in Chapter 1, a key factor in determining the in vivo anti-
resorptive activities of N-BPs is their binding affinity to bone minerals or, the main component of
bone mineral, hydroxyapatite (HAP). The strength of these interactions not only affects the uptake
and retention of N-BPs by the skeleton, but also their distribution within bone. Their bone binding
affinity may also influence the diffusion of these drugs through the osteocyte lacunar-canicular
network inside the bone. As shown in Figure 4.1B, the lower affinity phosphonocarboxylate
(RISPC) conjugates labeled with rhodamine red (RhR) diffuse deeper into the bone matrix. In
contrast, the higher affinity conjugates: carboxyfluorescein labeled risedronate (5,6-FAM-RIS),
accumulate at the surface of the bone.
1, 2, 3

Further implications of the differences in bone binding affinity of various N-BPs are their
different clinical dosages and dosing intervals. For instance, the recommended dosage of
zoledronate (ZOL) for osteoporotic patients is 5 mg intravenously once a year, whereas that of
ibandronate (IBN) is 3 mg every three months.
4
The stronger HAP binding affinity of ZOL in
comparison to IBN could explain the difference in dosage between the two bisphosphonates. The
strong HAP binding of N-BPs relative to their non-nitrogen-containing BPs may also explain the
long lasting effect of N-BPs months after discontinuation of the medication.
5, 6
Studies performed
by Khan et al. in patients treated with alendronate (ALN) administered via intravenous route for
postmenopausal osteoporosis estimated that the terminal elimination time of ALN was on the order
91

of 10 years.
7
Similar studies in animal models (rats, mice, and dogs) also show long terminal
elimination times on the order of several months.
8, 9

Figure 4.1. Relationship between the bone binding affinity of bisphosphonate drugs and their
uptake in bone. (A) Differences in binding affinities and their effects on mineral surface properties
of bisphosphonate drugs.
10
(B) Uptake of fluorescently labeled N-BP probes with different HAP
binding affinity.
1
Image of the cross section of the tibia of a 9-week-old male Sprague−Dawley rat
(0.2 kg) was taken 7 days after subcutaneous administration of a single dose containing 5(6)-FAM-
RIS (0.345 mg/kg) and 5(6)-RhR-RISPC (0.385 mg/kg). In the figure, nuclei were stained with
DAPI (blue), 5(6)-FAM-RIS was green, and 5(6)-RhR-RISPC was red. The arrows indicate newly
formed osteocytes. While the lower HAP binding affinity of 5(6)-RhR-RISPC (red) allows deeper
penetration of the compound into the bone matrix and their high localization in the osteocyte
lacunae (arrows). Higher HAP binding affinity 5(6)-FAM-RIS (green) concentrates at the surface
of the bone.
4.2 Methods used to determine the bone binding affinity of bisphosphonates
Many methods have been employed to determine the bone binding affinity of BPs. In the
1970s and 1980s, bone scintigraphy using radioactive labeled with
99m
Tc was the predominant
92

method in determining bone binding affinity, as well as the tissue distribution of BPs.
11, 12
Studies
were conducted in rats using non-nitrogen containing BPs, including methylenediphosphonic acid
(MDP), monomethyl-methylene bisphosphonate (Compound A), and clodronate (compound D)
(Figure 4.2).
11, 12
Results from these experiments demonstrated that the highest levels of BPs were
found in bones, such as the femur, and at much higher concentrations than blood, liver, and muscle
(Figure 4.2).
11, 12

Figure 4.2. Tissue distribution of
99m
Tc BP complexes.
12

Recently, several new techniques have been developed to determine the bone binding
affinity of N-BPs. These include (1) measuring their inhibition of HAP crystal growth; (2)
observing the competitive binding assays of [
14
C]-ALN against other BPs; (3) using
31
P NMR
spectroscopy to quantify the amount of BP bound to HAP powder; (4) determining the retention
time of BPs using a HAP column; (5) determining K d (the equilibrium dissociation constant) and
93

Bmax (the maximum number of binding sites) using HAP powder; and (6) binding studies using
calcium phosphate (CaP) coated discs. It is important to note that these methods do not provide an
absolute value of the HAP binding affinity of an individual BP, but rather a relative ranking of
BPs based on their binding affinity.
Nancollas et al. studied the inhibitory effect of BPs on HAP crystal growth at various
concentrations, which revealed a kinetic model of mineral-binding affinity of bisphosphonates at
the mineral surface.
10
The study focused on the zeta potential. It is defined as the electrical potential
of the shear plane at the crystal surfaces and was used to measure the absorption of BPs at the
mineral surface. A graph of zeta potential versus concentration provides the adsorption affinity
constant (KL). The ranking of BPs on inhibition of HAP growth based on K L values is shown in
Table 4.1. This method was also used to conduct another experiment, in which the in vitro
inhibition of ALN, RIS, and ZOL on carbonated apatite was evaluated.
13
Results from the study
indicated that ZOL > ALN > RIS in inhibiting carbonated apatite crystal growth. This means that
ZOL has the highest bone binding affinity, followed by ALN and RIS.
Leu et al. at Merck Research Laboratories employed another approach to determine the
bone binding affinity of BPs. His technique used [
14
C]-ALN in competitive binding assays against
other clinically used BPs to human bone particles at various concentrations.
14
The competitive
inhibition curves of [
14
C]-ALN against other BPs yielded relative Ki values (the equilibrium
inhibition constant). The N-BPs were ranked in the order of increasing binding affinity based on
the experimental Ki values. A potential drawback in this method is the poor displacement of BPs
once bound to bone mineral in the competitive binding assays, in which weaker HAP binding
affinity N-BPs were co-incubated with stronger HAP binding affinity N-BPs.
15
The Ki values of
94

these weakly bound BPs as determined from the competitive assays may be lower than they are
supposed to.
Ranking
of BP
(high to
low
affinity)
In vitro
inhibition of
crystal growth
induced by the
presence of
BPs
10

Competitive
binding
assay using
[
14
C]-ALN
14

31
P NMR
spectroscopy by
Mukherjee et al.
based on free
binding energy
(ΔGbinding)
16, 17

31
P NMR
spectroscopy
by Jahnke et
al. using
HAP
powder
18

FPLC
using
HAP
column
22

Determination
of Kd and Bmax
using HAP
powder
22

1 ZOL ALN ALN PAM PAM PAM
2 PAM ZOL PAM ALN ALN ALN
3 ALN PAM ETI ZOL NER ETI
4 IBN RIS ZOL RIS ETI NER
5 RIS ETI RIS IBN ZOL ZOL
6 ETI IBN CLO IBN RIS
7 CLO TIL MIN IBN
8 CLO RIS MIN
9 CLO CLO
Table 4.1. Ranking of clinically used BPs using various methods. PAM: pamidronate, IBN:
ibandronate, ETI: etidronate, NER: neridronate, MIN: minodronate, TIL: tiludronate, CLO:
clodronate.
NMR spectroscopy, particularly
31
P NMR, is a useful technique that can be used to
determine thermodynamic properties of the bone binding reaction of BPs. Multiple studies
performed by Mukherjee et al. using solid-state
31
P NMR attempted to determine the interaction
and binding mode of clinically used BPs to human bone powder.
16, 17
They also attempted to rank
them in the order of increasing binding affinity based on binding free energy (ΔG) derived from
isothermal calorimetry data.
17
On the other hand, Jahnke et al. employed
31
P NMR spectroscopy
to study the amount of clinically used BPs remaining in solution.
18
This method was utilized for
both competitive and non-competitive binding assays of BPs to HAP and bone powder. Results
from competitive assays provided a relative ranking of pamidronate (PAM), ZOL, ALN,
risedronate (RIS), and IBN (Table 4.1). Recent studies performed by Puljula et al. also used similar
techniques to study the binding affinities of BPs in various phosphoester forms in order to elucidate
the importance of OH in the phosphonic group.
19
These findings demonstrated that the degree of
95

esterification of BPs was crucial to their bone binding affinity, with affinity decreasing as the
number of esterified OH groups increased.

Figure 4.3. Determination of the HAP binding affinity using FPLC. (A) Chromatographs of
NE10790 (RISPC), RIS, and ZOL overlaid together. Higher binding affinity compounds (e.g., RIS,
ZOL) strongly interact with HAP powder, resulting in their broad peak. (B) Comparison of the
broadness of eluting peaks of RIS (an α-hydroxy BP), NE58043 (deoxy-RIS, or dRIS), and
NE10790 (RISPC, a phosphonocarboxylate). The red dashed circles indicate the differences in the
chemical structures of RIS, dRIS, and RISPC. Higher binding affinity compounds (e.g., RIS, dRIS)
have broad eluting peaks while that of RISPC, a weaker binding affinity compound, is sharp.
Another technique was developed to further simplify the quantification of bone binding
affinity of BPs. In this technique, the binding affinity was determined via the retention time of BP
in a fast protein liquid chromatography (FPLC) column packed with crystalline HAP.
20, 21

Detection of eluted BPs was performed via either UV-Vis absorption, chemical assay or tandem
LC/MS-MS with a known internal standard for quantification.
22
This method was further expanded
to other types of phosphate minerals, such as ceramic HAP and fluoroapatite. The HAP binding
affinities of the tested BPs were ranked based on their retention time. The longer a BP was retained
96

on the column, the stronger the presumed binding of that BP to HAP powder was. A major
drawback of this method was that strong interaction of N-BPs with HAP resulted in observation
of a characteristic broad peak, which contributes to the inaccurate determination of the retention
time (Figure 4.3A). In addition, HAP in either crystalline or powder forms was found to be partially
soluble in the eluting buffer, 1.5 M phosphate buffer.

Figure 4.4. HAP binding of ROX-RIS, ROX-RISPC, AF647-RIS, and AF647-RISPC determined
using two different methods.
1
(A) Determination of Kd and Bmax using HAP powder. (B) Relative
HAP binding using HAP column method. The Kd values of AF647-RIS and AF647-RISPC were
well correlated to their HAP binding affinity determined using the HAP column method, while in
contrast, the Kd values of ROX-RIS and ROX-RISPC did not correlate with their predicted
affinities from the HAP column method.
97

HAP powder was also employed to quantitatively determine K d and Bmax via constructing
the Langmuir isotherm curve of the % HAP binding of BPs versus concentration.
22
Various
concentrations of BPs were incubated with an excess amount of HAP powder. The amount of BP
absorbed by the HAP powder was determined using either tandem LC-MS/MS with a known
internal standard or UV-Vis spectroscopy for non-fluorescent and fluorescent BPs, respectively.
The small amount of HAP powder used can incur inconsistent results, and is a potential source of
error in this method.
Recently, in 2013, Hokugo et al. described a new method to observe the displacement of
fluorescently labeled BPs on calcium phosphate (CaP) coated discs by another fluorescently
labeled BP.
23
However, the method was conducted only using FAM-RIS and ROX-RIS, and the
experimental results were qualitative rather than quantitative. Therefore, this method is not suitable
for our purpose of determining the relative HAP binding affinity of our series of newly synthesized
fluorescently labeled BPs.
Several of these methods described above were previously used to study the bone binding
affinity of fluorescently labeled BPs, including 1) inhibition of HAP crystal growth assays,
24
2)
FPLC column packed with commercially available ceramic HAP,
1, 25
and 3) determination of Kd
using the Langmuir isotherm curves.
1
Method 1 required instruments that were not available in
our laboratory. Method 2 was convenient and simple to set up. However, significant drawbacks
were that inconsistency in the column lifetime could potentially interfere with our results, as well
as the high cost of commercially available ceramic HAP. Similar to Method 2, Method 3 had the
advantage of using commercially available ceramic HAP. However, it might have required a large
amount of fluorescently labeled BPs, which would have been cost prohibitive. Previously
98

published inconsistent data, as observed in the K d values of ROX-RIS and ROX-RISPC, was an
additional concern against this method.
These previously published techniques all had their individual advantages and drawbacks,
but were not applicable for our project due to various reasons as described above. Thus, a new
simple method to determine the bone binding affinity of fluorescently labeled BPs was required.
4. 3 Results and discussion
Instead of using HAP in powder or column form as described in the previously reported
methods, we used commercially available dentine discs purchased from IDS Osteosite. First,
preliminary studies were performed on the HAP discs incubated with fluorescent-labeled BP
compounds for 1.5 h. Aliquots of 50 µL were collected every 15 minutes. The studies were
conducted at 20 µM concentration of both 5-FAM-RIS and 5-FAM-ZOL (Figure 4.5) to ensure
that aliquot samples can be analyzed by UV-Vis spectroscopy. The data was processed and percent
binding of BP-dye conjugates to HAP discs was calculated.
Preliminary studies indicated that full binding equilibrium of dye-BP conjugates with HAP
was not complete after the initial 1.5 hours. Thus, a longer study time was required to fully assess
their HAP binding affinity. The new study would span 27 hours, with aliquots of 50 µL collected
every 2 hours for the first 10 hours, followed by two final collections at 24 and 27 hours. In addition,
preliminary experiments also indicated that at 20 µM concentration, the HAP discs were saturated.
Therefore, subsequent experiments with lower concentrations at 10 µM and 5 µM of both 5-FAM-
RIS and 5-FAM-ZOL were conducted in order to determine the optimal time and concentration
for HAP binding studies.
99

Figure 4.5. Chemical structure of 5-FAM-RIS, 5-FAM-ZOL, ROX-RISPC, ROX-RIS, AF647-
RIS, AND AF647-RISPC. FAM: carboxyfluorescein, ROX: rhodamine X, AF647: Alexa Fluor
647.
Our data showed that at concentrations of 10 µM and 20 µM, the HAP discs became
saturated after 2 hours. At 5 µM, the maximum % HAP binding reached almost 80% over the same
time period. This revealed that 5 µM was the optimal concentration for our binding studies. In
addition, the majority of FAM compounds bound to the HAP discs within the first 2 hours, and
the rate of binding slowed down substantially afterwards. These findings suggested that the
100

binding of BPs to HAP discs involved two separate processes: 1) saturation of disc surface and 2)
slow diffusion of BP conjugates into the dentine discs.
The previous studies on determining the in vitro binding of fluorescently labeled BPs were
performed on dentine discs that were available in-house and directly processed in the lab, where
the thickness and diameter could be manually controlled.
26
A measurement of a sample of 5
commercially purchased dentine discs indicated that mass of the discs varied within a 6% range.
Therefore, the mass, diameter, and thickness of each HAP disc used in the binding studies was
measured and accounted for in our calculations to minimize experimental errors.
Relative to 5-FAM-ZOL, 5-FAM-RIS was observed to have stronger HAP binding affinity
in our preliminary studies. For instance, after 2 hours, approximately 44% of 5-FAM-RIS bound
to the HAP disc while only 32% of 5-FAM-ZOL was bound over the same time course.
Additionally, 5-FAM-RIS also reached equilibrium much faster than 5-FAM-ZOL. This represents
another indicator of stronger HAP affinity of 5-FAM-RIS relative to 5-FAM-ZOL. This
observation was in agreement with previously published data.
1
The ratio of percent binding of 5-
FAM-RIS and 5-FAM-ZOL (1.26 versus 1.18, respectively), was also similar to that of the
previously published data.
The final parameters for our bone binding affinity experiments of fluorescently labeled BPs
were the following: 1) an optimal concentration of dye-BPs at 5 µM and 2) the experiment time
limited to 2 h, with aliquots of 50 µL collected every 15 min. The experiments were performed in
triplicate for every compound, and aliquot samples were diluted to a total volume of 100 µL with
1X PBS and analyzed using UV-Vis spectroscopy. The collected data was processed, and percent
HAP binding was normalized against the surface area (SA) of discs.
101

Figure 4.6. HAP binding of previously published fluorescently labeled RIS using the newly
established method (A). Comparison of HAP binding of previously published fluorescently labeled
RIS using HAP disc and HAP column methods (B).
Besides the 12 new RIS conjugates previously described in Chapter 3, binding studies for
previously reported compounds were also performed for comparison. These included 5-FAM-RIS,
5-FAM-ZOL, ROX-RISPC, ROX-RIS, AF647-RIS and AF647-RISPC (Figure 4.5). At 100 min,
saturation at the disc surface occurred, indicated by the plateau observed in binding curves (Figure
4.6A). In order to simplify and more easily compare the binding affinity data of BP conjugates,
HAP binding data at 100 min, where saturation occurred, for all compounds was collected and
normalized against that of 5-FAM-RIS (Figure 4.6B).
Order HAP Column HAP Discs
1 ROX-RIS ROX-RIS
2 5(6)-FAM-RIS ROX-RISPC
3 5-FAM-ZOL 5-FAM-RIS
4 ROX-RISPC 5-FAM-ZOL
5 AF647-RIS AF647-RIS
6 AF647-RISPC AF647-RISPC
Table 4.2. Relative ranking of the HAP binding affinity of previously published fluorescently
labeled BPs and PCs.
102

To establish the validity of our HAP Disc method, the previously reported HAP binding
affinities of these fluorescently labeled BPs were also normalized against 5-FAM-RIS and plotted
in the same graph as the relative HAP binding collected using our newly established method (Fig.
4.6B).
1
The HAP binding affinity of BP conjugates using these two different methods (HAP
Column and HAP Discs) showed similar trends with ROX-RIS and AF647-RISPC possessing the
highest and lowest binding affinities, respectively. Other reference compounds, such as 5-FAM-
RIS, 5-FAM-ZOL, and ROX-RISPC, showed a slightly different trend. While ROX-RISPC
exhibits stronger affinity than 5-FAM-RIS and 5-FAM-ZOL using dentine disc method, 5-FAM-
RIS and 5-FAM-ZOL have higher affinity than ROX-RISPC does using HAP column method. As
previously discussed, the relative HAP binding of ROX-RIS and ROX-RISPC determined by HAP
powder did not match what was observed in the HAP column method. Here, our newly established
method did reflect the similar relative HAP binding affinity of these compounds measured by HAP
column.
Several additional trends are notably apparent in our collected data. Regarding the effect
of the linker on binding affinity, there was no clear trend observed with different linker lengths
(Figure 4.7). The HAP binding affinities in BP and PC compounds are primarily determined by
the bisphosphonate, α-hydroxy, or phosphonocarboxylate groups. Structural modifications in the
fluorescently labeled dye conjugates did not occur at these moieties. Therefore, the HAP binding
affinity of these conjugates should be comparable to their parent compounds.
On the other hand, the HAP binding affinity of BP conjugates may depend on the charges
of the fluorescent dyes at a physiological pH of 7.4. For instance, ROX, FAM, and sulfo-Cy5 either
are neutral or have one negative charge at pH 7.4. Conjugation of ROX dye to BPs enhances its
HAP binding affinity. Particularly, this observation was notable in the data for ROX-RISPC.
103

Figure 4.7. HAP binding of fluorescently labeled RIS, ZOL, and RISPC determined using HAP column and HAP disc methods. All the
HAP binding affinities were normalized against 5-FAM-RIS.
104

RISPC has lower affinity due to the elimination of one phosphonate group.
15
However, when
conjugated to ROX dye, ROX-RISPC exhibited higher HAP binding affinity, even as comparable
as that of 5-FAM-RIS, determined using our newly established disc method. The effect of ROX
on HAP binding was not evident in the HAP column method (Figure 4.7). In contrast, conjugation
of AF647 to BPs resulted in the lower binding affinity of the conjugates. AF647 has three negative
charges under physiological pH. This may result in the electrostatic repulsion of the dye and the
negatively charged bone surface.
27
The low HAP binding affinity of AF647 conjugates was most
apparent in the data for AF647-RISPC, which was observed in both methods. Furthermore, the
binding affinity of AF647-RISPC determined using the dentine disc method was much lower than
that determined using the HAP column method. This could be due to the intrinsic differences in
the physical processes involved in the two analytical methods. While the HAP disc method
measures kinetics of surface saturation of the binding process, the HAP column surveys the
equilibrium of the process.
Ultimately, the HAP binding affinity of the fluorescently labeled BP conjugates depends
on two factors: 1) the parent drug N-BPs, and 2) fluorescent dyes. It is also important to note that
this method is only applicable for fluorescently labeled BPs, where the high molar extinction
coefficient of fluorescent dye allows the detection of samples at the low micromolar concentrations
using UV-Vis spectroscopy.
4.4 Conclusion
A new method to determine the relative HAP binding affinity of a series of newly
synthesized fluorescently labeled BPs was established using commercially available dentine discs.
For previously published compounds, this method resulted in a ranking order similar to those
obtained by other methods. While the different linker lengths did not affect the HAP binding of
105

the BP conjugates, the parent drug and the conjugated dye played key roles in determining their
HAP binding affinity. Our new method could be used to determine the relative HAP binding
affinity of other fluorescently labeled conjugates. A drawback of this method is that it relies on the
high molar extinction coefficient of fluorescent dyes. This property would make this method
unsuitable for determining the bone binding affinity of non-fluorescent BPs.
4.5 Experimental Section
4.5.1. Chemical Synthesis
The synthesis of novel fluorescently labeled BP conjugates was previously described in Chapter
3. Reference compounds, including 5-FAM-RIS, 6-FAM-RIS, 5-FAM-ZOL, ROX-RIS, ROX-
RISPC, AF647-RISPC were synthesized as previously reported.
1, 25

4.5.2. HAP Binding Experiments
Dentine discs were purchase from IDS Osteosite. The weight and dimensions of each disc were
measured using a Shimadzu Libror AEG-45SM and Leica Galen III microscope, respectively. The
discs were equilibrated overnight in 0.5 mL of 10 mM (1X) PBS buffer (pH 7.4) using a Corning
Costar 24 well cell culture plate. Each disc was equilibrated in an individual well in a custom built
water bath, whose temperature was kept at 37
o
C. The disc was subsequently transferred to a
different well that contained 0.55 mL of 5 μM solution of dye-BP conjugates prewarmed to 37
o
C,
and remained there for 10 minutes. An aliquot of 50µL was collected right before the disc was
added into the solution and labeled as T0 sample. Aliquots of 50µL were collected every 20 minutes
up to 140 minutes. After 1:2 dilution of the samples with 1X PBS (pH 7.4), they were analyzed
using a DU800 Beckman-Coulter spectrophotometer. Molar extinction coefficients used for the
dye-BP conjugates were the same as those of the unconjugated dyes, which were previously
described in Chapter 3. The collected data was calculated as percent HAP binding and normalized
106

with the surface area of the discs, assuming that the discs were cylindrical. All experiments were
triplicated. The evaporation rate of 1X PBS buffer was measured every 20 minutes up to 140 min
and that experiment was also triplicated. Volume correction was included during data processing
for every aliquot at each time point.
4.6 References
1. Sun, S.; Blazewska, K. M.; Kadina, A. P.; Kashemirov, B. A.; Duan, X.; Triffitt, J. T.;
Dunford, J. E.; Russell, R. G.; Ebetino, F. H.; Roelofs, A. J.; Coxon, F. P.; Lundy, M. W.;
McKenna, C. E., Fluorescent Bisphosphonate and Carboxyphosphonate Probes: A Versatile
Imaging Toolkit for Applications in Bone Biology and Biomedicine. Bioconjug Chem 2016, 27,
329-340.
2. Oldfield, E., Targeting isoprenoid biosynthesis for drug discovery: bench to bedside. Acc.
Chem. Res. 2010, 43, 1216-1226.
3. Russell, R. G.; Xia, Z.; Dunford, J. E.; Oppermann, U.; Kwaasi, A.; Hulley, P. A.;
Kavanagh, K. L.; Triffitt, J. T.; Lundy, M. W.; Phipps, R. J.; Barnett, B. L.; Coxon, F. P.; Rogers,
M. J.; Watts, N. B.; Ebetino, F. H., Bisphosphonates: an update on mechanisms of action and how
these relate to clinical efficacy. Ann. N.Y. Acad. Sci. 2007, 1117, 209-257.
4. Sambrook, P., Quarterly intravenous injection of ibandronate to treat osteoporosis in
postmenopausal women. Clin. Interv. Aging 2007, 2, 65-72.
5. Black, D. M.; Delmas, P. D.; Eastell, R.; Reid, I. R.; Boonen, S.; Cauley, J. A.; Cosman,
F.; Lakatos, P.; Leung, P. C.; Man, Z.; Mautalen, C.; Mesenbrink, P.; Hu, H.; Caminis, J.; Tong,
K.; Rosario-Jansen, T.; Krasnow, J.; Hue, T. F.; Sellmeyer, D.; Eriksen, E. F.; Cummings, S. R.;
Trial, H. P. F., Once-yearly zoledronic acid for treatment of postmenopausal osteoporosis. N. Engl.
J. Med. 2007, 356, 1809-1822.
6. Owens, G.; Jackson, R.; Lewiecki, E. M., An integrated approach: bisphosphonate
management for the treatment of osteoporosis. Am. J. Manag. Care 2007, 13 Suppl 11, S290-308;
quiz S309-212.
7. Khan, S. A.; Kanis, J. A.; Vasikaran, S.; Kline, W. F.; Matuszewski, B. K.; McCloskey, E.
V.; Beneton, M. N.; Gertz, B. J.; Sciberras, D. G.; Holland, S. D.; Orgee, J.; Coombes, G. M.;
Rogers, S. R.; Porras, A. G., Elimination and biochemical responses to intravenous alendronate in
postmenopausal osteoporosis. J. Bone Miner. Res. 1997, 12, 1700-1707.
8. Pentikainen, P. J.; Elomaa, I.; Nurmi, A. K.; Karkkainen, S., Pharmacokinetics of
clodronate in patients with metastatic breast cancer. Int. J. Clin. Pharmacol. Ther. Toxicol. 1989,
27, 222-228.
107

9. Lin, J. H.; Duggan, D. E.; Chen, I. W.; Ellsworth, R. L., Physiological disposition of
alendronate, a potent anti-osteolytic bisphosphonate, in laboratory animals. Drug Metab. Dispos.
1991, 19, 926-932.
10. Nancollas, G. H.; Tang, R.; Phipps, R. J.; Henneman, Z.; Gulde, S.; Wu, W.; Mangood, A.;
Russell, R. G.; Ebetino, F. H., Novel insights into actions of bisphosphonates on bone: differences
in interactions with hydroxyapatite. Bone 2006, 38, 617-627.
11. Wang, T. S.; Fawwaz, R. A.; Johnson, L. J.; Mojdehi, G. E.; Johnson, P. M., Bone-seeking
properties of Tc-99m carbonyl diphosphonic acid, dihydroxy-methylene diphosphonic acid and
monohydroxy-methylene phosphoinic acid: concise communication. J. Nucl. Med. 1980, 21, 767-
770.
12. Wang, T. S.; Mojdehi, G. E.; Fawwaz, R. A.; Johnson, P. M., A study of the relationship
between chemical structure and bone localization of Tc-99m diphosphonic acids: concise
communication. J. Nucl. Med. 1979, 20, 1066-1070.
13. Henneman, Z. J.; Nancollas, G. H.; Ebetino, F. H.; Russell, R. G.; Phipps, R. J.,
Bisphosphonate binding affinity as assessed by inhibition of carbonated apatite dissolution in vitro.
J. Biomed. Mater. Res. A 2008, 85, 993-1000.
14. Leu, C. T.; Luegmayr, E.; Freedman, L. P.; Rodan, G. A.; Reszka, A. A., Relative binding
affinities of bisphosphonates for human bone and relationship to antiresorptive efficacy. Bone
2006, 38, 628-636.
15. Ebetino, F. H.; Hogan, A. M.; Sun, S.; Tsoumpra, M. K.; Duan, X.; Triffitt, J. T.; Kwaasi,
A. A.; Dunford, J. E.; Barnett, B. L.; Oppermann, U.; Lundy, M. W.; Boyde, A.; Kashemirov, B.
A.; McKenna, C. E.; Russell, R. G., The relationship between the chemistry and biological activity
of the bisphosphonates. Bone 2011, 49, 20-33.
16. Mukherjee, S.; Huang, C.; Guerra, F.; Wang, K.; Oldfield, E., Thermodynamics of
bisphosphonates binding to human bone: a two-site model. J. Am. Chem. Soc. 2009, 131, 8374-
8375.
17. Mukherjee, S.; Song, Y.; Oldfield, E., NMR investigations of the static and dynamic
structures of bisphosphonates on human bone: a molecular model. J. Am. Chem. Soc. 2008, 130,
1264-1273.
18. Jahnke, W.; Henry, C., An in vitro assay to measure targeted drug delivery to bone mineral.
ChemMedChem 2010, 5, 770-776.
19. Puljula, E.; Turhanen, P.; Vepsalainen, J.; Monteil, M.; Lecouvey, M.; Weisell, J.,
Structural requirements for bisphosphonate binding on hydroxyapatite: NMR study of
bisphosphonate partial esters. ACS Med. Chem. Lett. 2015, 6, 397-401.
20. Lawson, M. A.; Xia, Z.; Barnett, B. L.; Triffitt, J. T.; Phipps, R. J.; Dunford, J. E.; Locklin,
R. M.; Ebetino, F. H.; Russell, R. G., Differences between bisphosphonates in binding affinities
for hydroxyapatite. J. Biomed. Mater. Res. B Appl. Biomater. 2010, 92, 149-155.
108

21. Marma, M. S.; Xia, Z.; Stewart, C.; Coxon, F.; Dunford, J. E.; Baron, R.; Kashemirov, B.
A.; Ebetino, F. H.; Triffitt, J. T.; Russell, R. G.; McKenna, C. E., Synthesis and biological
evaluation of alpha-halogenated bisphosphonate and phosphonocarboxylate analogues of
risedronate. J. Med. Chem. 2007, 50, 5967-5975.
22. Duan, X. PhD. Dissertation, Oxford, 2010.
23. Hokugo, A.; Sun, S. T.; Park, S.; McKenna, C. E.; Nishimura, I., Equilibrium-dependent
bisphosphonate interaction with crystalline bone mineral explains anti-resorptive
pharmacokinetics and prevalence of osteonecrosis of the jaw in rats. Bone 2013, 53, 59-68.
24. Roelofs, A. J.; Coxon, F. P.; Ebetino, F. H.; Lundy, M. W.; Henneman, Z. J.; Nancollas,
G. H.; Sun, S.; Blazewska, K. M.; Bala, J. L.; Kashemirov, B. A.; Khalid, A. B.; McKenna, C. E.;
Rogers, M. J., Fluorescent risedronate analogues reveal bisphosphonate uptake by bone marrow
monocytes and localization around osteocytes in vivo. J. Bone Miner. Res. 2010, 25, 606-616.
25. Kashemirov, B. A.; Bala, J. L.; Chen, X.; Ebetino, F. H.; Xia, Z.; Russell, R. G.; Coxon, F.
P.; Roelofs, A. J.; Rogers, M. J.; McKenna, C. E., Fluorescently labeled risedronate and related
analogues: "magic linker" synthesis. Bioconjug Chem. 2008, 19, 2308-2310.
26. Roelofs, A. J.; Stewart, C. A.; Sun, S.; Blazewska, K. M.; Kashemirov, B. A.; McKenna,
C. E.; Russell, R. G.; Rogers, M. J.; Lundy, M. W.; Ebetino, F. H.; Coxon, F. P., Influence of bone
affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo. J. Bone Miner.
Res. 2012, 27, 835-847.
27. Gross, D.; Williams, W. S., Streaming potential and the electromechanical response of
physiologically-moist bone. J. Biomech. 1982, 15, 277-295.

109

CHAPTER 5
Overview of the Anatomical Structure of the Human Ear
and Drug Delivery to the Ear
5.1 Anatomical structure of the human ear
The human ear can be segregated into three parts: the outer, middle, and inner ear (Fig.
5.1). The outer ear includes the pinna and the external auditory canal and is separated from the
middle ear by the tympanic membrane.
1, 2
The middle ear houses the three hearing bones—the
ossicular chain, comprised of the malleus, incus, and stapes. The Eustachian tube provides a
conduit between the middle ear and the upper airway to allow for equalization of middle ear
pressure.
2

Figure 5.1. Anatomical Structure of the Human Ear.
Adapted from 2, 3. RWM: round window membrane; OW: oval window; IHCs: inner hair cells;
OHCs: outer hair cells.
110

The inner ear consists of the cochlea and the vestibular organs with the semicircular canals,
which share fluid spaces (Fig. 5.1).
1
The bony structure of the cochlea can be separated into three
compartments: scala tympani, scala vestibule, and scala media.
3
Connected via the helicotrema,
the scala tympani and scala vestibule are both occupied by perilymph (Fig. 5.1).
2
The middle
compartment, the scala media or cochlear duct, is filled with endolymph. The organ of Corti is
located on the basilar membrane in the scala media and is the site where a sound signal is converted
into an electrochemical signal and transmitted to the brain via the auditory nerve.
2
The organ of
Corti harbors inner and outer hair cells (IHCs, OHCs),
3
the sensory cells responsible for hearing,
and their surrounding supporting cells (SCs). Cochlear HCs are arranged in a frequency-dependent
manner, in which HCs at the base are tuned to high frequencies while those at the apex are sensitive
to low frequencies.
2
The endolymph-filled, membranous portion of the vestibular organs
(vestibular labyrinth) harbors vestibular HCs and is surrounded by perilymph.
External sound signals reach the middle ear via the external ear canal and the tympanic
membrane (TM). The ossicular chain attaches to the TM within the middle ear and provides a
connection to the oval window, one of the two bony openings of the cochlea that separate the inner
ear from the middle ear. Coordinated movements of the TM and the ossicular chain transform a
mechanical sound wave into a sound pressure wave that propagates within the inner ear fluids.
2

This wave causes motion of the basilar membrane and the organ of Corti, with displacement of
hair cell (HC) bundles. Displacement of inner hair cell (IHC) bundles opens transduction channels
at the bundle. The subsequent influx of potassium ions depolarizes the HCs, causing Ca
2+
mediated
excitatory neurotransmitter release at the basal portion of the IHC, where the ribbon synapse
connects to the spiral ganglion neuron. This mechanoelectrical conversion of sound allows
111

propagation as an electrical signal along the central auditory pathway and eventual processing in
the auditory cortex.
2, 3

Generation of a transduction current depends on an electrochemical gradient between the
intracellular environment and extracellular fluids (endolymph and perilymph). The endolymph
(about 8 µL in the human) is unique in that it is rich in potassium ([K
+
],160 mM) and low in sodium
(Na
+
< 1 mM), while perilymph (about 70 µL) consists of nearly opposite concentrations of K
+
(4
mM) and Na
+
(150 mM).
2, 4
The resulting difference in electrical potential plays a key role in the
potassium-dependent depolarization of HCs and subsequent generation of a neuronal action
potential. Both HCs and neurons are sensitive to small changes in ionic composition, pH, osmotic
pressure, volume, and potential.
2

5.2 Clinical considerations for drug delivery to the human inner ear
Similar to the blood-brain barrier, the blood-perilymph barrier (BPB) serves as a physical
and biochemical barrier between the systemic circulation and the cochlea. From a drug delivery
perspective, its existence is considered to be both beneficial and problematic. On the one hand, the
BPB protects the ear from damage caused by exogenous and endogenous toxins in the systemic
circulation.
2
However, it also presents challenges for efficient drug delivery of therapeutic agents
from the systemic circulation to the cochlea for the treatment of inner ear disorders. This is
particularly relevant for highly charged molecules with high molecular weight and hydrophilicity.
4

Other barriers interfering with drug delivery to the inner ear include the tympanic
membrane, the round window and the oval window. The tympanic membrane has three layers,
(the outer epidermal layer, a middle fibrous layer, and an inner mucosal layer), with a total
thickness of 0.6 mm and surface area of 80 mm
2
in human adults.
2
The round window (RW) and
the oval window (OW) serve as the semi-permeable membranous barriers between the middle ear
112

and the scala tympani and scala vestibule, respectively. The round window membrane (RWM)
consists of three layers, including the outer epithelium, which is separated from the inner
epithelium by a layer of connective tissues containing fibroblasts, blood vessels, collagen, and
elastic fibers, with a total thickness of 70 µm and a surface area of 2.2 mm
2
in humans.
2
This elastic
structure accommodates the changes in intracochlear pressure created by stapes movement within
the OW.
2

5.3 Hydrogels as a drug delivery vehicle to the human inner ear
The location and complex structure of the inner ear presents unique challenges for drug
delivery. With systemic delivery, relatively high doses may be required to achieve effective
intracochlear concentrations of a chosen drug, increasing undesirable side effects. Local delivery
offers the potential to achieve higher local concentrations for a given dose while avoiding systemic
toxicity, although the oval and round windows present barriers to intracochlear delivery. Direct
intracochlear administration requires a cochleostomy, significantly increasing the risk of
permanent SNHL.
2
By contrast, intratympanic (IT) administration incurs very little risk of SNHL
and is routinely performed in adult patients on an outpatient basis.
5, 6
Delivery via IT injection
typically involves a narrow needle puncture of the TM, followed by filling of the middle ear space
with a solution of medication
5
—subsequent intracochlear penetration relies on permeability of the
OW and RW to the drug of interest.
4
Otic agents delivered via this method, such as steroids, are
formulated at high concentrations, which can result in their rapid clearance from the middle ear
via Eustachian tube.
4
Sustained IT delivery, therefore, typically requires multiple injections over
time.
5
Several commonly used hydrogels are therefore discussed here to evaluate their efficiency
at providing sustained release of drugs to the inner ear.
113

5.3.1 Hyaluronic acid (HA)
HA is a non-sulfated, polyanionic glycosaminoglycan with a molecular weight (MW) that
varies from 100 kDa to 8000 kDa. It contains a linear polysaccharide composed of a disaccharide
repeating unit, β-1,4-D-gluronic acid-β-1,3-N-acetyl-D-glycosamine (Fig. 4A).
7
HA is found in
many places in the human body, with highest concentration in the extracellular matrix (ECM) of
cartilage and synovial fluid. Its stability in tissues varies, depending on tissue type.
8
HA
degradation involves hyaluronidase (an endogenous enzyme), followed by metabolism by the
liver.
8
Biomedical applications of HA have been investigated in many fields, including
ophthalmology, orthopedic surgery, rheumatology, dermatology, plastic surgery, wound healing,
and drug delivery.
9
In the field of HL, HA in its ester form was formulated as a film and utilized
to promote wound healing of the tympanic membrane and re-epithelization while preventing
adhesion between layers of mucous tissues.
9
With respect to inner ear drug delivery, Auris Medical
AG disclosed the use of HA at 2.8% w/w formulated with S-ketamine, 2.5% w/w, in several
patients for sustained release to the inner ear.
10
The release profile of the formulation, described
both in vitro and in vivo, shows that the sustained release of S-ketamine lasts up to 72 hours.
10, 11

5.3.2 Chitosan (CS)
CS is a linear polysaccharide produced by deacetylating chitin, a natural polysaccharide
commonly found in the exoskeleton of many insects and arthropods (Fig. 4B).
12, 13
Its
physiochemical characteristics depend mainly on the degree of deacetylation and its MW. Low
deacetylation results in poor water solubility, even at neutral or weakly acidic pH, where the amino
group is in protonated form, aiding water solubility.
13
Approved by the FDA for usage in wound
dressing,
13
CS also possesses unique properties as a cationic, biodegradable polymer with in situ
gelling ability. It has therefore proven suitable for the prolonged delivery of anionic drugs (e.g.,
114

naproxen) and other polyanionic molecules, such as DNA and siRNA.
14
Sustained delivery using
CS for inner ear drugs, such as A-317491
15
and carbamazepine,
16
has also been disclosed.
Sustained drug release by CS can be further regulated using chitosanase, which degrades the
hydrogel.
17
Thus, formulation of CS (2-10% w/w) combined with glycerolphosphate (5-30% w/w)
has been reported for sustained release of gentamicin, an ototoxic antibiotic used in the ablative
treatment of Ménière’s disease. The process involves a two-step delivery: 1) slow release of drug
from the hydrogel, and 2) termination of release via degradation using chitosanase.
17

5.3.3 Poloxamer 407 (P407)
Poloxamer 407 is a polymer consisting of polyethylene oxide (PEO) and polypropylene
oxide (PPO), organized in a triblock pattern as EO m-POn-EOm, with m and n in the range of 95-
105 and 54-60, respectively (Fig. 4C). Its MW has an average of 12,600 Da and varies within the
range of 9840-14600 Da.
18
One feature of P407 that has been extensively exploited by researchers
is its thermoreversibility. P407 solutions remain liquid at low temperature and gel as the
temperature increases. Increasing the concentration of P407 in solution results in lowering of the
gelling temperature,
18
allowing for tuning of the formulation for a specific application.
The use of P407 hydrogel for delivery of dexamethasone (Dex) for treatment of inner ear
disorders was first reported in 2008.
19
Since then, Otonomy Inc. has disclosed a series of
formulations utilizing 16-18% w/w P407 with a wide range of otic agents at concentrations of 2-
50% w/w, including gacyclidine, CNXQ, AMN082, carbamazepine, D-methionine,
methylprednisolone, prednisolone, prednisone, and ciprofloxacin.
15, 16, 20, 21, 22
At concentrations of
16-20% w/w P407, hydrogel gels near body temperature (37
o
C). The hydrogel formulation can
extend the release of such drugs for at least 5 days or more, depending on the agent.
15, 16, 20, 21, 22

Formulation of P407 with Dex is in clinical Phase 2 trials for cisplatin-induced hearing loss (HL)
115

in pediatrics, and Phase 3 trials for Ménière’s disease (MD) (although the Phase 2b results in the
MD trial did not meet their primary endpoints).
23
Formulation with gacyclidine, an NMDA
receptor antagonist, is in clinical Phase 1 for tinnitus.
24
P407 has also been utilized in the treatment
of middle ear disorders.
25, 26
Formulation of P407 with ciprofloxacin is in clinical Phase 2 trials for
the treatment of acute otitis media with tubes and acute otitis externa.
26, 27
Auris Medical AG has
disclosed the use of 20% w/w P407 in formulation with Dex, gacyclidine, and fluoroquinolone for
the treatment of tinnitus and middle ear infections, respectively.
28, 29

Polymers Hyaluronic Acid Chitosan Poloxamer 407
Chemical
Structures

The number of –NCOCH3 varies

Related
Applications
Sustained drug delivery of
S-ketamine
Sustained and regulated drug
delivery of gentamicin to the
inner ear
Sustained drug delivery of other
therapeutic agents (e.g., A-
317491 and carbamazepine)
Sustained drug delivery of
various therapeutic agents (e.g.,
dexamethasone, ciprofloxacin,
gacyclidine, etc.)
Related
Disorders
Tinnitus Ménière’s disease
Acute otitis media and acute
otitis externa
Ototoxic related HL
Tinnitus
Ménière’s disease
Table 5.1. Summary of related applications, disorders, and patent references associated with
different polymer systems.
5.4 Device-based delivery technologies to the human inner ear
A comprehensive review of medical devices for drug delivery to the inner ear has been
published elsewhere.
4, 30
Here we survey novel, innovative designs reported in the recent literature.
116

Currently, most clinical methods of local delivery to the cochlea involve IT injection,
followed by passive diffusion across the RW and OW.
31, 32
Passive diffusion precludes regulated,
precise delivery. In addition, passive diffusion does not allow for delivery of larger
macromolecules or viral vectors. One way to address this issue is to incorporate an array of silicon
microneedles into the device
33
to create temporary perforation of the RWM, permitting diffusion
of therapeutic agents into the inner ear (Fig. 5.2A). The agents can be infused via a regulated
osmotic pump, or coated on the exterior of solid microneedles.
33

Figure 5.2. Design of New Medical Devices for Drug Delivery to the Ear.
A-General scheme for the position of the microneedles connected to a delivery device in the inner
ear. Retrieved and adapted from 33. B-General scheme of the position of the stapes prosthesis in
the ear and its detail structure. Retrieved and adapted from 34. C-Magnetic guided drug delivery
systems. Average distance from the outer ear to round window membrane in human is 3 cm. The
magnetic pushing force works in the range of 2.6-5.2 cm. SPNs: superparamagnetic iron oxide
nanoparticles; Fpushing: magnetic pushing force existing beyond the magnetic field cancellation
117

point. Retrieved and adapted from 31, 35. D-Microfluidic Pump implanted behind the pinna and
located in the mastoid cavity. Retrieved and adapted from 36. Note: Ear diagram was retrieved
from 37.
Another method for direct intracochlear delivery could involve placement through the OW,
an approach informed by significant surgical experience in the treatment of otosclerosis.
Otosclerosis is a disease of abnormal bone remodeling around the OW, resulting in fixation of the
stapes and conductive HL. Otosclerosis treatment therefore utilizes either a hearing aid or
stapedectomy surgery, which involves removal of the arch of the stapes and creation of a small
fenestration through the fixed stapes footplate, followed by placement of a replacement
prosthesis.
38, 39
Based on this technique, a modified stapes prosthesis has been proposed (Fig. 5.2B),
which contains a hollow piston into which a drug-polymer matrix may be placed to allow slow
release into the inner ear.
34
This method could potentially be generalizable for any drug, although
stapedectomy typically carries a 1% risk of significant SNHL, including complete deafness in the
operated ear. In a related approach, a microfluidic pump has been disclosed to infuse viral vector
particles through an OW fenestration without removal of the stapes arch.
40

Another intriguing proposal is the use of powerful magnets to deliver nanoparticles into
the inner ear. In rodents, delivery to the inner ear using superparamagnetic iron oxide nanoparticles
(SPNs) was achieved by placing the nanoparticles at the RWM and actively transporting them
inside the inner ear using a magnet field of ~0.3 tesla.
41
The magnet was placed on the contralateral
side to the injected ear, with a measured effective distance in rat and guinea pigs of 2.5 and 3 cm,
respectively.
41, 42
However, since strength of the magnetic field exponentially decreases as the
distance between the injected ear and the magnet increases, the magnetic field required for human
scale delivery would be approximately 13 tesla, which significantly surpasses the safety limit set
118

by the FDA (8 tesla for adults and 4 tesla for children).
31, 43
To address this issue, an innovative
design was reported using two magnets placed at an angle (Fig. 5.2C)
44
to create a magnetic
cancellation node, beyond which the magnetic field creates a magnetic force pushing the SPNs
away from the magnets. The pushing magnetic force was determined to be effective within a range
of 2.6 and 5.2 cm,
35
and therefore suitable for delivery in humans. This system may also be
beneficial for drug delivery to the middle ear, with the SPNs applied at the tympanic membrane
and subsequently magnetically guided into the middle ear.
31

Finally, an implantable microfluidic device has been described.
36
The device contains a
micropump, with a flow rate controlled by an electronic, battery-powered system. The micropump
is connected to a reservoir containing the drug of interest. Drug delivery to the inner ear is achieved
by the circulation of the drug solution through a double-lumen catheter to a cannula, where infusion
and withdrawal of fluids occur. While the device is implanted in a mastoid cavity behind the pinna,
the cannula is implanted into the vestibule of the human ear (Fig. 5.2D). This complex system
enables a well-regulated, active diffusion of drugs and removal of fluid from inner ear at a
considerably low flow rate, resulting in minimal change of volume of the inner ear fluid, which is
an important consideration for direct cochlear drug delivery.
5.5 Conclusion
The remote location of the human inner ear presents unique opportunities and unmet
challenges in the search for effective methods to deliver drugs to the inner ears. Continued progress
toward understanding the biology of the ear, coupled with improvements in local delivery
technologies, should ultimately provide optimal solutions for curing inner ear diseases.
119

5.6 References
1. Hoskison, E.; Daniel, M.; Al-Zahid, S.; Shakesheff, K. M.; Bayston, R.; Birchall, J. P.,
Drug delivery to the ear. Ther. Deliv. 2013, 4, 115-124.
2. El Kechai, N.; Agnely, F.; Mamelle, E.; Nguyen, Y.; Ferrary, E.; Bochot, A., Recent
advances in local drug delivery to the inner ear. Int. J. Pharm. 2015, 494, 83-101.
3. Muller, U.; Barr-Gillespie, P. G., New treatment options for hearing loss. Nat. Rev. Drug
Discov. 2015, 14, 346-365.
4. Swan, E. E.; Mescher, M. J.; Sewell, W. F.; Tao, S. L.; Borenstein, J. T., Inner ear drug
delivery for auditory applications. Adv. Drug Deliv. Rev. 2008, 60, 1583-1599.
5. Hu, A.; Parnes, L. S., Intratympanic steroids for inner ear disorders: a review. Audiol.
Neurootol. 2009, 14, 373-382.
6. Hamid, M.; Trune, D., Issues, indications, and controversies regarding intratympanic
steroid perfusion. Curr. Opin. Otolaryngol. Head Neck Surg. 2008, 16, 434-440.
7. Burdick, J. A.; Prestwich, G. D., Hyaluronic acid hydrogels for biomedical applications.
Adv. Mater. 2011, 23, H41-56.
8. Heldin, P.; Asplund, T.; Ytterberg, D.; Thelin, S.; Laurent, T. C., Characterization of the
molecular mechanism involved in the activation of hyaluronan synthetase by platelet-derived
growth factor in human mesothelial cells. Biochem. J. 1992, 283 ( Pt 1), 165-170.
9. Kogan, G.; Soltes, L.; Stern, R.; Gemeiner, P., Hyaluronic acid: a natural biopolymer with
a broad range of biomedical and industrial applications. Biotechnol. Lett. 2007, 29, 17-25.
10. Guitton, M.; Puel, J.-L.; Pujol, R.; Ruel, J.; Wang, J. Methods using NMDA receptor
antagonists for the treatment of tinnitus induced by cochlear excitotoxicity. US20150265552A1,
2015.
11. Lopez-Escamez, J. A.; Carey, J.; Chung, W. H.; Goebel, J. A.; Magnusson, M.; Mandala,
M.; Newman-Toker, D. E.; Strupp, M.; Suzuki, M.; Trabalzini, F.; Bisdorff, A., Diagnostic criteria
for Meniere's disease. Consensus document of the Barany Society, the Japan Society for
Equilibrium Research, the European Academy of Otology and Neurotology (EAONO), the
American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) and the Korean
Balance Society. Acta Otorrinolaringol. Esp. 2016, 67, 1-7.
12. Klouda, L., Thermoresponsive hydrogels in biomedical applications: A seven-year update.
Eur. J. Pharm. Biopharm. 2015, 97, 338-349.
13. Hu, L.; Sun, Y.; Wu, Y., Advances in chitosan-based drug delivery vehicles. Nanoscale
2013, 5, 3103-3111.
120

14. Bernkop-Schnurch, A.; Dunnhaupt, S., Chitosan-based drug delivery systems. Eur. J.
Pharm. Biopharm. 2012, 81, 463-469.
15. Lichter, J.; Vollrath, B.; Trammel, A. M.; Duron, S. G.; Piu, F.; Dellamary, L. A.; Ye, Q.;
Lebel, C.; Scaife, M. C.; Harris, J. P. Controlled-release ion channel modulator compositions and
methods for the treatment of otic disorders. US20130150410A1, 2015.
16. Lichter, J.; Vollrath, B.; Trammel, A. M.; Duron, S. G.; Piu, F.; Dellamary, L. A.; Ye, Q.;
Lebel, C.; Scaife, M. C.; Harris, J. P. Controlled release auris sensory cell modulator compositions
and methods for the treatment of otic disorders. US20150313839A1, 2015.
17. O'Malley, B. W.; Li, D. Chitosan-based matrixes for controlled-release delivery systems
for inner ear drug application. US20130189241A1, 2013.
18. Dumortier, G.; Grossiord, J. L.; Agnely, F.; Chaumeil, J. C., A review of poloxamer 407
pharmaceutical and pharmacological characteristics. Pharm. Res. 2006, 23, 2709-2728.
19. Wang, X.; Dellamary, L.; Fernandez, R.; Harrop, A.; Keithley, E. M.; Harris, J. P.; Ye, Q.;
Lichter, J.; LeBel, C.; Piu, F., Dose-Dependent Sustained Release of Dexamethasone in Inner Ear
Cochlear Fluids Using a Novel Local Delivery Approach. Audiol. Neurootol. 2009, 14.
20. Lichter, J.; Vollrath, B.; Trammel, A. M.; Duron, S. G.; Scaife, M. C.; Piu, F.; Harris, J. P.
Sustained release corticosteroid compositions for treatment of otic disorders. US20150313838A1,
2015.
21. Piu, F.; Ye, Q.; Dellamary, L.; Lebel, C. Prevention of and recovery from drug-induced
ototoxicity using corticosteroids and JNK inhibitors. US20130045957A1, 2013.
22. Ye, Q.; Dellamary, L. A.; Piu, F. Modulation of gel temperature of poloxamer-containing
sustained release formulations. US20120277199A1, 2012.
23. OTO-104. http://www.otonomy.com/pipeline/oto-104/ (accessed 25 April).
24. OTO-311. http://www.otonomy.com/pipeline/oto-311/ (accessed 25 April).
25. Lichter, J.; Vollrath, B.; Duron, S. G.; Lebel, C.; Piu, F.; Ye, Q.; Dellamary, L. A.; Trammel,
A. M.; Scaife, M. C.; Harris, J. P. Controlled release antimicrobial compositions and methods for
the treatment of otic disorders. US20150150793A1, 2015.
26. Piu, F.; Ye, Q.; Dellamary, L. A.; Lebel, C. Methods for the treatment of pediatric otic
disorders. US20150290124, 2015.
27. Clinical Trials. http://www.otonomy.com/pipeline/clinical-trials/ (accessed 25 April).
28. Meyer, T. Pharmaceutical compositions for the treatment of inner ear disorders.
US20150265532A1, 2015.
121

29. Meyer, T. Methods and composition for treating and preventing tinnitus.
US20150306178A1, 2015.
30. Ayoob, A. M.; Borenstein, J. T., The role of intracochlear drug delivery devices in the
management of inner ear disease. Expert Opin. Drug Deliv. 2015, 12, 465-479.
31. Shapiro, B.; Kulkarni, S.; Nacev, A.; Sarwar, A.; Preciado, D.; Depireux, D. A., Shaping
magnetic fields to direct therapy to ears and eyes. Annu. Rev. Biomed. Eng. 2014, 16, 455-481.
32. Shapiro, B.; Depireux, D.; Sarwar, A.; Nacev, A.; Preciado, D.; Hausfeld, J., Pre-Clinical
Development of Magnetic Delivery of Therapy to Middle and Inner Ears. ENT Audiol. News 2014,
23, 54-56.
33. Lalwani, A. K.; Kysar, J. W. System and method to locally deliver therapeutic agent to
inner ear. US20150265824A1, 2015.
34. Keegan, M. E.; McKenna, M. J. Drug-eluting staples prosthesis. US20150320551A1, 2015.
35. Sarwar, A.; Lee, R.; Depireux, D. A.; Shapiro, B., Magnetic Injection of Nanoparticles Into
Rat Inner Ears at a Human Head Working Distance. IEEE Trans. Magn. 2013, 49, 440-452.
36. Fiering, J. O.; Mescher, M. J.; Pararas, E. E.; Borenstein, J. T.; Sewell, W. F.; Kujawa, S.
G.; McKenna, M. J.; Ernest S. Kim Drug delivery apparatus US20150157837A1, 2015.
37. Humandiagram.info Blank Ear Diagram. http://humandiagram.info/how-important-ear-
diagram-labeled-is/blank-ear-diagram/#main (accessed April 26, 2016).
38. Liktor, B.; Szekanecz, Z.; Batta, T. J.; Sziklai, I.; Karosi, T., Perspectives of
pharmacological treatment in otosclerosis. Eur. Arch. Otorhinolaryngol. 2013, 270, 793-804.
39. Wegner, I.; Verhagen, J. J.; Stegeman, I.; Vincent, R.; Grolman, W., A systematic review
of the effect of piston diameter in stapes surgery for otosclerosis on hearing results. Laryngoscope
2016, 126, 182-190.
40. Klickstein, L. CGF166 Atonal Gene Therapy for Hearing Loss & Vestibular Dysfunction:
Review of NIH OBA protocol #1310-1260.
http://osp.od.nih.gov/sites/default/files/1_1260_CGF166_Klickstein.pdf (accessed April 25).
41. Kopke, R. D.; Wassel, R. A.; Mondalek, F.; Grady, B.; Chen, K.; Liu, J.; Gibson, D.;
Dormer, K. J., Magnetic nanoparticles: inner ear targeted molecule delivery and middle ear implant.
Audiol. Neurootol. 2006, 11, 123-133.
42. Dormer, K. J.; Awasthi, V.; Galbraith, W.; Kopke, R. D.; Chen, K.; Wassel, R.,
Magnetically-targeted, technetium 99m-labeled nanoparticles to the inner ear. J. Biomed.
Nanotechnol. 2008, 4, 174-184.
122

43. Sensenig, R.; Sapir, Y.; MacDonald, C.; Cohen, S.; Polyak, B., Magnetic nanoparticle-
based approaches to locally target therapy and enhance tissue regeneration in vivo. Nanomedicine
2012, 7, 1425-1442.
44. Shapiro, B.; Depireux, D. A.; Dormer, K.; Rutel, I. B. Device and methods for directing
agents to the middle ear and the inner ear including magnetic field. US20140073835A1, 2014.

123

CHAPTER 6
Local Cochlear Distribution of N-BP drugs in Animal and
Cadaveric Human Models Visualized using 6-FAM-ZOL for the
Treatment of Otosclerosis
6.1 Otosclerosis and the use of N-BP drugs in treating the disorder
The otic capsule is unique in that it normally exhibits little or no bone remodeling.
Otosclerosis is a bone disorder involving an abnormal remodeling of the otic capsule, resulting in
hearing impairment. Hearing impairment caused by otosclerosis is divided into two categories: 1)
conductive hearing loss, a result of the fixation of the stapes footplate, and 2) sensorineural hearing
loss (SSNHL), caused by otosclerotic lesions involving the cochlear endosteum and spiral
ligament.
1, 2, 3
SSNHL observed in cochlear otosclerosis has been hypothesized to originate from the
release of toxic metabolites into the inner ear and vascular shunts between the otosclerotic focus and
spiral capillaries,
4
although this remains unproven. The conductive hearing loss caused by otosclerosis
may be addressed by several approaches, including stapedectomy and installation of amplifying devices,
such as hearing aids. On the other hand, patients with cochlear otosclerosis may progress to a profound
loss and thus require cochlear implantation, which can present unique challenges.
5, 6, 7, 8

Notably, osteoporosis was observed to be more prevalent in patients diagnosed with
otosclerosis,
9
suggesting that they may be facilitated by a shared polymorphism in the COL1A1 gene.
10

Moreover, post-mortem histopathologic examination of temporal bone specimens from adult patients
with type I osteogenesis imperfecta revealed otosclerotic lesions.
11
In contrast, there is no association
found between otosclerosis and Paget’s disease, with the largest published study finding no evidence
124

for ossicular fixation in a histopathologic analysis of temporal bones derived from patients with Paget’s
disease.
12
In conclusion, these data suggest that the pathology of otosclerosis and other diseases of bone
metabolism may share some common features.
These studies imply that treatments for other metabolic bone disorders may, therefore, be
effective in addressing otosclerosis. In this regard, nitrogen-containing bisphosphonates (N-BPs),
such as risedronate (RIS) and zoledronate (ZOL)—potent inhibitors of bone remodeling and
common treatment for bone metabolic diseases such as osteoporosis, osteogenesis imperfecta, and
Paget’s disease
13
—are potentially useful to treat otosclerosis. In fact, a limited clinical trial with 10
patients has shown that systemic administration of ZOL is effective in halting the progression of
SSNHL in patients diagnosed with cochlear otosclerosis.
1

While no serious complication was observed in the small human trial, the systemic
administration of N-BPs has been sometimes associated with serious toxicities, including osteonecrosis
of the jaw (ONJ), atypical femur fracture, atrial fibrillation, erosive esophagitis, and renal failure,
14
as
discussed in Chapter 1, although the pathology of ONJ specifically associated to BPs is still under
debate.
15
Despite the low occurrence rate of these complications,
16
once a lowered risk of fracture has
been achieved through treatment with BPs for osteoporosis, patients are recommended to have “drug
holidays” prior to further treatments.
17
Furthermore, the presence of N-BPs within bone long after their
initial systemic administration potentially prolongs exposure of treated patients to risks of adverse side
effects associated with their use.
18
In addition, as discussed in Chapter 5, the existence of the blood
perilymph barrier presents challenges for systemic drug delivery to the cochlea. To achieve the desired
effective dose, a significantly high systemic dose is required, and thus the side effects related to exposure
to N-BPs are potential concerns.
Local delivery of BPs is a potential solution to the requirement of high local concentration at
125

the target site while avoiding potential systemic side effects. A series of experiments were conducted
using guinea pigs, a commonly used animal model employed for hearing studies, to investigate the
delivery of BPs to the cochlea. One of the key components is the use of fluorescently labeled 6-FAM-
ZOL to visualize the cochlear drug distribution via both systemic (intraperitoneal injections) and local
administrations, including passive diffusion of 6-FAM-ZOL via the round window membrane and
intracochlear delivery using a cochleostomy. The relative efficacy of systemic versus local cochlear
delivery of bisphosphonate was also compared. Furthermore, ototoxic evaluation of various doses of
ZOL locally delivered to the cochlea using a cochleostomy was also evaluated to determine the
maximum safe dose of ZOL. 6-FAM-ZOL (25% mol) was utilized as a trace to determine the cochlear
drug distribution. These findings provide important pre-clinical data in preparation for local
bisphosphonate delivery to the inner ear in humans, and may permit the rapid evaluation of other
cochlear drug delivery systems.
Although the in vivo guinea pig experiments provided crucial functional information
regarding our ability to deliver 6-FAM-ZOL locally to the cochlea, a better understanding of the
patterns of drug distribution following delivery to the human cochlea is of great interest. To this
end, 6-FAM-ZOL is an invaluable probe allowing visualization of the drug delivery to various
cochlear compartments, in addition to the quantification of the amount of drug accumulated in
each area. Studies of drug delivery to the human inner ear have been previously conducted in
cadaveric human temporal bones;
19, 20, 21, 22, 23
however, the cochlear distribution of drug from the
base to apex after administration has yet to be elucidated. In patients diagnosed with cochlear
otosclerosis, N-BPs might be locally administered either via an intratympanic injection or through
a drug-eluting stapes prosthesis, as previously described in Chapter 5. Notably, such delivery
methods might be used to improve the efficacy of inner ear delivery of other drugs, including
126

steroids for sudden hearing loss or, in the future, regeneration factors for spiral ganglion neurons
and hair cells. To better understand the extent of diffusion of substances placed within the oval
window and the scala vestibuli, the distribution of 6-FAM-ZOL in fresh cadaveric human temporal
bones delivered through the oval window was also studied.
6.2 Synthesis of 6-FAM-ZOL

Scheme 6.1. Synthesis of 5-FAM-ZOL (6.5) and 6-FAM-ZOL (6.6)
Unlike 5,6-FAM-RIS, 5,6-FAM-labeled ZOL was synthesized using another approach.
The reaction of ZOL with the NBoc-protected epoxide that was previously described in Chapter 3,
under aqueous conditions at pH 6.0, 40
o
C, was slow.
24
While the reaction rate is much faster at
pH 7.0-7.5, 40
o
C, the lack of chemoselectivity between N-alkylation and O-alkylation pointed to
a need for a more reactive epoxide linker precursor. Thus the synthesis of ZOL linker was explored
a q . N H
3
O H
N H
O
C O
2
H
H O O
O
p H 8 . 3
D r y D M F, H
2
O
H
2
O , p H 7 . 0 - 7 . 5
O H
N
H
O
C O
2
H
H O O
O
O
C l
O
C O
2
H
H O O
O
O
N
O
O
N
P
P
O H
O
O
H O
O H
O H
O H
N
O H
N H
2
N
P
P
O H
O
O
H O
O H
O H
O H
N
O H
C l
N
P
P
O H
O
O
H O
O H
O H
O H
N
N
P
P
O H
O
O
O H
O H
O H
O H
N
N
P
P
O H
O
O
H O O H
O H
O H
N
6 . 1
6 . 2
6 . 3 6 . 4
6 . 5 6 . 6
127

with epichlorohydrin. The reaction was performed at pH 7.5, room temperature, followed by amine
exchange in ammonia, to yield the desired ZOL-linker (85%).
24
ZOL-linker was purified from the
starting materials and its byproduct using strong anion exchange (SAX) reverse phase HPLC. The
purified linker was then reacted with NHS-5,6-FAM, resulting in the formation of 5,6-FAM-ZOL.
Isolated isomers, 5-FAM-ZOL and 6-FAM-ZOL, were obtained using C18 reverse phase HPLC.
6.3 Local delivery of 6-FAM-ZOL to the cochlea

Figure 6.1. A schematic diagram of the guinea pig cochlea viewed at a mid-modiolar section.
25

Fluorescence signal of the shaded areas of the modiolus and lateral cochlear wall at each half-turn
were measured.
Dose effects of 6-FAM-ZOL on its bone deposition via systemic administration were
evaluated for various amounts, including 1X or 3X of 6-FAM-ZOL relative to the human ZOL
dose. Quantification of fluorescence signals was performed along each cochlear half-turn at the
mid-modiolar section as illustrated in Figure 6.1. A statistically significant fluorescent signal
above the background signal was observed in the lateral wall of cochleae of both 1X and 3X 6-
128

FAM-ZOL -treated animals (Figures 6.2 A-C). The fluorescent signal increased in a dose-
dependent manner (Figure 6.2 G).

Figure 6.2. Systemic delivery of 6-FAM-ZOL and quantification of fluorescent signal.
25

Fluorescent photomicrographs taken at mid-modiolar cochlear and tibial sections are shown for
untreated cochlea (A and D, respectively), treatment with 1X of 6-FAM-ZOL (B and E,
respectively), and 3X of 6-FAM-ZOL relative to the human ZOL dose (C and F, respectively).
Quantifications of fluorescence after systemic 6-FAM-ZOL treatment are shown for cochleae (G)
(n = 7) and tibeae (H) (n = 3). Measurements of the cochlear fluorescent signal were taken at each
of six cochlear half-turns from base to apex, and then averaged. Using analysis of variance for
measurements of the lateral wall, the p values between control and 1X, control and 3X, and 1X
and 3X were 0.016, 0.0001, and 0.0352 respectively.
As a comparison for the cortical bone, the fluorescent green signals within the tibiae of
animals treated with various doses of 6-FAM-ZOL were also measured. Similar trends in dose-
dependency and statistical significance were observed (Figure 6.2 D-F). ABR and DPOAE
129

measurements were performed at the time of injection and immediately prior to sacrifice, with no
significant changes observed.
25
These findings demonstrated that systemic treatment with 6-FAM-
ZOL caused no ototoxicity. The fluorescent signal within otic capsule bone and appendicular
cortical bone increased in a dose-dependent manner, with more deposition detected in cortical bone
than in otic capsule bone.
Next, the distribution of 10% of the 1X systemic dose (0.1X) and 30% of the 1X systemic
dose (0.3X) of 6-FAM-ZOL locally delivered via diffusion through the RWM, was evaluated. At
a dose of 0.3X 6-FAM-ZOL, the fluorescent probe was distributed throughout the lateral wall of
the cochlea at levels comparable to the 1X systemic dose. The fluorescent signal at 0.1X of 6-
FAM-ZOL was lower but still detectable (Figure 6.3 A-C).

Figure 6.3. Local delivery of 6-FAM-ZOL via diffusion through the RWM and quantification of
fluorescent signal.
25
Fluorescent photomicrographs taken at mid-modiolar sections of the cochlea
are shown for untreated cochlea (A), 0.1X of systemic dose (B), and 0.3X of systemic dose (C).
130

The increased signal outside the RW niche in B was seen in some trials, but was not consistently
reproducible, indicating minor spillover from the niche. The fluorescence signal at each of six
cochlear half-turns from base to apex were quantified for both the modiolus (E) and lateral wall
(F). Quantified signals were plotted against the cochlear half-turns and the best fit regression was
determined. The p values for total average fluorescence between control and 1X, control and 3X,
and 1X and 3X for the modiolus were 0.0040, 0.0001, and 0.0194, respectively, whereas for the
lateral wall, the p values for total average fluorescence between control and 0.1X, control and 0.3X,
and 0.1X and 0.3X were 0.018, 0.0001, and 0.0038, respectively. Slopes of the 0.1X and 0.3X
regression lines were significantly different from the slope of the control along the modiolus (p <
0.0001), while the slopes for the lateral wall were not significantly different (p = 0.09).
While the fluorescent signals measured at the treated modiolus and the lateral wall were
significantly higher than the control (Figure 6.3 D and E), a steep gradient of fluorescent signal
from base to apex was observed along the osseous spiral lamina of the modiolus with difference
between regression line slopes p < 0.0001. However, within the lateral wall of the same cochleae,
no such gradient was observed (difference between regression line slopes p = 0.09). It is important
to note that no ototoxicity was observed in local delivery of 6-FAM-ZOL via diffusion through
RWM, proven by the lack of threshold shifts in ABR or DPOAE measured three weeks after
administration.
25
Furthermore, these experiments showed that the fluorescent signal of a dose of
0.3X of 6-FAM-ZOL delivered via this method was equivalent to that observed in 1X of systemic
dose detected on the lateral cochlear wall.
Finally, distribution of 6-FAM-ZOL infused directly into the cochlea via a cochleostomy,
using a microfluidic pump, was assessed. The fluorescent signal along both the osseous spiral
lamina of the modiolus and the lateral wall of the cochlea was detected after administration of
131

0.02X of systemic dose (Figure 6.4 A and B). Statistically significant steep gradients from base to
apex were observed at both the modiolus and the lateral wall compared to the controls (Figure 6.4
C and D) with a difference between regression line slopes (p < 0.0001). Data showed that a dose
at 0.02X locally delivered via intracochlear infusion was equivalent to 1X systemic dose
administered via intraperitoneal injection. In addition, stability in DPOAEs and CAPs
measurements (within 20 dB) during the experiments indicated that no immediate ototoxicity
occurred.
25

Figure 6.4. Intracochlear infusion of 6-FAM-ZOL.
25
Fluorescent photomicrographs taken at mid-
modiolar sections of the cochlea are shown for untreated cochlea (A) and 0.02X of systemic dose
(B). Fluorescent signal was quantified at each of six cochlear half-turns from base to apex, along
both the modiolus (C) and lateral wall (D). Data points from all experiments are shown, along with
the best-fit regression plot. Total average fluorescence at each turn was plotted and the best-fit
regression was determined. Data showed that fluorescent signal in 0.02X for both the modiolus
132

and the lateral wall (p = 0.0031 and 0.0124, respectively) was significantly different from the
control for both the modiolus and the lateral wall (p = 0.0003 and 0.0007, respectively).
These experiments show that BPs can be distributed in vivo to the cochlea using the three
different delivery methods of 6-FAM-ZOL described above. Most importantly, none of these
methods cause ototoxicity, as confirmed by consistency in hearing measurements. Systemic
delivery of fluorescently labeled BPs in animal models was previously reported with detailed
description on their skeletal delivery patterns, along with the length of their retention within
appendicular and mandibular bones.
26, 27
However, none of these studies has been carried out in
the cochlea. In this regard, this work is the first to evaluate the systemic delivery of BPs to the otic
capsule. This study further shows that the uptake of BPs by the otic capsule was lower than that
by the tibia (approximately 5,800 fluorescence units above control vs. 8,800 fluorescence units
above control for the 1X systemic dose; Figure 6.2). This possibly resulted from little-to-no bone
remodeling in the normal otic capsule relative to other skeletal bones, such as the tibia and other
appendicular bones. However, the assessment of the uptake of BP by an otic capsule bone actively
undergoing abnormal remodeling, as observed in otosclerosis, will require additional experiments.
Regarding this aspect, 6-FAM-ZOL could be a useful probe to identify areas of active bone
turnover in animal models for otosclerosis.
Among the three delivery methods, intracochlear infusion is the most efficient in delivering
the probe compared to diffusion via the RWM (0.02X vs. 0.3X) and systemic delivery (0.02X and
1X), as verified by the amount of fluorescent signal observed in the cochlear lateral wall. This
study also provides invaluable information in determining the effective dose required for local
delivery to the cochlea that would be equivalent to the effective systemic dose. The observed
difference in the uptake of BP by the osseous spiral lamina and the cochlear lateral wall in the
133

cochleostomy experiments, which may be of interest, would require additional studies to further
elucidate the physiologic difference.
Remarkably, the ability of 6-FAM-ZOL to diffuse through the RWM membrane and be
distributed throughout the cochlea is another surprising finding. While a notably steeper gradient
from cochlear base to apex was observed along the osseous spiral lamina, but not along the
cochlear lateral wall, following diffusion of 6-FAM-ZOL via the RWM, such a steep gradient was
observed in both the spiral lamina and lateral wall using intracochlear infusion via a cochleostomy.
A possible explanation for this difference in cochlear concentrations is that the presence of a
mucosal layer overlaying the cochlea in the RWM method may interfere with the diffusion of 6-
FAM-ZOL, resulting in a less steep gradient in the cochlear lateral wall. Nonetheless, this is
irrelevant in human models, given the much greater thickness of the otic capsule in the human ear.
Previously reported studies on the distribution of drugs, such as gentamicin and
dexamethasone, in the cochlea following RWM administration confirmed the observed
intracochlear concentration gradients in these experiments.
28, 29, 30
However, instead of determining
intracochlear concentrations at each time point by directly analyzing perilymph samples, this study
quantified the cumulative cochlear drug concentrations via the fluorescent signal detected from 6-
FAM-ZOL bound to the cochlea. A steeper gradient observed in analysis using the perilymph, as
well as a different shape of the concentration against cochlear turns, may be due to influence of
different modes of drug delivery.
The system model developed in this study potentially offers a direct and generalizable
evaluation method on cochlear drug delivery of other systems. The unique properties of BPs,
particularly their avid, strong bone binding affinity, permits direct visualization and quantification
of bound BP over the course of the experiment via undemineralized processing of otic capsule
134

bone. Besides intracochlear delivery, this system model may be applicable to other delivery
systems via round window systems, such as poloxamer,
31, 32
as well as other aqueous solution-
based systems.
33, 34
This system model is also useful for drug delivery to the middle ear, including
emerging transtympanic approaches.
35

An estimation of up to 0.3% of Caucasians experience conductive hearing loss as a result
of otosclerosis.
36
SNHL, associated with extensive lesions of cochlear otosclerosis, occurs in up
to 20-30% of these patients.
37
From a clinical perspective, findings from the study may contribute
to the development of safe and effective medical treatment for cochlear otosclerosis, especially the
development of therapies exploring new avenues for BP delivery. Particularly, utilization of a
drug-eluting stapes prosthesis to deliver BPs and other drugs including steroids to the inner ear is
a potential technique that is currently investigated by the MEEI collaborators.
6.4 Ototoxic evaluation of the local delivery of ZOL visualized using 6-FAM-ZOL

Figure 6.5. Fluorescent photomicrographs of the cochlea taken at mid-modiolar sections at various
time points after direct intracochlear infusion of artificial perilymph, 0.04X solution and 0.08X
4 hours
after
infusion
4 weeks
after
infusion
135

solution.
38
0.04X and 0.08X indicate 4% and 8%, respectively, of the human systemic ZOL dose
mixed with a tracing amount of 6-FAM-ZOL at corresponding molar concentrations.
As previously described, 0.02X systemic dose delivered via direct intracochlear delivery
induced no ototoxicity, verified by DPOAE and CAP measurements.
25
Here, the ototoxicity of 6-FAM-
ZOL was further assessed. Various doses of ZOL, such as 0.08X and 0.04X systemic dose, were
delivered to the cochlea via direct intracochlear infusion. A tracing amount of 6-FAM-ZOL (25% mol)
was included in ZOL samples as a fluorescent marker tracking cochlear distribution of ZOL. Consistent
with previous experiments described in section 6.2, the fluorescent signal was detected from the base to
apex along both the modiolus and lateral cochlear wall in all ZOL treated specimens, with the strongest
signal observed at the scala tympani of the first basal turn, adjacent to the delivery site (Figures 6.5 B,
C, E, and F). In contrast, only weak background auto-fluorescent signals were measured in the control
specimens (Figures 6.5 A and D). While surgical middle ear inflammation significantly waned after
four weeks, and a small amount of granulation tissue and mucosal synechiae were found around the
cochleostomy and the bullectomy sites in a few animals, changes in CAP and DPOAE measurements
were not associated with these anatomical changes.
In both acute and chronic (evaluated after four weeks) experiments, CAP and DPOAEs
measurements stayed within 10 dB and 20 dB of baseline, respectively, in control animals and those
treated with 0.04X ZOL (Figure 6.6 A, B, D and E). These results suggest that direct intracochlear
delivery of ZOL up to 4% of the human systemic dose does not induce ototoxicity.
136

Figure 6.6. DOAPEs and CAP measurements for the control, 0.04X, and 0.08X systemic doses at
various frequencies.
38

On the other hand, while DPOAEs remained within 20 dB of baseline in animals treated with
0.08X in both acute experiments and at four weeks (Figure 6.6 C), CAP measurement shifts between
12 and 16 kHz, in the 20-30 dB range in acute experiments (Figure 6.6 F). This cochlear region is where
the tubing was inserted for intracochlear delivery, thus resulting in the highest concentration of ZOL.
In acute experiments, the CAP shift was detected within 30 minutes following ZOL administration and
only partially resolved after 4 hours. The shift in CAP measurements recurred after 4 weeks within the
12-16 kHz region when evaluated for chronic experiments (Figure 6.6 F). Further analysis on several
representative frequencies with CAP shift over various time points demonstrates that the rapid CAP
shift occurred following initial drug delivery and remained for the 12-16 kHz region, the site where drug
delivery happened and where, consequently, the highest amount of ZOL resided (Figure 6.7 A-D).
137

Figure 6.7. CAP measurements after infusion of 0.08X systemic dose at the frequencies of 24 (A),
16 (B), 12 (C), and 8kHz (D) with the average values generated from independent experiments of
either acute (1 day) or chronic experiments (4 weeks).
38
The vertical and the horizontal standard
error bars represent CAP threshold shift and the time point when the CAP was measured,
respectively. The rectangular gray-shaded areas indicate the time period over which drug was
infused. 0.08X systemic dose contains a tracing amount of 6-FAM-ZOL (25% mol) at
corresponding molar concentrations.
The goal of this study is to determine the maximum local dose of ZOL without inducing
ototoxicity. Results from these experiments show that while 0.04X systemic dose locally delivered
caused no ototoxicity, 0.08X systemic dose induced shifts in CAP detected within 30 minutes following
administration, with the highest shifts occurring at the site of delivery (12-16 kHz). Despite a brief
recovery after 4 hours, this CAP shift reappeared after 4 weeks. In contrast, no change was observed in
DPOAE measurements induced by ZOL treatment.
Ototoxic sensitivity is species-dependent,
39
and guinea pigs are reported to be one of the more
sensitive rodent species.
40, 41
Despite the potential significant sensitivity difference between human and
138

animal models, many studies have employed guinea pigs as the model to predict drug ototoxicity in
humans. For instance, similar ototoxicity of cisplatin observed in guinea pigs and humans has been
reported.
42
Therefore, the use of the guinea pig model to relatively assess drug ototoxicity is reasonable.
The two methods used to evaluate hearing, CAP and DPOAE, measure different components
involved in hearing signal transduction. While CAPs evaluate the auditory nerve, DPOAEs assess the
outer hair cells, suggesting that ototoxicity observed at 0.08X ZOL systemic dose occurs at the inner
hair cells. It is remarkable that, despite reversible CAP shift at the site of drug delivery detected 4 hours
after initial drug delivery, ototoxicity recurs after four weeks, an observation that was not evident until
confirmed by CAP measurements. Free calcium ions (Ca
2+
) are critical in neurotransmitter release at
the hair cell synapse, as well as in other biological activities of the cochlea.
43, 44
The possibility that
ZOL, a strong calcium chelator,
45
may bind to free calcium (Ca
2+
) existing in the extracellular
environment around synapses, potentially leads to a decrease of free calcium ions in the cochlea and,
consequently, results in an attenuation in synaptic transmission and signaling between the inner hair cell
and auditory neurons. However, whether the acute and chronic CAP shifts are mechanistically related
is ambiguous.
As reported in previous clinical trials, due to concerns of renal toxicity, the dose of ZOL
prescribed for patients diagnosed with multiple myeloma and bone metastases was decreased from 8
mg to 4 mg.
46
As described in section 6.2, direct intracochlear infusion is superior to both local delivery,
via diffusion through the RWM (0.02X vs. 0.3X), and systemic delivery (0.02X vs. 1X), with systemic
dose of 4 mg of ZOL.
25
Through these ototoxic experiments, local delivery via intracochlear infusion
of ZOL can safely be achieved up to 0.04X systemic dose. This local delivery approach provides a
means to increase local concentration while bypassing systemic adverse effects associated with
administration of high doses of ZOL to achieve a high local concentration.
139

As demonstrated in section 6.2, 6-FAM-ZOL can be used as a non-ototoxic marker to evaluate
cochlear drug delivery. This study further demonstrates the advantage of using 6-FAM-ZOL over other
available cochlear sampling approaches, such as direct perilymph sampling from the apex,
47, 48
which
offers assessments of the amount of drugs delivered in real time. 6-FAM-ZOL, used as an internal
standard, allows not only direct, easy visualization of drug delivery, but also straightforward assessment
of the extent of cochlear drug exposure. Indeed, 6-FAM-ZOL could possibly be a useful tool to evaluate
the cumulative amount of drug delivered to the various regions of the cochlea.
6.5 Intracochlear delivery of 6-FAM-ZOL through the oval window in fresh cadaveric
human temporal bones
In this experiment, fresh, cadaveric human temporal bone specimens were used to evaluate
the distribution of 6-FAM-ZOL administered through the oval window. After application of 6-FAM-
ZOL through the oval window, strong fluorescent signals were detected at both the bony walls of the
vestibular capsule (Figure 2B) and cochlea (Figure 2D). Low background signals were observed in the
control samples, which were treated solely with artificial perilymph in the cochlea (Figure 2A and 2C).
As noted in the previous observations, in guinea pig models, a gradient of signal intensity appeared from
base to apex. Similar to previous experiments, the high bone affinity of 6-FAM-ZOL allows
quantification of the cumulative amount of compound diffused. The fluorescent signal associated
with 6-FAM-ZOL was determined for each of various areas within the lateral and modiolar walls of the
scala vestibuli and scala tympani, as indicated in Figure 6.8, in all specimens.
140

Figure 6.8. Human temporal bones after administration of 6-FAM-ZOL.
49
Fluorescent
photomicrographs of the cochlea taken at mid-modiolar sections for the vestibule of a cochlea
treated with artificial perilymph (A) and 6-FAM-ZOL (B), and for a cochlea treated with either
artificial perilymph (C) or 6-FAM-ZOL (D). The images are representative of three independent
experiments. The asterisk, the arrow, and the arrowhead indicate the stapes footplate, the lateral
wall of the vestibule, and the lateral wall of the basal cochlear turn, respectively.
A significant gradient signal from base to apex was detected at the bony wall of the scala
vestibule (SV) and at the bony wall of the scala tympani (ST) in specimens treated with 6-FAM-
ZOL. In contrast, the control specimens showed minimal fluorescence signal (Figure 6.9). A higher
signal was observed at the wall of the SV, where the drug was administered, than that of the ST.
Apical spread was not quite as strong in the ST (Figure 6.9).

Figure 6.9. Quantification of the fluorescent signal after intracochlear administration.
49

Measurements were taken at each of five cochlear half-turns from base to apex in the human
temporal bone specimens, along both the lateral cochlear wall (A and B) and the modiolus (C and
141

D). The average values were generated from three independent experiments, and standard error
bars are shown. The fluorescent signals along both the lateral cochlear wall and the modiolus
contacting the SV and ST are statistically significant when compared to the control. The p values
for specimens treated with 6-FAM-ZOL are 0.04 in the SV and 0.03 in the ST for the lateral
cochlear wall, whereas for the modiolus, the p values for the effect of 6-FAM-ZOL are 0.001 in
the SV and 0.002 in the ST.
In conclusion, the fluorescent dye-BP conjugate did, indeed, label the lateral wall and
modiolus of both the scala tympani and scala vestibuli to the apex after administration to the oval
window. The observed steep baso-apical gradient in both scalae suggests that direct diffusion from
the higher concentration within the scala vestibuli into the scala tympani occurred, facilitated by
inter-scalar communication between the two scalae. In fact, this notion is consistent with work
previously demonstrating radial diffusion between the scalae.
50, 51, 52

While previous guinea pig experiments indicated that 6-FAM-ZOL appeared to label the
modiolus more than the lateral wall when the probe was applied directly into the scala tympani,
the fluorescent signals detected in the lateral wall and osseous spiral lamina in this study appear to
be similar.
25
Together, the data suggest that bisphosphonates could be used to facilitate drug
delivery throughout the cochlea, including to the spiral ganglion neurons within the modiolus,
through communication routes between perilymph spaces and the osseous spiral lamina.
53, 54

This study using fresh cadaveric human temporal bones offers a better understanding of
drug diffusion through the native human inner ear anatomy. However, the model may not be
suitable to postulate the complex dynamics within the sealed living human cochlea and therefore
may confound efforts at description in a cadaveric system. In the guinea pig model, a diluting
effect on drugs delivered through the round window has been reported, which may stem from two
142

possible processes. In the first process, cerebrospinal fluid (CSF) enters into the scala tympani
through the cochlear aqueduct. In the second process, drugs leak into the CSF space,
55
with CSF
entering at a very slow rate in a sealed, normal cochlea.
55, 56
This phenomenon likely contributes
to the slow, apically-directed flow within the normal guinea pig cochlea, measured to be
approximately 1.6 nL/min.
56
However, it is unknown whether the human cochlear aqueduct would
have a similar effect as noted in the guinea pig, or what the rate of intracochlear perilymph flow is
in the human cochlea. Nonetheless, relative to what would be expected for simple diffusion, drug
level at the apex would greatly benefit from any apically-directed flow of perilymph following
administration either at the round or oval window.
6.6 In vitro sustained delivery of N-BP drugs using Poloxamer 407
As discussed above, local delivery of ZOL is favored over systemic delivery due to concern
over systemic side effects. One actively investigated approach for the local delivery of ZOL to
treat cochlear otosclerosis is the utilization of a prosthetic stapes containing a hollow body, into
which the drug could be embedded. The design of this prosthetic stapes has been described in
detail in Chapter 5. The goal of this experiment is to develop a sustained release formulation of
ZOL to be included in the hollow body of the medical device. Utilization of Poloxamer 407 in
sustaining local drug delivery in the ear was discussed in detail in Chapter 5. Since the thermo-
reversible polymer is commonly used in local delivery of drugs to the cochlea and many of its
formulations are currently in clinical trials, P407 was the primary choice for this project.

143

Figure 6.10. (A) Standard curve of the absorbance of ZOL in 10 mM PBS buffer (pH 7.4) and (B)
UV-Vis Spectra of Bisphosphonate in solution with P407.
Since the molar extinction coefficient of ZOL was not available in the literature, a
calibration curve of the absorption of ZOL was first constructed and the molar extinction
coefficient was established at λmax = 206 nm (ε206 = 4186.2 M
-1
cm
-1
). Preliminary data on the
release profile of 2% w/w of ZOL and RIS in the presence of 17% w/w P407 indicated that P407
did, indeed, have an effect in prolonging the release of ZOL and RIS. The release profiles of both
compounds were similar and completed within 2 hours. The release of RIS from P407 was further
validated with
31
P NMR spectroscopy with 85% phosphoric acid as the internal standard. Since
the relaxation time of phosphorus nuclei is pH-dependent, the samples were adjusted to basic pH
(~10) to ensure consistency in
31
P NMR spectra.
Preliminary data also showed that the release of N-BPs was dependent on how many
aliquots were being collected, suggesting that the rate of clearance or the amount of sample being
removed would be a significant factor in deciding the equilibrium and that the duration of the
release and the concentration of RIS also played an important role in its release. Thus the procedure
was subsequently modified to induce equilibrium of the release of RIS from P407. Instead of
144

collecting the minimum amount of sample for quantitative analysis (2% of the total buffer volume),
a sample volume of 20% of the total buffer in the reservoir was collected and low concentration
of RIS was utilized. Instead of 37 mM (1% w/w), the concentration of RIS for the release studies
would be at 5 mM (~0.02X systemic dose of ZOL administered in patients to treat postmenopausal
osteoporosis).

Figure 6.11. Release profiles of RIS (5 mM) and ZOL (5 mM) at the presence of 17% w/w P407
(A) before and (B) after normalization.
Again, data showed that the release of both compounds was completed after 2 hours. At
concentration of 5 mM, the release of RIS reached 100% while that of ZOL was above 100%
(Figure 6.11). This could be explained by the fact that λ max of ZOL (λmax = 206 nm) was relatively
close to the light scattering peak of P407 (λmax = 202 nm) (Figure 6.10). The release profiles of
both N-BP drugs were similar once the data was normalized. To avoid the effect of P407 on UV-
Vis analysis of the release of BP drugs, RIS was chosen as a replacement for ZOL in the sustained
release studies.
Previous literature reported that the release profile of drug in the ear should ideally range
from 6 to 8 hours.
57, 58, 59
This suggested that interaction of RIS with P407 needed to be enhanced,
possibly via hydrophobic interaction, in order to prolong the release of RIS from P407.
145

Tributylammonium salt (TBA) of RIS (TBA-RIS) was formed and the release profile of the
compound from 17% w/w P407 was constructed. Experimental results indicated that RIS in
tributylammonium salt form had the same release profile as that of the sodium salt form.
Furthermore, various salt forms of RIS with different equivalents of TBA did not change the drug
release profile from P407. The release profiles of RIS from 17% w/w P407 in the presence of
magnesium stearate, a common additive in drug formulation, or benzathine, an FDA approved
additive that was previously used to sustain the release of penicillin, were also established. Similar
to TBA, these additives had no effect in prolonging the release of RIS from P407 (Fig. 6.12).

Figure 6.12. In vitro release profile of various salt forms of RIS with 17% w/w P407.
To validate our experimental setup, the release profile of methylprednisolone
hemisuccinate (MPS) was established and compared with literature data. Due to the commercial
unavailability of 6.5 mm diameter Snapwells, all experiments described above used 12 mm
diameter Snapwells. However, once the 6.5 mm diameter Snapwells were commerically available,
experiments using similar setup as previously described performed with 6.5 mm membrane
diameter and methylprednisolone hemisuccinate (MPS) (2% w/w) with 17% w/w P407. In addition,
the concentration of P407 was also increased to 25% w/w to examine whether the literature data
146

could be reproduced at a higher concentration of P407. At 25% w/w P407 and using 6.5 mm
diameter membrane, the release profile established by our setup was similar to that reported by
Wang et al
57, 58, 59
at 17% w/w P407 (Fig. 6.13). These results validated the current experimental
setup, method of analysis, and the optimal concentration of P407 at 25% w/w. Furthermore, data
shown in Figure 6.13 A suggests that the diameter of the membrane made a significant contribution
to the release of MPS.

Figure 6.13. Experimental (A, B) and Literature (C) Kinetic Release Profile of 2% w/w MPS with
P407.
As the conditions for the release experiments were determined, the release of RIS with
various concentration of P407, such as 17% and 25.5%, were performed using 6.5 mm membrane
diameter, and the results were compared with the release profile of ZOL in formulation with 25%
w/w P407. The collected data also suggested that 25% w/w P407 did sustain the release of RIS,
increased from 2 h to 4 h (Figure 6.14 A). Similar increase in release time was observed in the
profile of ZOL with 25% w/w P407, and the release studies for ZOL and RIS exhibited similar
behavior (Figure 6.14 B).
147

Figure 6.14. Kinetic Release Profiles of RIS and ZOL under Various Experimental Conditions.
The release profile of 5-FAM-ZOL with 25% w/w P407 was also performed to examine
the effect of carboxyfluorescein on the interaction of bisphosphonate with P407. Experimental
data indicated that the release profiles of 5-FAM-ZOL and 6-FAM-ZOL were similar using 12
mm membrane system (Figure 6.15 A). While the release of 5-FAM-ZOL was similar to that of
RIS from 17% w/w P407, the release of 5-FAM-ZOL was sustained for 5 hours, an hour longer
than that of RIS at similar concentration of P407, when the concentration of P407 was increased
to 25% w/w (Figure 6.15 B). This suggested that the attachment of carboxyfluorescein did have a
moderate effect on the release of 5-FAM-ZOL when formulated with 25% w/w/ P407.
In summary, Poloxamer 407 did delay the in vitro release of bisphosphonate drugs, such
as Risedronate and Zoledronate, up to 4 hours. Experimental results indicate that factors, including
concentration of drug, volume of each collected aliquot, and surface area of the porous membrane,
significantly affect the release time of the drugs. Various salt forms of bisphosphonate with
different amines, such as tributyl-amine and benzathine, did not prolong the release of
bisphosphonates using P407. In addition, modification of bisphosphonates with fluorescent dye
148

(FAM-RIS) did not influence its kinetic properties with P407. In addition, the in vitro experimental
results indicated that the optimal formulation of P407 and ZOL or RIS is 25% w/w P407 and 5
mM, respectively, for a 4-hour sustained release.

Figure 6.15. (A) Release Profiles of 5-FAM-ZOL and 6-FAM-ZOL from 17 % w/w P407 using
12 mm membrane. (B) Release Profiles of 5-FAM-ZOL and RIS from 17% and 25% w/w P407
using 6.5 mm membrane.
6.7 Conclusion
Absorption of 6-FAM-ZOL to the cochlea, delivered both locally and systemically in
guinea pigs, increased in a dose-dependent manner and caused no ototoxicity. All three methods
were able to deliver 6-FAM-ZOL throughout the cochlea, and a fluorescent signal gradient was
observed from the base to the apex in the cochlea. Non-ototoxic delivery via direct intracochlear
infusion (DII) was most efficient in delivering the compound: 1) 50 times vs. systemic
administration (0.02X vs. 1X), and 2) 15 times vs. diffusion through the RW (0.02X vs. 0.3X).
The safety profile for ZOL was 4:1 therapeutic index for toxic vs. effective dose. 6-FAM-ZOL can
be used as an internal standard to evaluate cochlear drug delivery. Further investigation of the
149

cochlear distribution of 6-FAM-ZOL in fresh, cadaveric human temporal bones confirmed that the
probe could be delivered throughout the cochlea, with a steep baso-apical gradient observed.
Formulation of N-BPs with poloxamer 407 was also developed, with 25% w/w of P407 sustaining
the drug release for 4 hours. Findings from these studies may contribute to the development of
methods to assess the cochlear distribution of other drugs and treatment of cochlear otosclerosis
using local delivery of N-BPs.
6.8 Experimental Section
6.8.1 Chemical Synthesis of 6-FAM-ZOL
Details of the synthesis of 6-FAM-ZOL was previously described.
24
ZOL in acid form was
purchased from Molekula, UK. TEA was freshly distilled over KOH. All other chemicals were
purchased from either Sigma-Aldrich or Alfa Aesar. Thin layer chromatography (TLC) was
performed on Merck Silica Gel 60 F254 plates, and the developed plates were visualized under a
UV lamp at 354 nm. HPLC separations were performed on a Rainan Dynamax Model SD-200
system with a Rainan Dynamax absorbance detector Model UV-DII. NMR spectra were recorded
on either 400 MHz Varian, or 500 MHz Varian. UV−vis spectra were performed on a DU 800
spectrometer, and fluorescence emission spectra were taken on a Jobin Yvon Horiba FluoroMax-
3 fluorimeter equipped with a DataMax Software version 2.20 (Jobin Yvon Inc.). Mass spectra
were obtained on a Thermo-Finnigan LCQ DECA XP MAX Ion Trap LC/MS/MS spectrometer
operated in ESI mode. 5-FAM-ZOL and 6-FAM-ZOL, obtained as triethylammonium salts, was
quantified using UV-VIS spectroscopy, assuming that the molar extinction coefficient of FAM-
ZOL is similar to that of the fluorescence dye itself. Characterization of FAM-ZOL was performed
using NMR spectroscopy (
1
H and
31
P), fluorescence spectroscopy, and mass spectrometry.
150

3-(3-chloro-2-hydroxypropyl)-1-(2-hydroxy-2,2-diphosphonoethyl)-1H-imidazol-3-ium (6.3).
Dissolved in 3 mL of water was ZOL in acid form (i.e., [1-Hydroxy-2-(1H-imidazol-1-yl)ethane-
1,1-diyl]bis(phosphonic acid), (53.1 mg, 0.19 mmol, 1.00 equiv), and the solution was adjusted
to basic pH (~7.5) using sodium carbonate (s), followed by the addition of 80.0 μL of 6.2 (1.02
mmol, 5 equiv). The reaction mixture was stirred at rt overnight, and its progress was monitored
by
31
P NMR. After 23.5 h, 84% of 6.3 was obtained with ∼15% of side products and less than 1%
of 6.1. The aqueous layer was washed with diethyl ether (3X). 6.3 was used for the next step
without further purification. (84%)
3-(3-amino-2-hydroxypropyl)-1-(2-hydroxy-2,2-diphosphonoethyl)-1H-imidazol-3-ium (6.4).
Compound 6.3 was dissolved in 3 mL of ammonium hydroxide solution and the reaction mixture
was left stirred at room temperature overnight. Solvent was removed and the reaction mixture was
dissolved in mL of water. Purification of the crude reaction mixture was performed using SAX
RP-HPLC (Macherey-Nagel 21.4 mm × 150 mm SP15/25 Nucleogel column) with flow rate at 9
mL/min and UV–vis detection was set at 230 nm. Buffer A and B were water and 0.5 M TEAB
pH 7.5, respectively. Gradient for the purification was 0%-30% over 10 min and stayed at 30%
from 10 to 15 min, followed by increasing to 100% of B from 15 to 35 min. The desired product
was eluted between 10-13 min and collected. Solvent was removed and 6.4 was used for the next
step without further purification. (83%)
1
H NMR (500 MHz, Deuterium Oxide) δ 8.75 (s, 1H), 7.45 (s, 1H), 7.34 (s, 1H), 4.53 (s, 2H), 4.33
(d, J = 14.7 Hz, 1H), 4.09 (d, J = 13.9 Hz, 2H), 3.18 (s, 1H), 2.88 (s, 1H).

31
P NMR (202 MHz,
Deuterium Oxide) δ 13.60 (s).
3-(3-(3-carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzamido)-2-hydroxypropyl)-1-(2-hydro-
xy-2,2-diphosphonoethyl)-1H-imidazol-3-ium (6.5) (5-FAM-ZOL) and 3-(3-(4-carboxy-3-(6-
151

hydroxy-3-oxo-3H-xanthen-9-yl)benzamido)-2-hydroxypropyl)-1-(2-hydroxy-2,2-diphosphono
ethyl)-1H-imidazol-3-ium (6.6) (6-FAM-ZOL). In a small 10 mL flask, 59.4 mg of 6.4, obtained as
TEA
+
salt, (0.17 mmol, 3.7 equiv.) was dissolved in 0.75 mL of water and pH of the solution was
adjusted to 8.4 with Na2CO3 (s). To the solution, 21.85 mg of 5(6)-FAM, SE (0.046 mmol, 1 equiv.)
dissolved in 350 µL anhydrous DMF was added and pH of the reaction mixture was readjusted to
8.3 or until all precipitates dissolved. The mixture was left stirred at room temperature overnight.
The product was first purified using TLC to remove the leftover dyes, followed by C18 RP-HPLC
purification. The purification used a Beckman Ultrasphere ODS C18 (250 x 10 mm, 5 μm, 80 Å
pore size) with flow rate 4.0 mL/min and UV-vis detection was set at 230 nm for the first 10 min
and at 492 nm for the rest of the process. Buffer A and B were 0.1 M TEAC (pH 7.0 – 7.8)
containing 10% and 75% MeOH, respectively. Gradient of the method included a linear increase
up to 40% of B in 25 min, followed by increasing to 100% of B from 25 - 100 min. Isolated of 5-
and 6-FAM isomers were collected in separate fractions and solvent was removed. Compounds
6.5 and 6.6 were obtained as triethylammonium salts, and their amount was determined using UV-
Vis spectroscopy. (65%)
(6.5), as triethylammonium salts.
1
H NMR (500 MHz, Deuterium Oxide) δ 8.76 (s, 1H), 8.12 (s,
1H), 7.89 (d, J = 9.2 Hz, 1H), 7.53 – 7.19 (m, 3H), 7.15 – 7.03 (m, 2H), 6.59 (d, J = 8.3 Hz, 4H),
4.51 (s, 2H), 4.39 (d, J = 12.7 Hz, 1H), 4.23 – 4.07 (m, 2H), 3.60 (dd, J = 13.9, 4.6 Hz, 1H), 3.46
(dd, J = 13.9, 6.7 Hz, 1H).
31
P NMR (202 MHz, Deuterium Oxide) δ 13.89 (s).
ESI-MS (negative ion, M-): calcd 704.10 m/z, found [M-2H]
2-
= 351.2 m/z.
(6.6), as triethylammonium salts.
1
H NMR (500 MHz, Deuterium Oxide) δ 8.86 (s, 1H), 8.03 (s,
1H), 7.95 (s, 1H), 7.63 (s, 1H), 7.57 (s, 1H), 7.47 (s, 1H), 7.19 (d, J = 9.3 Hz, 2H), 6.82 – 6.64 (m,
152

4H), 4.69 (d, J = 17.9 Hz, 2H), 4.47 (s, 1H), 4.22 (s, 2H), 3.62 (d, J = 14.1 Hz, 1H), 3.46 (d, J =
14.0 Hz, 1H).
31
P NMR (202 MHz, Deuterium Oxide) δ 13.67 (s).
ESI-MS (negative ion, M-): calcd 704.10 m/z, found [M-2H]
2-
= 351.2 m/z.
6.8.2 Animal Experiments
Animal experiments were conducted by Drs. Woo Seok Kang, David H. Jung, William
Sewell, and Michael McKenna at Massachusetts Eye and Ear Infirmary (MEEI) and statistical
analysis was provided by Doug Hayden, PhD. This work was supported by NIDCD grant R01
DC009837. Details of the experiments were described below.
Animal models. Male albino guinea pigs (~350 g/each) (Hartley strain; Charles River
Laboratories, Inc., Wilmington, MA) were used. The guinea pigs were anesthetized using
Nembutal (12.5 mg/kg intraperitoneally), Fentanyl (0.1 mg/kg intramuscularly), and Haloperidol
(5 mg/kg intramuscularly). Supplemental doses of 0.1 mg/kg Fentanyl and 5 mg/kg Haloperidol
alternating every hour with 8.3 mg/kg Nembutal were administered as needed. Fatal-Plus, a highly
concentrated pentobarbital solution, was intraperitoneally injected for euthanizing animals (390
mg/kg). All animal experiments were approved by the Massachusetts Eye and Ear Infirmary
Institutional Animal Care and Use Committee.
Preparation of ZOL samples for ototoxic evaluation. ZOL was dissolved in AP at two different
molar concentrations. Previous experiments showed that 6-FAM-ZOL with a concentration of 0.25
µg/µL could be infused at a flow rate of 1 µL/min over a minute five times, spaced 9 minutes apart,
into the scala tympani of guinea pigs without shifts in CAP or DPOAE, which corresponds in total
to 2% of human systemic dose of ZOL adjusted by weight.
25
This concentration of 6-FAM-ZOL
(M.W.= 704.5) corresponds to a molar concentration of 354.9 µM. The ototoxic experiment
evaluated twofold and fourfold molar concentrations of ZOL to look for ototoxicity. As an internal
153

control for delivery to the cochlea, 6-FAM-ZOL was included as a tracer (25 molar %) in the dose
escalation experiments.
225 µL of 0.193 µg/µL ZOL (M.W.= 272.1) was mixed with 75 µL of 0.5 µg/µL 6-FAM-ZOL to
form the 0.02X ZOL (709.7 µM). Five microliters of this mixed solution equals 4% of the human
systemic molar dose of ZOL (i.e., 0.04X ZOL). For the fourfold molar concentration (1419.4 µM),
262.5 µL of 0.386 µg/µL ZOL was mixed with 37.5 µL of 1 µg/µL 6-FAM-ZOL. Five microliters
of this mixed solution yields 8% of the human systemic molar dose of ZOL (i.e., 0.08X ZOL).
Systemic drug delivery of 6-FAM-ZOL via intraperitoneal injection. Various doses of 6-FAM-
ZOL that corresponded by molar weight to either one or three times the human systemic dose (5
mg injection) of ZOL recommended for osteoporosis, were administered. 6-FAM-ZOL was
dissolved into 1 ml of artificial perilymph (AP) or phosphate buffered saline (PBS) for injection
at 0.185 mg/kg (1X 6-FAM-ZOL) and 0.555mg/kg (3X 6-FAM-ZOL). AP and PBS were both
used as carriers in different experiments, and no differences were observed. Animals were
anesthetized, and a 0.5-cm incision made in the abdomen through which 6-FAM-ZOL was injected
into the peritoneum. In the control animals, 1 ml of vehicle alone, either AP or PBS, was injected.
All guinea pigs underwent DPOAEs and ABRs both at the time of treatment and immediately
before being sacrificed. Four independent experiments, each with one vehicle-only control, one1X,
and one 3X animal, were performed with AP and three independent experiments with PBS. The
content of AP was 130 mmol/L NaCl, 3.5 mmol/L KCl, 1.5 mmol/L CaCl 2, 5.5 mmol/L glucose,
20 mmol/L HEPES, pH 7.4 and that of PBS was 137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L
Na2HPO4, 1.8 mmol/L KH2PO4, pH 7.4.
Local delivery of 6-FAM-ZOL via diffusion through the round window membrane. 2% (w/v)
solution of sodium alginate was prepared in PBS. 10% (0.1X) and 30% (0.3X) of the human
154

systemic dose by molar weight were tested. For 0.1X 6-FAM-ZOL, 1.3 µL of 4 µg/µL 6-FAM-
ZOL was mixed with 1.3 µL of 2% sodium alginate solution. For 0.3X 6-FAM-ZOL, 1 µL of 15
µg/µL 6-FAM-ZOL was mixed with 1.3 µL of 2% sodium alginate solution. A few drops of 0.2 M
CaCl2 were placed over this mixture using a pipette, which then formed a hydrogel with a diameter
of 1-2 mm. Each hydrogel was made immediately before use.
The alginate beads were place on the round window membrane (RWM) under direct
microscopic visualization. Animals were sacrificed 3 weeks later, after middle ear inflammation
from surgery had subsided. DPOAEs and ABRs were measured immediately prior to surgery and
3 weeks later immediately prior to analysis.
Local delivery of 6-FAM-ZOL and ZOL via infusion into the scala tympani using a
cochleostomy. A Harvard Apparatus PHD 2000 Infusion Syringe Pump was used to introduce the
6-FAM-ZOL or ZOL/6-FAM-ZOL mixture into the scala tympani of guinea pigs via a
cochleostomy approximately 0.5mm distal to the round window. A 500 µL glass syringe was
connected via polyether ether ketone (PEEK; Upchurch Scientific) tubing to an 11 mm length of
PTFE (Teflon) tubing (201 µm od, 101 µm id). A small bleb was made 3 mm from the distal end
of the tubing using methyltriacetoxysilane to prevent over-insertion.
A cochleostomy were performed at distant of approximately 0.5mm distal to the round
window via a bullectomy approach and inserted the Teflon tubing. Dental cement was used to seal
the cochleostomy around the tubing. After cannula insertion, DPOAEs and CAPs were measured,
and repeated several times during the experiment. 1uL of 6-FAM-ZOL at a concentration of
0.25µg/µL were infused 5 times, each spaced 10 minutes apart, corresponding in total to 2% by
molar weight of the human systemic dose. Animals were euthanized approximately 4 hours after
155

the first injection. In parallel, untreated animals also had DPOAEs and CAPs measured and were
sacrificed for control tissue analysis.
In ototoxic studies, similar infusion method was used, 1 µL over one minute five times,
spaced nine minutes apart. Animals were administered with ZOL solution mixed with 6-FAM-
ZOL either at 0.04X or 0.08X while the control animals were treated with AP alone. In chronic
experiments, the infusion line was removed, a blood clot was used to seal the cochleostomy, the
surgical site was closed, and the animals were allowed to recover for 4 weeks before reanalysis of
hearing. Both the acute and chronic ototoxicity experiments had three subgroups, which are control
animals, animals treated with 0.04X ZOL and 0.08X ZOL, respectively.
Hearing measurements. Distortion product otoacoustic emissions (DPOAEs) and auditory
brainstem responses (ABRs) were measured in the systemic and RWM delivery experiments. For
the cochleostomy experiments, cochlear action potentials (CAPs) were substituted for ABRs due
to its greater speed and better signal-to-noise ratio. Hearing was measured at 2.78, 4, 5.6, 8, 12,
16, 24, and 32 kHz.
In the ototoxic experiment, DPOAE and CAP were measured multiple times over 4 hours
following administration of solution, after which the animals were euthanized, to evaluate acute
ototoxicity. In the chronic ototoxicity experiments, DPOAE and CAP were measured during the
drug infusion, after which the animals were allowed to emerge from anesthesia. Hearing was
measured again 4 weeks later and the animals were euthanized.
Sample analyses. ZOL administered via intravenous route was rapidly cleared from plasma in
humans via two main processes, including bone deposition and renal excretion, with two half-lives
at 0.2 and 1.4hours, and less than 1% of the drug remaining in plasma after 24 hours.
60
All animals
were therefore analyzed after maximal bisphosphonate deposition. Systemic delivery animals were
156

analyzed at 48 hours to account for potential differences between intraperitoneal (our system) and
intravenous (human) delivery. RW-delivery animals were analyzed at 3 weeks to allow middle ear
inflammation to subside before hearing analysis and to account for transit across the RWM.
Intracochlear delivery animals were analyzed at 3 hours as this time point allowed for high levels
of delivery while avoiding the potential confounding long-term effects of a cochleostomy.
Sample processing and analysis using confocal microscopy. Temporal bones were harvested
and fixed in an excess volume of neutral buffered formalin (10%, Fisher Scientific, Pittsburgh PA)
at room temperature for 24 hours. The fixation continued in fresh neutral buffered formalin for
another 24 hours. Specimens were dehydrated in ethanol, followed by xylene, after which they
were infiltrated and embedded in methyl methacrylate (Dorn and Hart, Villa Park, IL). Curing of
the specimens was performed at room temperature in the dark to prevent photobleaching. The
hardened block was ground to a mid-modiolar section of the cochlea.
High-resolution images (1360 by 1024; 16 bit) were taken under fluorescein isothiocyanate
illumination (470/40 excitation, 495lp dicroic; 525/50 emission) using a Carl Zeiss Axiovert 200
Inverted Microscope. ImageJ was used to quantify the amount of fluorescence within the cochlear
wall and modiolus at each half-turn (Fig. 6.1).
61
Background fluorescence, defined as the average
amount of fluorescence in an area of the resin block containing no sample, was subtracted from all
measurements. Fluorescence remained stable over at least 2 weeks after grinding if samples were
kept in the dark at room temperature. Some images (1024 by 1024; 8bit) were obtained using a
Leica TCS-SP2 confocal microscope over a depth of 500 µm to produce maximum fluorescence
images with the section of the strongest signal selected in the middle. The same microscope
settings were used in all experiments and all specimens from a single experiment were
photographed at the same sitting.
157

Statistical analysis. Statistical data analysis was performed and null hypotheses were rejected at
p < 0.05. Mixed model analysis of variance was used to analyze the effect of dose on fluorescent
response in the cochlea and in the tibia. A separate model was fit for each experiment. The model
included a fixed effect of dose and a random effect of dose within each replicate. For each dose
level, the reported estimated response is the model-based least-squares mean and standard error of
the mean.
6.8.3 Cadaveric Human Temporal Bone Experiments
Animal experiments were conducted by Drs. Woo Seok Kang, David H. Jung, William
Sewell, and Michael J. McKenna at Massachusetts Eye and Ear Infirmary (MEEI). This work was
supported by NIDCD grant R01 DC009837. Details of the experiments were described below.
Preparation of 6-FAM-ZOL solution. The compound was dissolved in AP at concentration of
10 µg/µL.
Delivery of 6-FAM-ZOL in human cadaveric temporal bones via injection through the oval
window. Six freshly harvested cadaveric human temporal bones were obtained within 24 hours after
death by the human temporal bone histopathology lab at Massachusetts Eye and Ear Infirmary. The
Pathology Quality Assurance Committee at Massachusetts General Hospital approved procurement of
the specimens. The squamous and mastoid portions were drilled away, leaving the labyrinth. In three
temporal bones, 1 µL of 10µg/µL 6-FAM-ZOL through the OW was injected using a micropipette after
lifting the stapes footplate. The injection was performed very slowly and we did not pipette “up and
down.” The footplate was carefully laid back down. The other three temporal bones were treated with
AP as controls. All specimens were placed in a sealed, humidified plastic container and left in the dark
at room temperature for 4 days.
158

Our previous study showed that one injection of 1.25 µg of 6-FAM-ZOL over 40 minutes
into the cochlea of guinea pigs did not cause any observed ototoxicity and damaging hearing.
25
With
the inner ear fluid space being approximately 10 times larger in the human than in the guinea pig
(204.5 µL vs. 20.9 µL),
62, 63
ten times of the amount of 6-FAM-ZOL used in guinea pigs (10 µg) was
utilized in this study.
Sample processing. All subsequent steps were performed in covered vials to limit light exposure and
prevent 6-FAM-ZOL bleaching. Specimens were fixed in an excess volume of neutral buffered formalin
(10%, Fisher Scientific, Pittsburgh PA) at room temperature for one week, with one change of fixative
at mid-week. They were then dehydrated in an ascending series of ethanol (Fisher Scientific, 2 days at
70%, 1 hr at 70%, 1 hr at 85%, 40min at 95%, 80 min at 100%) followed by a wash in xylene (Fisher
Scientific) and a 30minute immersion in xylene. They were cleared of xylene by a 1-hour infiltration in
methyl methacrylate (MMA, Acrylosin soft, Dorn and Hart, Villa Park, IL) under vacuum. The MMA
solution was changed and specimens were infiltrated for 24 hours under vacuum. The infiltration
solution was changed and the process was repeated for another 2 days. Specimens were embedded in a
solution of MMA containing 0.25% w/v (2.5 g/L) of perkadox-16 (Dorn and Hart). Specimens were
embedded for several hours under vacuum. After several hours, specimens were sealed in 50ml
polypropylene tubes and maintained at room temperature in a heat sink until fully cured (approximately
5 days). The hardened block was ground to a mid-modiolar section of the cochlea using a 12” table
mounted rotary wheel.
Analysis using confocal microscopy and statistical analysis. Using a Leica TCS-SP2 confocal
microscope, images (1024x1024, 8 bit) were collected to quantify the fluorescence associated with the
labeled bisphosphonate. To identify the surface of each specimen, the point of maximum fluorescence
in the z-axis was first identified. A stack of images to a depth of 500 µm from this point of maximum
159

fluorescence was collected. Images were obtained from control and experimental specimens in parallel
at the same sitting under identical microscope settings. ImageJ was used to quantify the amount of
fluorescence within the lateral cochlear wall and the modiolus of the scala tympani and scala vestibuli
at each half-turn (Figure 1).
61
Mixed model analysis of variance was performed to analyze the effect of
dose on fluorescent response in the cochlea with null hypotheses rejected at p < 0.05.
6.8.4 In Vitro Experiments of the Release of N-BP drugs using P407
Materials. Risedronate in monosodium salt form was a kind gift from Warner Chilcott
Pharmaceuticals (formerly P&G Pharmaceuticals). ZOL (acid form) was commercially available
from Molekula, UK. 5-FAMRIS, 5-FAM-ZOL, and 6-FAM-ZOL were synthesized using
previously reported method.
24, 64
Snapwells of 6.5 mm and 12.0 mm diameter polycarbonate
membrane with pore size 0.4 μm were purchased from Corning. The commercial plates were
incubated in a custom built water jacket set at 37
o
C. Poloxamer P407 and methylprednisolone
hemi succinate were purchased from Sigma-Aldrich.
Procedure. The stock hydrogel solution (34% w/w) was prepared by slowly dissolving powder
P407 into cold, sterilized 10 mM PBS buffer (pH 7.4). The desired concentration of drug with
P407 mixture was obtained by diluting the stock solution of the drug of interest in10 mM PBS
buffer (pH 7.4) in the stock P407 solution. The mixture was brought up to the needed total volume
using 10 mM PBS buffer (pH 7.4)
Stock solution of ZOL was prepared by dissolving ZOL in acid form (~10 mg) in 1 mL of
MilliQ water. The pH of the sample was adjusted to pH 7.0 using 1 M NaOH. The sample was
mixed with 1X PBS (pH 7.4) and P407 stock solution to obtain the concentration of 1% w/w ZOL.
Similarly, stock solution of Risedronate was prepared by dissolving RIS in sodium form (~10 mg)
in 1 mL of MilliQ water. The sample was mixed with 1X PBS (pH 7.4) and P407 stock solution
160

to obtain the concentration of 1% w/w RIS (37 mM) and 5 mM RIS. FAM-RIS sample was
prepared by mixing approximately 1.6 mg of FAM-RIS in 350 μL 1X PBS (pH 7.4) and 350 μL
of P407 stock solution. 5-FAM-ZOL, 5-FAMRIS, and 6-FAM-ZOL samples were prepared by
diluting stock solution of 50 mM.

Figure 6.16. Experimental Setups for In vitro Release Studies of Bisphosphonate in Formulation
with P407
The in vitro kinetic release profile was performed at 37
o
C using the setup shown in Figure
1. Unless specified, kinetic studies on the release of bisphosphonate drugs with P407 were
performed using Method 1. In addition, some experiments were performed using either 6.5 or 12
mm diameter Snapwells and unless specified, all in vitro release studies were performed with 12
mm diameter Snapwells. All experiments were triplicated.
A volume of 0.3 mL of bisphosphonate-hydrogel sample was transferred into a Snapwell
and left at room temperature for 5-10 min to gel. Placed into the reservoir was 1.5 mL of 10 mM
PBS and the well plate was shaken at 70 rpm using an orbit shaker with the ambiance temperature
being 37
o
C. Aliquots of 300 μL were taken out every 60 minutes up to 6 hours and 300 μL of pre-
warmed buffer was added to preserve the original volume of 1.5 mL. UV-Vis analysis of the
sample was performed to determine the concentration and the percentage of compound released
was calculated.
161

6.9 References
1. Quesnel, A. M.; Seton, M.; Merchant, S. N.; Halpin, C.; McKenna, M. J., Third-generation
bisphosphonates for treatment of sensorineural hearing loss in otosclerosis. Otol. Neurotol. 2012,
33, 1308-1314.
2. Chole, R. A.; McKenna, M., Pathophysiology of otosclerosis. Otol. Neurotol. 2001, 22,
249-257.
3. Doherty, J. K.; Linthicum, F. H., Spiral ligament and stria vascularis changes in cochlear
otosclerosis: effect on hearing level. Otol. Neurotol. 2004, 25, 457-464.
4. Balle, V.; Linthicum, F. H., Histologically proven cochlear otosclerosis with pure
sensorineural hearing loss. Ann. Otol. Rhinol. Laryngol. 1984, 93, 105-111.
5. Rama-Lopez, J.; Cervera-Paz, F. J.; Manrique, M., Cochlear implantation of patients with
far-advanced otosclerosis. Otol. Neurotol. 2006, 27, 153-158.
6. Marshall, A. H.; Fanning, N.; Symons, S.; Shipp, D.; Chen, J. M.; Nedzelski, J. M.,
Cochlear implantation in cochlear otosclerosis. Laryngoscope 2005, 115, 1728-1733.
7. Ramsden, R.; Rotteveel, L.; Proops, D.; Saeed, S.; van Olphen, A.; Mylanus, E., Cochlear
implantation in otosclerotic deafness. Adv. Otorhinolaryngol. 2007, 65, 328-334.
8. Semaan, M. T.; Gehani, N. C.; Tummala, N.; Coughlan, C.; Fares, S. A.; Hsu, D. P.;
Murray, G. S.; Lippy, W. H.; Megerian, C. A., Cochlear implantation outcomes in patients with
far advanced otosclerosis. Am. J. Otolaryngol. 2012, 33, 608-614.
9. Clayton, A. E.; Mikulec, A. A.; Mikulec, K. H.; Merchant, S. N.; McKenna, M. J.,
Association between osteoporosis and otosclerosis in women. J. Laryngol. Otol. 2004, 118, 617-
621.
10. McKenna, M. J.; Nguyen-Huynh, A. T.; Kristiansen, A. G., Association of otosclerosis
with Sp1 binding site polymorphism in COL1A1 gene: evidence for a shared genetic etiology with
osteoporosis. Otol. Neurotol. 2004, 25, 447-450.
11. Santos, F.; McCall, A. A.; Chien, W.; Merchant, S., Otopathology in Osteogenesis
Imperfecta. Otol. Neurotol. 2012, 33, 1562-1566.
12. Khetarpal, U.; Schuknecht, H. F., In search of pathologic correlates for hearing loss and
vertigo in Paget's disease. A clinical and histopathologic study of 26 temporal bones. Ann. Otol.
Rhinol. Laryngol. Suppl. 1990, 145, 1-16.
13. Lipton, A., New therapeutic agents for the treatment of bone diseases. Expert Opin. Biol.
Ther. 2005, 5, 817-832.
14. Papapetrou, P. D., Bisphosphonate-associated adverse events. Hormones (Athens) 2009, 8,
96-110.
162

15. Silverman, S. L.; Landesberg, R., Osteonecrosis of the jaw and the role of bisphosphonates:
a critical review. Am. J. Med. 2009, 122, S33-45.
16. Reid, I. R., Efficacy, effectiveness and side effects of medications used to prevent fractures.
J. Intern. Med. 2015, 277, 690-706.
17. Whitaker, M.; Guo, J.; Kehoe, T.; Benson, G., Bisphosphonates for osteoporosis--where
do we go from here? N. Engl. J. Med. 2012, 366, 2048-2051.
18. Russell, R. G., Bisphosphonates: mode of action and pharmacology. Pediatrics 2007, 119
Suppl 2, S150-162.
19. Roy, S.; Glueckert, R.; Johnston, A. H.; Perrier, T.; Bitsche, M.; Newman, T. A.; Saulnier,
P.; Schrott-Fischer, A., Strategies for drug delivery to the human inner ear by multifunctional
nanoparticles. Nanomedicine 2012, 7, 55-63.
20. Paasche, G.; Gibson, P.; Averbeck, T.; Becker, H.; Lenarz, T.; Stover, T., Technical report:
modification of a cochlear implant electrode for drug delivery to the inner ear. Otol. Neurotol.
2003, 24, 222-227.
21. Peters, G.; Lin, J.; Arriaga, M. A.; Nuss, D. W.; Schaitkin, B.; Walvekar, R. R., Middle-
ear endoscopy and trans-tympanic drug delivery using an interventional sialendoscope: feasibility
study in human cadaveric temporal bones. J. Laryngol. Otol. 2010, 124, 1263-1267.
22. Roy, S.; Glueckert, R.; Johnston, A. H.; Perrier, T.; Bitsche, M.; Newman, T. A.; Saulnier,
P.; Schrott-Fischer, A., Strategies for drug delivery to the human inner ear by multifunctional
nanoparticles. Nanomedicine (Lond) 2012, 7, 55-63.
23. Peters, G.; Lin, J.; Arriaga, M. A.; Nuss, D. W.; Schaitkin, B.; Walvekar, R. R., Middle-
ear endoscopy and trans-tympanic drug delivery using an interventional sialendoscope: feasibility
study in human cadaveric temporal bones. J Laryngol Otol 2010, 124, 1263-1267.
24. Sun, S.; Blazewska, K. M.; Kadina, A. P.; Kashemirov, B. A.; Duan, X.; Triffitt, J. T.;
Dunford, J. E.; Russell, R. G.; Ebetino, F. H.; Roelofs, A. J.; Coxon, F. P.; Lundy, M. W.;
McKenna, C. E., Fluorescent Bisphosphonate and Carboxyphosphonate Probes: A Versatile
Imaging Toolkit for Applications in Bone Biology and Biomedicine. Bioconjug Chem 2016, 27,
329-340.
25. Kang, W. S.; Sun, S.; Nguyen, K.; Kashemirov, B.; McKenna, C. E.; Hacking, S. A.;
Quesnel, A. M.; Sewell, W. F.; McKenna, M. J.; Jung, D. H., Non-Ototoxic Local Delivery of
Bisphosphonate to the Mammalian Cochlea. Otol. Neurotol. 2015, 36, 953-960.
26. Kozloff, K. M.; Volakis, L. I.; Marini, J. C.; Caird, M. S., Near-infrared fluorescent probe
traces bisphosphonate delivery and retention in vivo. J. Bone Miner. Res. 2010, 25, 1748-1758.
27. Wen, D.; Qing, L.; Harrison, G.; Golub, E.; Akintoye, S. O., Anatomic site variability in
rat skeletal uptake and desorption of fluorescently labeled bisphosphonate. Oral. Dis. 2011, 17,
427-432.
163

28. Plontke, S. K.; Biegner, T.; Kammerer, B.; Delabar, U.; Salt, A. N., Dexamethasone
concentration gradients along scala tympani after application to the round window membrane. Otol.
Neurotol. 2008, 29, 401-406.
29. Mynatt, R.; Hale, S. A.; Gill, R. M.; Plontke, S. K.; Salt, A. N., Demonstration of a
longitudinal concentration gradient along scala tympani by sequential sampling of perilymph from
the cochlear apex. J. Assoc. Res. Otolaryngol. 2006, 7, 182-193.
30. Plontke, S. K.; Mynatt, R.; Gill, R. M.; Borgmann, S.; Salt, A. N., Concentration gradient
along the scala tympani after local application of gentamicin to the round window membrane.
Laryngoscope 2007, 117, 1191-1198.
31. Wang, X.; Dellamary, L.; Fernandez, R.; Ye, Q.; LeBel, C.; Piu, F., Principles of inner ear
sustained release following intratympanic administration. Laryngoscope 2011, 121, 385-391.
32. Wang, X.; Fernandez, R.; Tsivkovskaia, N.; Harrop-Jones, A.; Hou, H. J.; Dellamary, L.;
Dolan, D. F.; Altschuler, R. A.; Lebel, C.; Piu, F., OTO-201: Nonclinical Assessment of a
Sustained-Release Ciprofloxacin Hydrogel for the Treatment of Otitis Media. Otol. Neurotol. 2014,
35, 459-469.
33. Plontke, S. K.; Biegner, T.; Kammerer, B.; Delabar, U.; Salt, A. N., Dexamethasone
concentration gradients along scala tympani after application to the round window membrane. Otol
Neurotol 2008, 29, 401-406.
34. Lajud, S. A.; Han, Z.; Chi, F. L.; Gu, R.; Nagda, D. A.; Bezpalko, O.; Sanyal, S.; Bur, A.;
Han, Z.; O'Malley, B. W.; Li, D., A regulated delivery system for inner ear drug application. J.
Control Release 2013, 166, 268-276.
35. Khoo, X.; Simons, E. J.; Chiang, H. H.; Hickey, J. M.; Sabharwal, V.; Pelton, S. I.;
Rosowski, J. J.; Langer, R.; Kohane, D. S., Formulations for trans-tympanic antibiotic delivery.
Biomaterials 2013, 34, 1281-1288.
36. Ealy, M.; Smith, R. J., Otosclerosis. Adv. Otorhinolaryngol. 2011, 70, 122-129.
37. Quesnel, A. M.; Seton, M.; Merchant, S. N.; Halpin, C.; McKenna, M. J., Third-generation
bisphosphonates for treatment of sensorineural hearing loss in otosclerosis. Otol Neurotol 2012,
33, 1308-1314.
38. Kang, W. S.; Nguyen, K.; McKenna, C. E.; Sewell, W. F.; McKenna, M. J.; Jung, D. H.,
Measurement of Ototoxicity Following Intracochlear Bisphosphonate Delivery. Otol. Neurotol.
2016, 37, 621-626.
39. Poirrier, A. L.; Van den Ackerveken, P.; Kim, T. S.; Vandenbosch, R.; Nguyen, L.;
Lefebvre, P. P.; Malgrange, B., Ototoxic drugs: Difference in sensitivity between mice and guinea
pigs. Toxicol. Lett. 2010, 193, 41-49.
164

40. Sockalingam, R.; Freeman, S.; Cherny, T. L.; Sohmer, H., Effect of high-dose cisplatin on
auditory brainstem responses and otoacoustic emissions in laboratory animals. Am. J. Otol. 2000,
21, 521-527.
41. McWilliams, M. L.; Chen, G. D.; Fechter, L. D., Characterization of the ototoxicity of
difluoromethylornithine and its enantiomers. Toxicol. Sci. 2000, 56, 124-132.
42. Blakley, B. W.; Hochman, J.; Wellman, M.; Gooi, A.; Hussain, A. E., Differences in
Ototoxicity across Species. J. Otolaryngol. Head Neck Surg. 2008, 37, 700-703.
43. Han, D. Y.; Harada, N.; Tomoda, K.; Yamashita, T., Characterization of the calcium influx
induced by depolarization of guinea pig cochlear spiral ganglion cells. ORL J. Otorhinolaryngol.
Relat. Spec. 1994, 56, 125-129.
44. Ceriani, F.; Mammano, F., Calcium signaling in the cochlea - Molecular mechanisms and
physiopathological implications. Cell Commun. Signal 2012, 10, 20.
45. Russell, R. G., Bisphosphonates: the first 40 years. Bone 2011, 49, 2-19.
46. Ibrahim, A.; Scher, N.; Williams, G.; Sridhara, R.; Li, N.; Chen, G.; Leighton, J.; Booth,
B.; Gobburu, J. V.; Rahman, A.; Hsieh, Y.; Wood, R.; Vause, D.; Pazdur, R., Approval summary
for zoledronic acid for treatment of multiple myeloma and cancer bone metastases. Clin. Cancer.
Res. 2003, 9, 2394-2399.
47. Hahn, H.; Salt, A. N.; Biegner, T.; Kammerer, B.; Delabar, U.; Hartsock, J. J.; Plontke, S.
K., Dexamethasone levels and base-to-apex concentration gradients in the scala tympani
perilymph after intracochlear delivery in the guinea pig. Otol. Neurotol. 2012, 33, 660-665.
48. Mikulec, A. A.; Plontke, S. K.; Hartsock, J. J.; Salt, A. N., Entry of substances into
perilymph through the bone of the otic capsule after intratympanic applications in guinea pigs:
implications for local drug delivery in humans. Otol. Neurotol. 2009, 30, 131-138.
49. Kang, W. S.; Nguyen, K.; McKenna, C. E.; Sewell, W. F.; McKenna, M. J.; Jung, D. H.,
Intracochlear Drug Delivery Through the Oval Window in Fresh Cadaveric Human Temporal
Bones. Otol. Neurotol. 2016, 37, 218-222.
50. Salt, A. N.; Ohyama, K.; Thalmann, R., Radial communication between the perilymphatic
scalae of the cochlea. II: Estimation by bolus injection of tracer into the sealed cochlea. Hear. Res.
1991, 56, 37-43.
51. Salt, A. N.; King, E. B.; Hartsock, J. J.; Gill, R. M.; O'Leary, S. J., Marker entry into
vestibular perilymph via the stapes following applications to the round window niche of guinea
pigs. Hear. Res. 2012, 283, 14-23.
52. Zou, J.; Pyykko, I.; Bjelke, B.; Dastidar, P.; Toppila, E., Communication between the
perilymphatic scalae and spiral ligament visualized by in vivo MRI. Audiol. Neurootol. 2005, 10,
145-152.
165

53. Kucuk, B.; Abe, K.; Ushiki, T.; Inuyama, Y.; Fukuda, S.; Ishikawa, K., Microstructures of
the bony modiolus in the human cochlea: a scanning electron microscopic study. J. Electron
Microsc. 1991, 40, 193-197.
54. Rask-Andersen, H.; Schrott-Fischer, A.; Pfaller, K.; Glueckert, R., Perilymph/modiolar
communication routes in the human cochlea. Ear Hear. 2006, 27, 457-465.
55. Salt, A. N.; Gill, R. M.; Hartsock, J. J., Perilymph Kinetics of FITC-Dextran Reveals
Homeostasis Dominated by the Cochlear Aqueduct and Cerebrospinal Fluid. J. Assoc. Res.
Otolaryngol. 2015, 16, 357-371.
56. Ohyama, K.; Salt, A. N.; Thalmann, R., Volume flow rate of perilymph in the guinea-pig
cochlea. Hear. Res. 1988, 35, 119-129.
57. Wang, X.; Dellamary, L.; Fernandez, R.; Harrop, A.; Keithley, E. M.; Harris, J. P.; Ye, Q.;
Lichter, J.; LeBel, C.; Piu, F., Dose-Dependent Sustained Release of Dexamethasone in Inner Ear
Cochlear Fluids Using a Novel Local Delivery Approach. Audiology and Neuro-Otology 2009, 14.
58. Wang, X.; Fernandez, R.; Dellamary, L.; Harrop, A.; Ye, Q.; Lichter, J.; Lau, D.; Lebel,
C.; Piu, F., Pharmacokinetics of Dexamethasone Solution following Intratympanic Injection in
Guinea Pig and Sheep. Audiol. Neurootol. 2011, 16.
59. Wang, X.; Dellamary, L.; Fernandez, R.; Ye, Q.; LeBel, C.; Piu, F., Principles of Inner Ear
Sustained Release Following Intratympanic Administration. Laryngoscope 2011, 121.
60. Chen, T.; Berenson, J.; Vescio, R.; Swift, R.; Gilchick, A.; Goodin, S.; LoRusso, P.; Ma,
P.; Ravera, C.; Deckert, F.; Schran, H.; Seaman, J.; Skerjanec, A., Pharmacokinetics and
pharmacodynamics of zoledronic acid in cancer patients with bone metastases. J. Clin. Pharmacol.
2002, 42, 1228-1236.
61. Rasband, W. S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA,
http://imagej.nih.gov/ij/, 1997-2014.
62. Igarashi, M.; Ohashi, K.; Ishii, M., Morphometric comparison of endolymphatic and
perilymphatic spaces in human temporal bones. Acta Otolaryngol. 1986, 101, 161-164.
63. Shinomori, Y.; Spack, D. S.; Jones, D. D.; Kimura, R. S., Volumetric and dimensional
analysis of the guinea pig inner ear. Ann. Otol. Rhinol. Laryngol. 2001, 110, 91-98.
64. Kashemirov, B. A.; Bala, J. L.; Chen, X.; Ebetino, F. H.; Xia, Z.; Russell, R. G.; Coxon, F.
P.; Roelofs, A. J.; Rogers, M. J.; McKenna, C. E., Fluorescently labeled risedronate and related
analogues: "magic linker" synthesis. Bioconjug Chem 2008, 19, 2308-2310.

166

CHAPTER 7
Bisphosphonates Prodrugs for Targeted Drug Delivery of
Neurotrophic Agents for the Treatment of Hearing Loss
7.1 Hearing Loss (HL)
Hearing loss (HL) affects more than 17% of adults in the U.S.
1
Senior adults are
disproportionately affected, as two-thirds of the senior population (70 years old or older) live with
presbycusis, or age-related hearing loss. HL can be segregated into conductive (impaired
conduction to the inner ear), sensorineural (damage to sensory and non-sensory inner ear
structures), or mixed. Genetic defects can contribute to HL.
2
Aging, noise exposure, infection, and
exogenous toxins have all been implicated as causes of sensorineural hearing loss (SNHL).
3

Previous human temporal bone histopathologic studies conducted by Schuknecht
demonstrated that presbycusis is correlated with loss of cochlear hair cells (HCs), cochlear spiral
ganglion neurons (SGNs), and cells of the stria vascularis (a structure in the lateral cochlear wall
critical for maintaining ionic gradients in endolymph), with many specimens demonstrating mixed
loss of all three populations.
4
More recent studies suggest that the mere presence or absence of cell
populations may underestimate the true extent of SNHL. Thus cochlear synaptopathy, the loss of
synapses between inner hair cells (IHCs) and neurons without overt IHC or neuronal loss, has been
described after noise exposure.
5
Cochlear synaptopathy also may be part of the aging process in
humans.
6
Although noise exposure has been shown to accelerate the loss of synapses in animal
models,
7
SGNs may persist for years—or even decades—following synaptic loss,
8
a finding that
has intensified interest in therapies to regenerate synaptic function.
9

167

Though a recent study demonstrated that the intake of antioxidant vitamins, such as vitamin
A and vitamin C, along with magnesium, is associated with lowering the risks of hearing loss,
10

the origin of the phenomenon, neural cell death, has yet to be fully addressed. Therefore, the search
for neurotrophic reagents to stimulate the growth of hair cells and spiral ganglion cells is of great
interest.
7.2 Neurotrophic factors and their receptors
Members of the tropomyosin receptor kinase (trk) family, including trkA, trkB, and trkC,
are mainly found in neural cells. Through their highly specific interactions with neurotrophins,
they help regulate the development and maintain the well-being of the peripheral and central
nervous systems.
11
In particular, trkB’s interaction with brain-derived neurotrophic factor (BDNF)
is currently drawing great interest from researchers. Many neurodegenerative diseases, such as
Alzheimer’s disease,
12
Huntington’s disease,
13
and Parkinson’s disease,
14
have been correlated to
changes in the expression of BDNF, which indicates that the interaction of BDNF with trkB may
play a key role in neurodegeneration. Delivery of BDNF using gene therapy in mice was reported
to increase the survival rate of damaged auditory neurons and prevent their degeneration after
exposure to neomycin, an ototoxic drug.
15
In 2010, Jang et al. reported high neurotrophic activities
of 7,8-dihydroxyflavone, which acts as a highly selective agonist of trkB, and thus protects neuron
from apoptosis.
16
In the same year, Massa et al. discovered high neurotrophic efficacy at nanomolar
concentrations in rodents induced by a set of small molecules that binds and activates trkB.
17
This
suggests that trkB and its agonists could potentially be the solution in slowing neurodegeneration
and possibly inducing neural cell growth, particularly in the ear.
Our previous studies on otosclerosis using fluorescently labeled zoledronate (6-FAM-ZOL)
demonstrated that 6-FAM-ZOL, delivered via the round window membrane (RWM), was diffused
168

throughout the cochlea, and no ototoxicity was observed at potential therapeutic levels. Given the
great bone affinity of bisphosphonate drugs (BPs) to the cochlear wall, they offer a promising and
innovative approach to promote nerve cell growth via in situ target delivery of neurotrophic agents
to cochlear nerves. Here, a new approach in addressing neural cell death in the ear was proposed:
local delivery of TrkB agonist in the ear using BPs to target the cochlear wall. The agents will
interact with TrkB of the spiral ganglion cells and activate them. Specifically, a TrkB agonist-BP
conjugate was synthesized, and the neurotrophic activities of the conjugates were evaluated.

Figure 7.1. Illustration of the organ of Corti before and after exposure to cDNA BDNF.
7.3 Molecular docking using Autodock Vina
In this project, the main goal is to synthesize a BP conjugate containing a BP drug, such as
risedronate, connected to a trkB agonist via a sizeable linker in order to maintain the activities of
both BP and the neurotrophic reagent. Here, 7,8-DHF (Figure 7.2), a well-known trkB agonist,
was chosen due to its structural simplicity and the availability of inexpensive starting materials.
First, in order to determine which position is suitable for modification, molecular docking
using AutoDock Vina
18
of 7,8-dihydroxyflavone (7,8-DHF) was performed with the crystal
structure of trkB (PDB code 4AT3).
19
However, the protein is co-crystallized in complex with
Cpd5n, a trkB inhibitor, and the crystal structure of the receptor with an agonist has yet to be
169

available. Docking results show interactions of 7,8-DHF in the binding site, including 7-OH
hydrogen bonding with GLU604 and π-stacking of the phenyl ring with PHE565 (Figure 7.2 A
and B). The interactions are fairly similar to those of Cpd5n to trkB (Figure 7.2 C). Therefore, it
is uncertain whether these interactions could be similar to the real interactions of 7,8-DHF with
trkB. Nevertheless, the orientation of the compound in the pocket could be potentially useful in
suggesting the possible modifying position on 7,8-DHF. The phenyl ring of 7,8-DHF is facing
toward the entrance of the binding pocket. This suggests that modifications at the phenyl ring
(highlighted in red) can be tolerated by the active binding site of trkB.

Figure 7.2. Molecular docking of 7,8-DHF in trkB crystal structure (PDB code: 4AT3).
(A) General overview of 7,8-DHF at the binding site of trkB. (B) A zoomed-in view of the
interaction of 7,8-DHF with trkB. (C) A zoomed-in view of the interaction of 7,8-DHF, Cpd5n
with trkB at the active site.
Thus, a 7,8-DHF derivative containing a carboxylic group (COOH-DHF) at the para
position was chosen to be our target molecule. The DHF derivative was also docked with trkB to
170

examine its interaction at the binding site. The addition of the carboxylic group, indeed, did not
interfere with the orientation of the rest of molecule. While docking results provided a low binding
energy for COOH-DHF, the energy is not the conclusive factor in determining whether the
compound is active toward trkB.

Figure 7. 3. Structure of the DHF-derivative 7.1.
7.4 Synthesis of a DHF derivative
The syntheses of flavones similar to 7,8-DHF have been previously reported.
20
Similarly,
the synthesis of 7.1 would proceed using the previously reported approach (Scheme 7.1). However,
instead of benzaldehyde, 4-carboxybenzaldehyde (7.4) was utilized. Initially, the synthesis of 7.5a,
the key precursor for 7.1, was thought to be straightforward with a good yield using route A (in
blue). Selective benzyl deprotection using TFA was previously reported with any aromatic systems
that had a carboxyl group at the ortho position to the O-protected benzyl group.
21
However, despite
many attempts to improve the yield, including increasing the temperature (up to 70
o
C), along with
enhancing the solubility and the nucleophilicity of 7.4 by protecting the carboxyl group with either
benzyl or tert-butyl group, the yield of 7.6 remained stagnant at 20-25%. Thus, switching the steps
in route A (in blue) to route B (in red), resulted in a significant improvement in the yield of 7.5b
(64%). Similar conditions to those employed to obtain 7.5a were used to selectively deprotect the
benzyl group of 7.5b. Cyclization of 7.6 in DMSO to produce O-benzyl protected flavone, 7.7, is
surprisingly efficient in the presence of a catalytic amount of iodine (0.1 equivalent) and heating
171

up to 120
o
C. However, the work-up of this reaction was not as straightforward, since 7.7 had low
solubility in organic solvents and aqueous buffer solution (e.g., 0.1M TEAB, 50% MeOH, pH 7.5).
The compound was easily precipitated from DMSO using ice cold water. The fine, white crystal
7.7 was collected and utilized for the next step without further purification.

Scheme 7.1 Synthesis of 7.1
To obtain 7.1, benzyl deprotection of 7.7 using hydrogenolysis was performed. Since 7.7
is insoluble in methanol, a common choice of solvent for hydrogenolysis, a mixture of methanol
and other solvents, including both aqueous and organic solvents, was examined. The mixture of
methanol and freshly distilled tetrahydrofuran (1:1 v/v) proved to be the best solvent system for
hydrogenolysis. Recrystallization in methanol were performed in methanol, yielding pure 7.1. To
examine whether the extra negative charge attributed to the addition of carboxyl group would
172

ultimately affect their binding to trkB, methyl ester form of 7.1, 7.8, was also synthesized via
esterification using catalytic amount of concentrated sulfuric acid in methanol.
7.5 Synthesis of DHF-linker
Since the biological activities of 7.9 would determine the need for a cleavable linker, DHF-
linker needed to be synthesized first using approach as shown in Scheme 7.2. Linker 7.7 was
chosen for the synthesis since it was readily available in the lab.

Scheme 7.2. Synthesis of 7,8-DHF-Linker (7.11)
Compound 7.10 was formed through a direct coupling reaction of 7.6 and 7.9 using various
coupling reagents (e.g., EDC, HATU). Among them, HATU gave the best yield of 35%. The
synthesis of the target compound 7.11 from 7.10 was thought to be easily achieved using a well-
studied reaction such as hydrogenolysis catalyzed by Pd/C catalysts. Attempts were made using
many Pd catalysts, such as Pd/C, Pd(OH)2/C, and a 1:1 w/w mixture of both,
22
or hydrogen transfer
reagents (e.g., ammonium formate),
23
yielding inconsistent results. However, when examining
carefully various conditions of successful and failed experiments, it is worth noting that
173

hydrogenolysis was highly sensitive to the concentration of the substrate. When the concentration
of substrate was around 2mM, hydrogenolysis of 7.10 using a catalytic amount (30% w) of a 1:1
w/w mixture of Pd/C and Pd(OH)2/C occurred. With the issue of hydrogenolysis resolved, the
purification of 7.11 using flash column chromatography was found to be another troublesome step.
In particular, during the purification step of employing the flash column, the target compound 7.11
was tailing on the silica column and much of the product was lost. Previous experiences indicated
that this issue could be addressed by the addition of a small amount of acetic acid (0.5% v/v in
DCM). Finally, the desired 7.11 was successfully synthesized and purified using flash column
chromatography, yielding of 16.7 mg. Based on the
1
H NMR spectrum in a 1:1 v/v mixture of d-
chloroform and methanol-d4, an unknown impurity, which signal appears at ~7.9ppm, was
recorded. However, LC-MS analysis of 7.11 showed that the compound was pure without other
impurity detected. Both linker 7.9 and DHF-linker 7.11 were sent for biological assays to evaluate
their neurotrophic activities.
7.6 Synthesis of a BP-DHF conjugate
With DHF-linker 7.11 successfully synthesized, attempts to make BP-DHF were
conducted using approach shown in Scheme 7.3. Risedronate (RIS) was the BP of choice due to
its availability. Compound 7.13 can be easily synthesized using analogous approach of zoledronate
(ZOL) with epichlorohydrin, which was previously reported in Chapter 6.
24
The reaction progress
was monitored using
31
P NMR, and 7.13 was purified using SAX RP-HPLC. Despite many
attempts, including utilization of phase transfer reagents (e.g., tertrabutyl ammonium bromide), to
address the solubility of 7.12 in aqueous solvents, no formation of 7.14 was detected via MS. Thus,
approach using NHS-activated form of 7.16 was employed as shown in Scheme 7.4.
174

Scheme 7.3. Synthesis of RIS-DHF using Route 1
Compound 7.6 was first activated to NHS ester 7.16 in the presence of EDC and N-
hydroxysuccinimide in anhydrous THF (Scheme 7.4). RIS-V2 linker (7.17) was chosen for the
synthesis of the conjugate due to availability, and its synthesis was reported using procedure
previously described in Chapter 3. The coupling reaction between 7.16 and 7.17 was conducted to
yield 7.18. Since solubility of 7.16 in a mixture of water and DMF is significantly low, none of the
expected product, 7.18, was detected. Thus, another approach was required to obtain the target
compound, 7.19.
175

Scheme 7.4. Synthesis of RIS-DHF conjugate using Route 2
Instead of using 7.16, hydrogenolysis was first performed on 7.16 to yield 7.20 (Scheme
7.5). This was followed by the reaction of 7.20 with 7.17 to obtain the desired 7.19. However, the
solubility of 7.20 remained problematic. In a mixture of solvents (e.g., 1:1 v/v DMF and water),
7.20 precipitated from the solution. When the solution became a clear, dark yellow after 30 minutes,
this signaled that 7.20 was hydrolyzed and became 7.1 instead of reacting with 7.17 and forming
the desired RIS-DHF. This observation was confirmed via MS analysis of the reaction mixture.
Scheme 7.5. Synthesis of RIS-DHF (7.19) conjugate using Route 3
176

Since the solubility of 7.16 and 7.20 were problematic in a mixture of organic and aqueous
solvents, the addition of a PEG linker, such as (15-amino)-4,7,10,13-tetraoxapentadecanoic acid,
was hypothesized to improve their solubility in organic/aqueous solvent mixture commonly used
for the coupling of BP with NHS-activated dye (Scheme 7.6). (15-amino)-4,7,10,13-
tetraoxapentadecanoic acid is commercially available in N-Boc protected form, 7.21.

Scheme 7.6. Synthesis of 7.25
The linker 7.21 was needed to be first N-Boc deprotected using TFA in 1:1 v/v in DCM.
The reaction finished after 2 hours and gave quantitative yield. The deprotected linker 7.22 was
then reacted with 7.16, NHS-Bn-DHF to yield 7.23. Unreacted 7.22 can be easily removed from
product 7.22 by extraction with 0.1 M HCl solution. NHS-activated 7.24 was synthesized via
reacting 7.23 with EDC and N-hydroxysuccinimide in the presence of 3 equivalent of distilled
177

TEA. The reaction was washed twice with water and product 7.24 was obtained without further
purification (82%). Purity of the product 7.24 was confirmed via
1
H NMR and MS.
As previously discussed, hydrogenolysis for compounds similar to 7.24 was successfully
achieved when the concentration of substrate was approximately 2 mM in a 1:1 v/v solvent mixture
of MeOH: THF and 30% w/w of a 1:1 w/w mixture of Pd/C and Pd(OH) 2/C. Similar conditions
were utilized to obtain 7.25, verified by
1
H NMR and MS. Due to the unstable nature of 7.25, the
compound was used for the next step without further purification.
Previous attempts using routes 2 and 3 to synthesize 7.19 are summarized in route A
(Scheme 7.7). With previous approaches yielding no success in the formation of 7.19, route 4 was
proposed (Scheme 7.7). Synthesis of 7.19 was achieved by the coupling reaction of RIS-linker
7.26 and 7.25 in a 4:1 v/v mixture of DMF: water at 50
o
C. RIS-linker 7.26 was synthesized using
method previously described in Chapter 3. The compound was dissolved in water and pH of the
solution was adjusted to 8.3 using either sodium carbonate or cesium carbonate.

Scheme 7.7. Synthesis of RIS-DHF (7.19) using route 4.
178

Formation of 7.19 in the reaction mixture was verified via MS. LC-MS analysis of the
reaction mixture was also performed to ensure that extra ions, such as sodium and cesium, do not
interfere with the signal of 7.19. Decomposition of 7.25 in basic, aqueous conditions occurs at a
faster rate than that of 7.25 and 7.26. Solubility of 7.26 in polar organics solvents (e.g., DMF,
DMSO) with different bases (e.g., tetrabutylammonium hydroxide DIEA, DBU) was investigated
to overcome the solubility issue. Despite many attempts, no peak of product 7.19 was detected.
When sodium carbonate was utilized to adjust pH and a mixture of 1:1 v/v water and THF
was employed, the reaction successfully formed the desired product 7.19 (17%). Purification of
the target compound was successfully performed using C18 RP-HPLC with 0.1M
triethylammonium bicarbonate buffers (pH 7.0) containing 10% and 75% of acetonitrile. However,
the compound could not be well characterized using
1
H NMR spectroscopy, especially in the
aromatic area, where most signal peaks were broad. This could be due to the proton of 7-OH
moiety being deprotonated at pH 7 due to its significantly low pKa of a calculated 6.6 (Figure 7.4).
Dissolving the compounds with 70% MeOH, 0.1M ammonium acetate buffer (pH 5.0) solved this
problem. RIS-DHF (7.19) was fully characterized via MS,
1
H and
31
P NMR spectroscopy.

Figure 7.4. pKa calculation of RIS-DHF (7.19) using MarvinSketch.
179

7.7 Neurotrophic activities of a DHF derivative and RIS-DHF conjugate
In vitro neurite outgrowth was conducted at Massachusetts Eye and Ear Infirmary (MEEI)
by Drs. David H. Jung and Judith S. Kempfle. The assays were performed on PLPCreER-tomato
culture of spiral ganglion neurons with the goals of 1) validating the neurotrophic activities of
DHF and their derivatives on hair cells, and (2) quantifying their activities. Nuclei and neurons
were stained with DAPI (blue) and TuJ (red), respectively.

Figure 7.5. In vitro neurite outgrowth. (A) Images of neurite outgrowth in the presence of DHF
and its derivatives, including 7.1 (AX), 7.8 (MX), 7.9 (Carb linker), and 7.11 (DHF linker). (B)
Maximum neurite outgrowth in the presence of DHF and its derivatives at various concentrations
(e.g., 200 and 400 nM). AX, MX, CL, and DL signify 7.1, 7.8, 7.9, 7.11, respectively. (C)
180

Quantification of % neurite outgrowth vs. degree of outgrowth. The degree of outgrowth was
evaluated as the distance that the neural fibers reached.
The nerve cell culture assays were performed at various concentrations, such as 200 and
400 nM. Photomicrograph of the neurons indicated that DHF, its acid derivatives, and DHF linker
did, indeed, induce neural growth (Figure 7.5). This effect was observed in the increasing number
of neuron nuclei, the sprouting of neural fiber, and inner ear nerves, in comparison to the control,
which was treated solely with DMSO (Figure 7.5 A and B). Average maximum neurite outgrowth
indicated that DHF linker exhibited similar neurotrophic effect as seen for DHF and COOH-DHF
at both 200 and 400 nM. The in vitro neurite outgrowth was further quantified at the concentration
of 200 nM. In this experiment, the amount of neurite outgrowth for a set amount of distance was
determined. While the control and the linker did not induce much neurite outgrowth, in the
presence of DHF (7.1) and DHF-linker (7.11), a statistically significant increase of neural fibers
reaching for 200, 300, and 500 µwas observed (Figure 7.5 C).

Figure 7.6. In vitro neurite outgrowth. (A) Images of neurite outgrowth in the presence of DHF
and its derivatives, including 7.11 (DL) and 7.19 (RIS-DHF). (B) Quantification of % neurite
outgrowth vs. degree of outgrowth. The degree of outgrowth was evaluated as the distance that the
neural fibers reached.
181

The assay was further evaluated for RIS-DHF, and its activities were compared with DHF
and 7.11 (DL). The in vitro assay indicated that modifications on DHF, such as the addition of a
carboxyl moiety at the para position (7.1), the attachments of linker (7.11) and linker-RIS (7.19),
still retain the neurotrophic activities of DHF.
7.8 Conclusion
A new derivative of DHF (COOH-DHF) (7.1) and a DHF-RIS (7.19) conjugate were
successfully synthesized. Optimal concentration for in vitro neurite outgrowth is 200 nM. In vitro
experiments performed on SGN culture indicated that the new COOH-DHF (7.1) and RIS-DHF
(7.19) retain neurotrophic activity when compared to the original DHF. The effect on neurite
outgrowth of DHF is still maintained when a linker and a linker-BP are attached to DHF.
7.9 Experimental section
Reagents and Instrumentation
All chemical compounds were commercially available and purchased from either Aldrich
or Alfa Aesar. Risedronate monosodium was a kind gift from Warner Chilcott (formerly known
as P&G Pharmaceuticals). Triethylamine (TEA) was distilled from KOH, and dioxane and THF
were distilled from sodium. 15-(Boc-amino)-4,7,10,13-tetraoxapentadecanoic acid was purchased
from Chem-Impex International, Inc. Other compounds were used as supplied by the manufacturer.
Flash chromatography purification were performed using a Teledyne CombiFlashRf
+
Lumen Isco
system. Thin layer chromatography (TLC) was performed on Merck Silica Gel 60 F254 plates,
and the developed plates were visualized under a UV lamp at 354 nm. HPLC separations were
performed on a Rainan Dynamax Model SD-200 system with a Rainan Dynamax absorbance
detector Model UV-DII. NMR spectra were recorded on either 400 MHz or 500 MHz Varian
182

spectrometers. LC-MS of several of the new dye-risedronate conjugates were obtained on a
Thermo-Finnigan LCQ DECA XP MAX Ion Trap LC/MS/MS spectrometer operated in ESI mode.
Chemical Syntheses
4-(7,8-dihydroxy-4-oxo-4H-chromen-2-yl) benzoic acid (7.1). 69 mg of 7.7 and catalytic
amount of 10% Pd/C (0.1 equivalent) was stirred in 15 mL of a 1:1 v/v mixture of MeOH: THF.
The reaction mixture was heated up to 40
o
C, flushed with N2 gas (3x) and stirred overnight under
H2 at atmospheric pressure. Solid Pd/C was filtered and solvent was removed from the filtrate.
Solid product 7 was recrystallized in methanol and 10.6 mg of pure 7.1 was collected (33%).
1
H NMR (400 MHz, DMSO-d6) δ 13.25 (s, 1H), 10.34 (s, 1H), 9.51 (s, 1H), 8.26 (d, J = 8.7 Hz,
2H), 8.08 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.7 Hz, 1H), 7.09 – 6.86 (m, 2H).
LC-MS (negative ion, M-): tretention = 1.85 min, calcd 298.05 m/z, found [M-H]
-
= 297.11 m/z.
2,3,4-Trisbenzyloxyacetophenone (7.3). In a dry 100mL round bottom flask, 1.7g (10 mmol) of
7.2 was added into a suspension containing 6.08g (35.0 mmol, 3.5 equivalent) of benzyl bromide,
9.0g (58.0 mmol) of potassium carbonate, and 0.3g (1.5 mmol) of potassium iodide, stirred in
20mL of anhydrous DMF. The reaction mixture was heated up to 50
o
C and stirred for 8 hours.
After cooling down to room temperature, solid potassium carbonate was filtered and the reaction
mixture was diluted with 100mL of ethylacetate. The organic layer was washed with water (1x),
brine (1x), and dried over Na2SO4. Purification of 7.3 was performed using column
chromatography with gradient up to 50% ethylacetate in hexane, yielding 3.53g (80%).
1
H NMR (400 MHz, Chloroform-d) δ 7.50 (d, J = 8.8 Hz, 1H), 7.47 – 7.19 (m, 17H), 6.81 (d, J =
8.9 Hz, 1H), 5.16 (d, J = 3.7 Hz, 4H), 5.06 (s, 2H), 2.52 (s, 3H).
13
C NMR (101 MHz, Chloroform-d) δ 198.29, 156.70, 137.12, 136.17, 128.72, 128.64, 128.53,
128.51, 128.32, 128.23, 128.14, 127.52, 127.04, 125.67, 108.83, 76.37, 75.60, 70.91.
183

1-(3,4-bis(benzyloxy)-2-hydroxyphenyl)ethan-1-one (7.5a). In a 100mL round bottom flask, 7.3
was dissolved in a mixture of 20mL of toluene and 4 mL of trifluoroacetic acid (5:1 v/v). The
reaction mixture was stirred for 1.5 hours. Neutralization of the mixture using 2.5M NaOH was
performed. Extraction was performed using ethyl acetate. The organic layer was washed with
water (1X), brine (1x) and dried over Na2SO4. Solvent was removed, and the product was
recrystallized in hexane, yielding 1.06g of 7.5a (90%).
1
H NMR (400 MHz, Chloroform-d) δ 12.64 (s, 1H), 7.83 – 7.12 (m, 10H), 6.50 (d, J = 9.0 Hz,
1H), 5.16 (s, 2H), 5.12 (s, 2H), 2.56 (s, 3H).
13
C NMR (101 MHz, Chloroform-d) δ 203.22, 157.85, 157.52, 136.19, 128.61, 128.55, 128.17,
128.12, 127.89, 127.21, 126.90, 115.40, 104.49, 74.75, 70.73, 26.40.
(E)-4-(3-oxo-3-(2,3,4-tris(benzyloxy)phenyl)prop-1-en-1-yl)benzoic acid (7.5b). Compound
7.3, 476mg (1.09 mmol), was dissolved in 50mL of MeOH and 15mL of 20% KOH was added.
The suspension was heated up to 70
o
C and stirred until the compound was completely dissolved.
207 mg of 4-carboxybenzaldehyde (7.4) (1.31 mmol, 1.2 equivalent), was added into the heated
reaction mixture and stirred for 8 hours. The mixture was cooled down to room temperature and
filtered. The pH of the solution was adjusted to pH 4 with acetic acid. Extraction with ethyl acetate
was performed. The organic layer was washed with water (1x), brine (1x), and dried over Na 2SO4.
Purification of 7.5b were performed using column chromatography with gradient up to 50%
methanol in dichloromethane mixed with 0.5% trifluoroacetic acid, yielding 395mg (64%).
1
H NMR (600 MHz, Chloroform-d) δ 9.56 (s, 4H), 8.03 (d, J = 8.5 Hz, 2H), 7.63 (s, 2H), 7.58 (d,
J = 8.8 Hz, 1H), 7.49 – 7.27 (m, 14H), 7.24 – 7.13 (m, 3H), 6.88 (d, J = 8.9 Hz, 1H), 5.19 (s, 2H),
5.12 (s, 2H), 5.09 (s, 2H).
184

13
C NMR (101 MHz, Chloroform-d) δ 189.90, 171.24, 141.73, 140.97, 136.53, 136.13, 130.55,
130.35, 129.25, 128.73, 128.68, 128.38, 128.30, 128.19, 127.55, 126.98, 126.56, 109.32, 77.00,
70.97.
(E)-4-(3-(3,4-bis(benzyloxy)-2-hydroxyphenyl)-3-oxoprop-1-en-1-yl) benzoic acid (7.6).
Synthesis of 7.6 could be achieved using either route A (in blue) or route B (in red). Here, route
B, which provides higher yield of 7.6, was described. In a 100 mL round bottom flask, 395 mg of
7.5b was dissolved in 20 mL of toluene, followed by the addition of trifluoroacetic acid (TFA)
(5:1 Toluene: TFA v/v). The reaction was stirred for 1.5 hours. Solvent was removed, and
purification of the reaction mixture was performed using flash column chromatography with
gradient up to 50% methanol in dichloromethane mixed with 0.5% trifluoroacetic acid, yielding
242 mg (73%).
1
H NMR (500 MHz, DMSO-d6) δ 13.18 (s, 1H), 8.22 – 8.08 (m, 2H), 8.06 – 7.95 (m, 4H), 7.87
(d, J = 15.4 Hz, 1H), 7.51 – 7.21 (m, 11H), 6.85 (d, J = 9.2 Hz, 1H), 5.30 (s, 2H), 5.00 (s, 2H).
13
C NMR (101 MHz, DMSO-d6) δ 192.81, 167.27, 158.41, 158.11, 143.29, 139.00, 137.98, 135.43,
130.15, 129.57, 128.91, 128.62, 128.51, 128.27, 128.17, 128.04, 123.84, 115.66, 105.40, 74.38,
70.63.
4-(7,8-bis(benzyloxy)-4-oxo-4H-chromen-2-yl) benzoic acid (7.7). 242 mg of 7.6 (0.5 mmol, 1
equivalent) was dissolved in 15 ml of DMSO and catalytic amount of iodine (12.7 mg, 0.1
equivalent) was added into the reaction. The reaction was heated up to 120
o
C and stir for 12 hours.
The reaction mixture was cooled down to room temperature, followed by addition of ice cold water.
Slight yellow precipitate appeared and was filtered. Extraction with EtOAc was performed, and
the organic layer was washed with saturated Na2S2O3, then brine (1x). Solvent was removed, and
185

slight yellow solid (6) was collected and combined with the yellow precipitate, yielding 133.4 mg
(55%). The compound was clean and no further purification is needed.
1
H NMR (400 MHz, DMSO-d6) δ 8.03 (s, 4H), 7.77 (d, J = 9.0 Hz, 1H), 7.54 – 7.50 (m, 2H), 7.47
– 7.29 (m, 9H), 7.01 (s, 1H), 5.35 (s, 2H), 5.19 (s, 2H).
13
C NMR (101 MHz, DMSO-d6) δ 177.02, 162.30, 156.17, 150.61, 137.47, 136.77, 136.04, 129.99,
126.18, 118.53, 79.86, 79.53, 79.20, 75.69, 71.03.
Methyl 4-(7,8-dihydroxy-4-oxo-4H-chromen-2-yl) benzoate (7.8). Unpurified 7.1 recovered
from recrystallization, 30.0 mg, was refluxed in methanol with a catalytic amount of concentrated
H2SO4. The esterified product, 7.8, was precipitated using ethyl acetate, yielding quantitative
amount of pure 7.8, 31.4 mg.
1
H NMR (600 MHz, Methanol-d4) δ 8.22 (d, J = 8.8 Hz, 2H), 8.17 (d, J = 8.8 Hz, 2H), 7.55 (d, J
= 8.7 Hz, 1H), 6.98 (d, J = 8.7 Hz, 1H), 6.90 (s, 1H), 3.94 (s, 3H).
LC-MS (negative ion, M-): tretention = 1.85 min, calcd 312.06 m/z, found [M-H]
-
= 311.4 m/z.
Tert-butyl (6-(4-(7,8-bis(benzyloxy)-4-oxo-4H-chromen-2-yl)benzamido)hexyl)carbamate
(7.10). In a 50 mL round bottom flask, 199.1 mg of 7.6 (0.42 mmol) was dissolved in 24 mL of a
mixture of DCM:THF (1/1 v:v), followed by the addition of 199.6 mg (0.52 mmol, 1.25 eq) of
HATU, and subsequently, 83.7 mg of 7.9 (0.39 mmol, 0.9 eq) was added dropwise. The reaction
mixture was stirred for overnight. Solvent was removed, and the reaction mixture was dissolved
in chloroform, followed by extraction. The organic layer was washed with water (2X), 4.5% citric
acid (5X), saturated sodium bicarbonate (2X), brine (2X) and dried over Na 2SO4. Solvent was
removed, and the product was purified using flash column chromatography with EtOAc: MeOH,
yielding 100 mg of 7.10 (35%).
186

1
H NMR (400 MHz, Chloroform-d) δ 7.90 (d, J = 8.9 Hz, 3H), 7.85 (d, J = 8.6 Hz, 2H), 7.49 –
7.27 (m, 10H), 7.11 (d, J = 9.1 Hz, 1H), 6.80 (s, 1H), 6.73 (s, 1H), 5.27 (s, 2H), 5.18 (s, 2H), 4.60
(s, 1H), 3.46 (s, 2H), 3.14 (s, 2H), 1.82 (s, 1H), 1.71 – 1.57 (m, 2H), 1.57 – 1.31 (m, 17H).
Tert-butyl (6-(4-(7,8-dihydroxy-4-oxo-4H-chromen-2-yl)benzamido)hexyl)carbamate (7.11).
Compound 7.10, 100 mg (0.15 mmol), was dissolved in 70mL of 1:1 v/v mixture of THF and
EtOH, followed by the addition of 30 mg of Pd catalysts, a 1:1 w/w mixture of Pd(OH) 2/C and
Pd/C (30% w/w of reactants). The reaction mixture was flushed under N 2 three times and H2 once
before left stirring under H2 overnight. Catalysts were filtered, and the crude product 7.11 was
collected. Purification was performed using flash column chromatography with gradient up to 50%
methanol in dichloromethane mixed with 0.5% acetic acid, yielding 16.7 mg (23%).
1
H NMR (400 MHz, Methanol-d4) δ 8.12 (d, J = 8.6 Hz, 2H), 7.94 (d, J = 8.6 Hz, 2H), 7.54 (d, J
= 8.8 Hz, 1H), 6.96 (d, J = 8.8 Hz, 1H), 6.79 (s, 1H), 3.39 (d, J = 8.1 Hz, 2H), 3.04 (t, J = 7.0 Hz,
3H), 1.61 (s, 2H), 1.40 (s, 19H).
ESI-MS: calcd 496.22 m/z; found [M-H]
-
= 495.34 m/z.
LC-MS (negative ion, M-): tretention = 1.99 min, calcd 496.22 m/z, found [M-H]
-
= 495.34 m/z.
N-(6-aminohexyl)-4-(7,8-bis(benzyloxy)-4-oxo-4H-chromen-2-yl)benzamide (7.12). In a 25
mL round bottom flask, 31.2 mg of 7.10 (0.046 mmol, 1 equivalent) was dissolved in 6 mL of
DCM and 3 mL of TFA was added into the solution. The reaction was left stirring overnight.
Solvent was removed yielding 25 mg of crude 7.12. (94%) The crude compound was used for the
next step without any further purification.
1
H NMR (400 MHz, Methanol-d4) δ 7.91 (q, J = 8.8 Hz, 5H), 7.80 (d, J = 9.0 Hz, 1H), 7.50 (d, J
= 6.6 Hz, 3H), 7.43 – 7.34 (m, 6H), 7.30 – 7.21 (m, 4H), 6.84 (s, 0H), 6.80 (s, 1H), 5.27 (s, 2H),
187

5.14 (s, 2H), 3.42 (t, J = 7.2 Hz, 3H), 2.94 (t, J = 7.6 Hz, 4H), 1.68 (d, J = 5.8 Hz, 6H), 1.52 – 1.43
(m, 6H), 1.27 (s, 2H).
1-(3-chloro-2-hydroxypropyl)-3-(2-hydroxy-2,2-diphosphonoethyl)pyridin-1-ium (7.13). In a
25 mL round bottom flask, 51.1 mg of RIS monosodium salt (0.168 mmol, 1 equivalent) was
dissolved 1 mL of water and the pH was adjusted to 6.5 using solid sodium carbonate. 66 µL of
epichlorohydrin (0.842 mmol, 5 equivalent) was added to the solution. Upon completion, the
reaction was left stirring overnight and reaction progress was monitored using
31
P NMR. Water
was added into the reaction mixture, and the aqueous layer was washed with ether (3X). Solvent
was removed and the crude reaction mixture was purified using preparative SAX HPLC with
gradient as followed: 0-30% of B for 15 minutes, isocratic at 30% of B for 15 minutes. Flow rate
and detection were set at 8.0 mL/min and 260 nm, respectively. Buffer A and B were water and
0.5 M TEAB (pH 7.8), respectively. Compound 7.13 was collected as triethylammonium salt after
purification (49.7 mg, 1.5 eq of TEA, 56%).
1
H NMR (500 MHz, Deuterium Oxide) δ 8.70 (s, 1H), 8.51 (d, J = 6.3 Hz, 1H), 8.44 (d, J = 8.0
Hz, 1H), 7.81 (dd, J = 7.9, 6.1 Hz, 1H), 4.74 (d, J = 16.4 Hz, 1H), 4.47 (dd, J = 13.6, 9.2 Hz, 1H),
4.33 – 4.22 (m, 1H), 3.66 (dd, J = 8.7, 4.8 Hz, 1H), 3.31 (t, J = 12.5 Hz, 2H).
31
P NMR (162 MHz, Deuterium Oxide) δ 17.46 – 17.29 (m), 16.57 – 15.94 (m), 15.27 (d, J = 18.3
Hz).
2,5-dioxopyrrolidin-1-yl 4-(7,8-bis(benzyloxy)-4-oxo-4H-chromen-2-yl)benzoate (7.16). In a
dry 50 mL flask, 198.9 mg of 7.7 (0.42 mmol, 1 equivalent), 101.6 mg of EDC.HCl (0.53 mmol,
1.25 equivalent), and 64.6 mg of N-hydroxysuccinimide (0.560.56/ mmol, 1.33 equivalent) was
dissolved in 30 ml anhydrous THF. The reaction mixture was heated up to 40
o
C and stirred
188

overnight. Solvent was removed. The collected solid was washed with isopropanol and ether, and
dried under vacuum, yielding 179.6 mg of 8 (79%).
1
H NMR (400 MHz, DMSO-d6) δ 8.25 – 8.13 (m, 4H), 7.78 (d, J = 9.0 Hz, 1H), 7.58 – 7.25 (m,
11H), 7.11 (s, 1H), 5.36 (s, 2H), 5.20 (s, 2H), 2.91 (s, 4H).
1-(1-amino-18-hydroxy-15-oxo-3,6,9,12-tetraoxa-16-azanonadecan-19-yl)-3-(2-hydroxy-
2,2diphosphonoethyl) pyridin-1-ium (7.17). Compound 7.17 was synthesized using previously
described method reported in Chapter 3.
1
H NMR (500 MHz, D2O) δ 8.72 (s, 1H), 8.48 (d, J = 6.1 Hz, 1H), 8.44 (d, J = 8.0 Hz, 1H), 7.80
(dd, J = 7.9, 6.3 Hz, 1H), 4.29 (dd, J = 13.5, 9.6 Hz, 1H), 4.12 (q, J = 6.5 Hz, 1H), 3.70 (t, J = 6.1
Hz, 2H), 3.58 (d, J = 7.1 Hz, 14H), 3.52 – 3.35 (m, 2H), 3.38 – 3.19 (m, 3H), 3.10 (t, J = 5.6 Hz,
1H), 2.96 – 2.88 (m, 2H), 2.49 (t, J = 6.0 Hz, 2H).
31
P NMR (202 MHz, Deuterium Oxide) δ 17.21 – 16.25 (br, 2P).
1-(1-(4-(7,8-dihydroxy-4-oxo-4H-chromen-2-yl)phenyl)-20-hydroxy-1,17-dioxo-5,8,11,14-te-
traoxa-2,18-diazahenicosan-21-yl)-3-(2-hydroxy-2,2-diphosphonoethyl)pyridin-1-ium
(7.19).` In a 25 mL flask, 17.0 mg of 7.25 (0.026 mmol) was dissolved in 800 µL of anhydrous
THF. In a 10 mL flask, 34.4 mg of 7.26 (0.060 mmol, 2.3 equivalent) was dissolved in 800 µL
water and pH was adjusted to pH 8.3 using solid sodium carbonate. Solution of 7.25 was added
dropwise into the solution of 7.26. The reaction mixture was stirred overnight. Solvent was
removed and the crude mixture was dissolved in water. The final DHF-RIS conjugate was purified
using gradient, reversed-phase (RP) HPLC on a C18 column (21.2 mm x 250 mm, 5 μ, 100 A pore
size), flow rate 8.0 mL/min, using 10% MeCN 0.1 M TEAC (pH 7.0) as buffer A and 75% MeCN
0.1 M TEAC (pH 7.8) as buffer B with the gradient increasing to 40% of buffer B for 25 minutes,
then 100% of Buffer B for 75 minutes. UV detection was set at 260nm. Product 7.19 was obtained
189

(3.03 mg, 13%) and redissolved with 70% MeOH, 0.1 M ammonium acetate buffer (pH 5.0).
Characterization of the compound was performed using mass spectrometry,
1
H NMR, and
31
P
NMR spectroscopy.
1
H NMR (400 MHz, D2O) δ 8.82 (s, 1H), 8.60 (dd, J = 26.4, 7.0 Hz, 2H), 8.27 (d, J = 6.9 Hz, 2H),
8.08 (d, J = 7.5 Hz, 2H), 8.01 – 7.86 (m, 1H), 7.39 (s, 1H), 7.26 (d, J = 8.6 Hz, 1H), 6.90 (d, J =
8.5 Hz, 1H), 6.61 (s, 1H), 4.41 (dd, J = 13.2, 9.7 Hz, 1H), 4.21 (s, 1H), 3.85 – 3.02 (m, 26H), 2.91
(d, J = 22.6 Hz, 4H), 2.73 (s, 3H), 2.46 (t, J = 5.9 Hz, 2H).
31
P NMR (162 MHz, Deuterium Oxide) δ 15.6-16.7 (br, 2P).
ESI-MS: calcd for C37H48N3O18P2
+
: 884.74 m/z; found [M-2H]
-
= 882.4 m/z.
2,5-dioxopyrrolidin-1-yl 4-(7,8-dihydroxy-4-oxo-4H-chromen-2-yl)benzoate (7.20). In a dry
100 mL three neck round bottom flask, Compound 6, 82.5 mg (0.143 mmol), was dissolved in 80
mL of 1:1 v/v mixture of THF and EtOH, followed by the addition of 24.3 mg of Pd catalysts, a
1:1 w/w mixture of Pd(OH)2/C and Pd/C (30% w/w of reactants). The reaction mixture was flushed
under N2 (3X) and H2 (1X) before stirring under H2 overnight. Catalysts were filtered and the
crude product 7.20 was collected, yielding 36.0 mg of crude product, which was used for the next
step without purification. (63%)
1
H NMR (400 MHz, DMSO-d6) δ 8.53 – 8.42 (m, 2H), 8.32 (d, J = 8.6 Hz, 2H), 8.26 (s, 1H), 7.48
(d, J = 8.7 Hz, 1H), 7.14 (s, 1H), 7.08 (d, J = 8.6 Hz, 1H), 2.98 (s, 6H).
ESI-MS: 395.06 m/z; found [M-H]
-
= 394.1 m/z.
1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid (7.22). In a 25 mL flask, 40.3 mg (0.11 mmol)
of 7.21 was dissolved in 3 mL of DCM, followed by the addition of 3 mL of trifluoroacetic acid
(DCM:TFA 1:1 v/v) . The reaction mixture was stirred over 2 hours and solvent was removed. The
190

1
H NMR spectrum of product 7.22 was collected to ensure that N-Boc deprotection was completed
(quantitative yield).
1
H NMR (400 MHz, Acetone-d6) δ 12.27 (s, 1H), 7.49 (s, 1H), 3.92 – 3.81 (m, 2H), 3.80 – 3.56
(m, 14H), 3.38 (h, J = 5.7 Hz, 2H), 2.58 (t, J = 30.4, 6.1 Hz, 2H).
1-(4-(7,8-bis(benzyloxy)-4-oxo-4H-chromen-2-yl)phenyl)-1-oxo-5,8,11,14-tetraoxa-2 azahep-
tadecan-17-oic acid (7.23). In a 25 mL flask, 29.1 mg of 7.22 (0.11 mmol) was dissolved in 1 mL
of anhydrous DMF, and pH of the solution was adjusted to basic using freshly distilled TEA. In a
test tube, 61.6 mg of 7.16 (0.107 mmol, 1 equivalent) was dissolved in 2.5 mL of anhydrous DMF.
The solution of 7.16 was added dropwise into solution of 7.22 and stirred for 6 hours. Solvent was
removed, and the reaction mixture was dissolved in 10 mL of chloroform. The organic layer was
washed with 30 mL 0.1M HCl (3X), followed by a wash with brine. The organic layer was dried
over sodium sulfate. Solvent was removed, yielding 17.2 mg of 7.23 (21.5%). The product was
verified via
1
H NMR spectroscopy and was used for the subsequent reaction without further
purification.
1
H NMR (400 MHz, CDCl3) δ 7.92 – 7.79 (m, 4H), 7.47 – 7.21 (m, 11H), 7.06 (d, J = 9.0 Hz, 1H),
6.84 (s, 1H), 5.21 (s, 2H), 5.14 (s, 2H), 3.74 – 3.49 (m, 18H), 2.51 (t, J = 5.8 Hz, 2H).
2,5-dioxopyrrolidin-1-yl-1-(4-(7,8-bis(benzyloxy)-4-oxo-4H-chromen-2-yl)phenyl)-1-oxo-
5,8,11,14-tetraoxa-2-aza-heptadecan-17-oate (7.24). In a 25 mL flask, 17.2 mg of 7.23 (0.024
mmol) was dissolved in 5 mL distilled THF, followed by the addition of 2 equivalents of N-
hydroxysuccinimide (6.25 mg, 0.054 mmol) and EDC.HCl (14.55 mg, 0.076 mmol). The reaction
mixture was stirred overnight. Solvent was removed, and the crude reaction mixture was dissolved
in 15 mL of chloroform. The organic layer was washed with 30 mL of water (1X), followed by a
wash with brine. The organic layer was dried over sodium sulfate and solvent was removed,
191

yielding 16.0 mg of 7.24 (82%). Product 7.24 was verified via
1
H NMR spectroscopy, MS, and
was used for the subsequent reaction without further purification.
1
H NMR (400 MHz, CDCl3) δ 7.89 – 7.78 (m, 5H), 7.44 – 7.21 (m, 10H), 7.11 (s, 1H), 7.05 (d, J
= 9.0 Hz, 1H), 6.70 (s, 1H), 5.21 (s, 2H), 5.14 (s, 2H), 3.71 (t, J = 6.4 Hz, 2H), 3.66 – 3.51 (m,
16H), 2.75 (m, 6H).
ESI-MS: calcd for C45H46N2O13: 822.86 m/z; found [M + Na]
-
= 845.4 m/z
2,5-dioxopyrrolidin-1-yl-1-(4-(7,8-dihydroxy-4-oxo-4H-chromen-2-yl)phenyl)-1-oxo-5,8,11,
14-tetraoxa-2-azaheptadecan-17-oate (7.25). In a 25 mL flask, 16.0 mg of 7.24 (0.019 mmol)
was dissolved in 20 mL of 1:1 v/v mixture of distilled THF and ethanol, followed by the addition
of 30% w/w of Pd catalyst containing a 1:1 w/w mixture of Pd/C and Pd(OH) 2/C (5.0 mg). The
reaction mixture was frozen, thawed, and flushed with nitrogen (3X) and hydrogen (1X) before
left stirred overnight. The catalyst was filtered from the reaction mixture and solvent was removed,
yielding 7.25 (quantitative yield). Formation of 7.25 was verified via
1
H NMR spectroscopy and
MS, and was used for the subsequent reaction without further purification.
1
H NMR (400 MHz, CD3OD) δ 8.04 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.7
Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H), 6.73 (s, 1H), 3.64 – 3.53 (m, 18H), 2.84 – 2.72 (m, 6H).
ESI-MS: calcd for C31H34N2O13: 642.61 m/z; found [M + Na]
-
= 665.5 m/z.
1-(3-amino-2-hydroxypropyl)-3-(2-hydroxy-2,2-diphosphonoethyl)pyridin-1-ium(6).
Compound 7.26 was synthesized using previously reported method.
25
Detail synthesis of the
compound can be found for compound 3.6 in Chapter 3.
1
H NMR (500 MHz, D2O) δ 8.83 (s, 1H), 8.74 (s, 0H), 8.66 (d, J = 6.0 Hz, 1H), 8.56 (d, J = 8.0
Hz, 1H), 7.98 – 7.93 (m, 1H), 4.82 (s, 1H), 4.53 – 4.44 (m, 1H), 4.36 (t, J = 9.7 Hz, 1H), 3.56 (d,
192

J = 7.1 Hz, 1H), 3.48 (t, J = 12.3 Hz, 2H), 3.37 (d, J = 15.2 Hz, 1H), 3.13 – 3.03 (m, 1H).
31
P{
1
H}
NMR (D2O) (202 MHz, D2O): δ 16.77 – 16.48 (m, 2P).
7.10 References
1. Schiller, J. S.; Lucas, J. W.; Ward, B. W.; Peregoy, J. A., Summary health statistics for U.S.
adults: National Health Interview Survey, 2010. Vital. Health Stat. 2012, 252, 1-207.
2. Angeli, S.; Lin, X.; Liu, X. Z., Genetics of hearing and deafness. Anat. Rec. (Hoboken)
2012, 295, 1812-1829.
3. Muller, U.; Barr-Gillespie, P. G., New treatment options for hearing loss. Nat Rev Drug
Discov 2015, 14, 346-365.
4. Schuknecht, H. F.; Gacek, M. R., Cochlear pathology in presbycusis. Ann. Otol. Rhinol.
Laryngol. 1993, 102, 1-16.
5. Kujawa, S. G.; Liberman, M. C., Acceleration of age-related hearing loss by early noise
exposure: evidence of a misspent youth. J. Neurosci. 2006, 26, 2115-2123.
6. Viana, L. M.; O'Malley, J. T.; Burgess, B. J.; Jones, D. D.; Oliveira, C. A.; Santos, F.;
Merchant, S. N.; Liberman, L. D.; Liberman, M. C., Cochlear neuropathy in human presbycusis:
Confocal analysis of hidden hearing loss in post-mortem tissue. Hear. Res. 2015, 327, 78-88.
7. Kujawa, S. G.; Liberman, M. C., Adding insult to injury: cochlear nerve degeneration after
"temporary" noise-induced hearing loss. J. Neurosci. 2009, 29, 14077-14085.
8. Nadol, J. B., Jr., Patterns of neural degeneration in the human cochlea and auditory nerve:
implications for cochlear implantation. Otolaryngol. Head Neck Surg. 1997, 117, 220-228.
9. Wan, G.; Gomez-Casati, M. E.; Gigliello, A. R.; Liberman, M. C.; Corfas, G.,
Neurotrophin-3 regulates ribbon synapse density in the cochlea and induces synapse regeneration
after acoustic trauma. eLife 2014, 3.
10. Choi, Y. H.; Miller, J. M.; Tucker, K. L.; Hu, H.; Park, S. K., Antioxidant vitamins and
magnesium and the risk of hearing loss in the US general population. Am. J. Clin. Nutr. 2014, 99,
148-155.
11. Snider, W. D., Functions of the Neurotrophins during Nervous-System Development -
What the Knockouts Are Teaching Us. Cell 1994, 77, 627-638.
12. Schindowski, K.; Belarbi, K.; Buee, L., Neurotrophic factors in Alzheimer's disease: role
of axonal transport. Genes Brain Behav. 2008, 7 Suppl 1, 43-56.
13. Zuccato, C.; Cattaneo, E., Role of brain-derived neurotrophic factor in Huntington's disease.
Prog. Neurobiol. 2007, 81, 294-330.
193

14. Fumagalli, F.; Racagni, G.; Riva, M. A., Shedding light into the role of BDNF in the
pharmacotherapy of Parkinson's disease. Pharmacogenomics J. 2006, 6, 95-104.
15. Staecker, H.; Gabaizadeh, R.; Federoff, H.; Van De Water, T. R., Brain-derived
neurotrophic factor gene therapy prevents spiral ganglion degeneration after hair cell loss.
Otolaryngol. Head Neck Surg. 1998, 119, 7-13.
16. Jang, S. W.; Liu, X.; Yepes, M.; Shepherd, K. R.; Miller, G. W.; Liu, Y.; Wilson, W. D.;
Xiao, G.; Blanchi, B.; Sun, Y. E.; Ye, K., A selective TrkB agonist with potent neurotrophic
activities by 7,8-dihydroxyflavone. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 2687-2692.
17. Massa, S. M.; Yang, T.; Xie, Y.; Shi, J.; Bilgen, M.; Joyce, J. N.; Nehama, D.; Rajadas, J.;
Longo, F. M., Small molecule BDNF mimetics activate TrkB signaling and prevent neuronal
degeneration in rodents. J. Clin. Invest. 2010, 120, 1774-1785.
18. Trott, O.; Olson, A. J., Software News and Update AutoDock Vina: Improving the Speed
and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and
Multithreading. J. Comput. Chem. 2010, 31, 455-461.
19. Bertrand, T.; Kothe, M.; Liu, J.; Dupuy, A.; Rak, A.; Berne, P. F.; Davis, S.; Gladysheva,
T.; Valtre, C.; Crenne, J. Y.; Mathieu, M., The crystal structures of TrkA and TrkB suggest key
regions for achieving selective inhibition. J. Mol. Biol. 2012, 423, 439-453.
20. Sun, H.; Chen, F.; Wang, X.; Liu, Z.; Yang, Q.; Zhang, X.; Zhu, J.; Qiang, L.; Guo, Q.;
You, Q., Studies on gambogic acid (IV): Exploring structure-activity relationship with IkappaB
kinase-beta (IKKbeta). Eur. J. Med. Chem. 2012, 51, 110-123.
21. Fletcher, S.; Gunning, P. T., Mild, efficient and rapid O-debenzylation of ortho-substituted
phenols with trifluoroacetic acid. Tetrahedron Lett. 2008, 49, 4817-4819.
22. Li, Y.; Manickam, G.; Ghoshal, A.; Subramaniam, P., More efficient palladium catalyst
for hydrogenolysis of benzyl groups. Synth. Commun. 2006, 36, 925-928.
23. Quai, M.; Repetto, C.; Barbaglia, W.; Cereda, E., Fast deprotection of phenoxy benzyl
ethers in transfer hydrogenation assisted by microwave. Tetrahedron Lett. 2007, 48, 1241-1245.
24. Sun, S.; Blazewska, K. M.; Kadina, A. P.; Kashemirov, B. A.; Duan, X.; Triffitt, J. T.;
Dunford, J. E.; Russell, R. G.; Ebetino, F. H.; Roelofs, A. J.; Coxon, F. P.; Lundy, M. W.;
McKenna, C. E., Fluorescent Bisphosphonate and Carboxyphosphonate Probes: A Versatile
Imaging Toolkit for Applications in Bone Biology and Biomedicine. Bioconjug Chem. 2016, 27,
329-340.
25. Kashemirov, B. A.; Bala, J. L.; Chen, X.; Ebetino, F. H.; Xia, Z.; Russell, R. G.; Coxon, F.
P.; Roelofs, A. J.; Rogers, M. J.; McKenna, C. E., Fluorescently labeled risedronate and related
analogues: "magic linker" synthesis. Bioconjug Chem. 2008, 19, 2308-2310.

194

Bibliography
OTO-104. http://www.otonomy.com/pipeline/oto-104/ (accessed 25 April).
OTO-311. http://www.otonomy.com/pipeline/oto-311/ (accessed 25 April).
Clinical Trials. http://www.otonomy.com/pipeline/clinical-trials/ (accessed 25 April).
AM-101. http://www.aurismedical.com/product-candidates/am-101 (accessed 25 April).
Abhishek, A.; Doherty, M., Pathophysiology of articular chondrocalcinosis--role of ANKH. Nat.
Rev. Rheumatol. 2011, 7, 96-104.
Amin, D.; Cornell, S. A.; Gustafson, S. K.; Needle, S. J.; Ullrich, J. W.; Bilder, G. E.; Perrone, M.
H., Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and
cholesterol biosynthesis. J. Lipid Res. 1992, 33, 1657-1663.
Amin, D.; Cornell, S. A.; Perrone, M. H.; Bilder, G. E., 1-Hydroxy-3-(methylpentylamino)-
propylidene-1,1-bisphosphonic acid as a potent inhibitor of squalene synthase.
Arzneimittelforschung 1996, 46, 759-762.
Anderson, H. C.; Harmey, D.; Camacho, N. P.; Garimella, R.; Sipe, J. B.; Tague, S.; Bi, X.;
Johnson, K.; Terkeltaub, R.; Millan, J. L., Sustained osteomalacia of long bones despite major
improvement in other hypophosphatasia-related mineral deficits in tissue nonspecific alkaline
phosphatase/nucleotide pyrophosphatase phosphodiesterase 1 double-deficient mice. Am. J.
Pathol. 2005, 166, 1711-1720.
Angeli, S.; Lin, X.; Liu, X. Z., Genetics of hearing and deafness. Anat. Rec. (Hoboken) 2012, 295,
1812-1829.
Artz, J. D.; Dunford, J. E.; Arrowood, M. J.; Dong, A.; Chruszcz, M.; Kavanagh, K. L.; Minor,
W.; Russell, R. G.; Ebetino, F. H.; Oppermann, U.; Hui, R., Targeting a uniquely nonspecific
prenyl synthase with bisphosphonates to combat cryptosporidiosis. Chem. Biol. 2008, 15, 1296-
1306.
Artz, J. D.; Wernimont, A. K.; Dunford, J. E.; Schapira, M.; Dong, A.; Zhao, Y.; Lew, J.; Russell,
R. G.; Ebetino, F. H.; Oppermann, U.; Hui, R., Molecular characterization of a novel
geranylgeranyl pyrophosphate synthase from Plasmodium parasites. J. Biol. Chem. 2011, 286,
3315-3322.
Aryal, S.; Grailer, J. J.; Pilla, S.; Steeber, D. A.; Gong, S. Q., Doxorubicin conjugated gold
nanoparticles as water-soluble and pH-responsive anticancer drug nanocarriers. J. Mater. Chem.
2009, 19, 7879-7884.
Assael, L. A., Oral bisphosphonates as a cause of bisphosphonate-related osteonecrosis of the jaws:
clinical findings, assessment of risks, and preventive strategies. J. Oral Maxillofac. Surg. 2009,
67, 35-43.
195

Ayoob, A. M.; Borenstein, J. T., The role of intracochlear drug delivery devices in the management
of inner ear disease. Expert Opin. Drug Deliv. 2015, 12, 465-479.
Azuma, Y.; Sato, H.; Oue, Y.; Okabe, K.; Ohta, T.; Tsuchimoto, M.; Kiyoki, M., Alendronate
distributed on bone surfaces inhibits osteoclastic bone resorption in vitro and in experimental
hypercalcemia models. Bone 1995, 16, 235-245.
Balle, V.; Linthicum, F. H., Histologically proven cochlear otosclerosis with pure sensorineural
hearing loss. Ann. Otol. Rhinol. Laryngol. 1984, 93, 105-111.
Baron, R.; Ferrari, S.; Russell, R. G. G., Denosumab and bisphosphonates: Different mechanisms
of action and effects. Bone 2011, 48, 677-692.
Baron, R. A.; Tavare, R.; Figueiredo, A. C.; Blazewska, K. M.; Kashemirov, B. A.; McKenna, C.
E.; Ebetino, F. H.; Taylor, A.; Rogers, M. J.; Coxon, F. P.; Seabra, M. C., Phosphonocarboxylates
inhibit the second geranylgeranyl addition by Rab geranylgeranyl transferase. J. Biol. Chem. 2009,
284, 6861-6868.
Bassett, C. A.; Donath, A.; Macagno, F.; Preisig, R.; Fleisch, H.; Francis, M. D., Diphosphonates
in the treatment of myositis ossificans. Lancet 1969, 2, 845.
Benford, H. L.; Frith, J. C.; Auriola, S.; Monkkonen, J.; Rogers, M. J., Farnesol and
geranylgeraniol prevent activation of caspases by aminobisphosphonates: biochemical evidence
for two distinct pharmacological classes of bisphosphonate drugs. Mol. Pharmacol. 1999, 56, 131-
140.
Benford, H. L.; McGowan, N. W.; Helfrich, M. H.; Nuttall, M. E.; Rogers, M. J., Visualization of
bisphosphonate-induced caspase-3 activity in apoptotic osteoclasts in vitro. Bone 2001, 28, 465-
473.
Bergstrom, J. D.; Bostedor, R. G.; Masarachia, P. J.; Reszka, A. A.; Rodan, G., Alendronate is a
specific, nanomolar inhibitor of farnesyl diphosphate synthase. Arch. Biochem. Biophys. 2000, 373,
231-241.
Bernkop-Schnurch, A.; Dunnhaupt, S., Chitosan-based drug delivery systems. Eur. J. Pharm.
Biopharm. 2012, 81, 463-469.
Bertrand, T.; Kothe, M.; Liu, J.; Dupuy, A.; Rak, A.; Berne, P. F.; Davis, S.; Gladysheva, T.;
Valtre, C.; Crenne, J. Y.; Mathieu, M., The crystal structures of TrkA and TrkB suggest key regions
for achieving selective inhibition. J. Mol. Biol. 2012, 423, 439-453.
Bhushan, K. R.; Tanaka, E.; Frangioni, J. V., Synthesis of conjugatable bisphosphonates for
molecular imaging of large animals. Angew. Chem., Int. Ed. 2007, 46, 7969-7971.
Black, D. M.; Delmas, P. D.; Eastell, R.; Reid, I. R.; Boonen, S.; Cauley, J. A.; Cosman, F.; Lakatos,
P.; Leung, P. C.; Man, Z.; Mautalen, C.; Mesenbrink, P.; Hu, H.; Caminis, J.; Tong, K.; Rosario-
Jansen, T.; Krasnow, J.; Hue, T. F.; Sellmeyer, D.; Eriksen, E. F.; Cummings, S. R.; Trial, H. P.
196

F., Once-yearly zoledronic acid for treatment of postmenopausal osteoporosis. N. Engl. J. Med.
2007, 356, 1809-1822.
Black, D. M.; Kelly, M. P.; Genant, H. K.; Palermo, L.; Eastell, R.; Bucci-Rechtweg, C.; Cauley,
J.; Leung, P. C.; Boonen, S.; Santora, A.; de Papp, A.; Bauer, D. C.; Fracture Intervention Trial
Steering, C.; Committee, H. P. F. T. S., Bisphosphonates and fractures of the subtrochanteric or
diaphyseal femur. N. Engl. J. Med. 2010, 362, 1761-1771.
Black, D. M.; Reid, I. R.; Boonen, S.; Bucci-Rechtweg, C.; Cauley, J. A.; Cosman, F.; Cummings,
S. R.; Hue, T. F.; Lippuner, K.; Lakatos, P.; Leung, P. C.; Man, Z.; Martinez, R. L.; Tan, M.;
Ruzycky, M. E.; Su, G.; Eastell, R., The effect of 3 versus 6 years of zoledronic acid treatment of
osteoporosis: a randomized extension to the HORIZON-Pivotal Fracture Trial (PFT). J. Bone
Miner. Res. 2012, 27, 243-254.
Black, D. M.; Schwartz, A. V.; Ensrud, K. E.; Cauley, J. A.; Levis, S.; Quandt, S. A.; Satterfield,
S.; Wallace, R. B.; Bauer, D. C.; Palermo, L.; Wehren, L. E.; Lombardi, A.; Santora, A. C.;
Cummings, S. R.; Group, F. R., Effects of continuing or stopping alendronate after 5 years of
treatment: the Fracture Intervention Trial Long-term Extension (FLEX): a randomized trial. JAMA
2006, 296, 2927-2938.
Blakley, B. W.; Hochman, J.; Wellman, M.; Gooi, A.; Hussain, A. E., Differences in Ototoxicity
across Species. J. Otolaryngol. Head Neck Surg. 2008, 37, 700-703.
Boonekamp, P. M.; van der Wee-Pals, L. J.; van Wijk-van Lennep, M. M.; Thesing, C. W.; Bijvoet,
O. L., Two modes of action of bisphosphonates on osteoclastic resorption of mineralized matrix.
Bone Miner. 1986, 1, 27-39.
Breuil, V.; Cosman, F.; Stein, L.; Horbert, W.; Nieves, J.; Shen, V.; Lindsay, R.; Dempster, D. W.,
Human osteoclast formation and activity in vitro: effects of alendronate. J. Bone Miner. Res. 1998,
13, 1721-1729.
Briner, W. W.; Francis, M. D., In vitro and in vivo evaluation of anti-calculus agents. Calcif. Tissue
Res. 1973, 11, 10-22.
Burdick, J. A.; Prestwich, G. D., Hyaluronic acid hydrogels for biomedical applications. Adv.
Mater. 2011, 23, H41-56.
Canfield, R.; Rosner, W.; Skinner, J.; McWhorter, J.; Resnick, L.; Feldman, F.; Kammerman, S.;
Ryan, K.; Kunigonis, M.; Bohne, W., Diphosphonate therapy of paget's disease of bone. J. Clin.
Endocrinol. Metab. 1977, 44, 96-106.
Carano, A.; Teitelbaum, S. L.; Konsek, J. D.; Schlesinger, P. H.; Blair, H. C., Bisphosphonates
directly inhibit the bone resorption activity of isolated avian osteoclasts in vitro. J. Clin. Invest.
1990, 85, 456-461.
Ceriani, F.; Mammano, F., Calcium signaling in the cochlea - Molecular mechanisms and
physiopathological implications. Cell Commun. Signal 2012, 10, 20.
197

Chen, T.; Berenson, J.; Vescio, R.; Swift, R.; Gilchick, A.; Goodin, S.; LoRusso, P.; Ma, P.; Ravera,
C.; Deckert, F.; Schran, H.; Seaman, J.; Skerjanec, A., Pharmacokinetics and pharmacodynamics
of zoledronic acid in cancer patients with bone metastases. J. Clin. Pharmacol. 2002, 42, 1228-
1236.
Choi, Y. H.; Miller, J. M.; Tucker, K. L.; Hu, H.; Park, S. K., Antioxidant vitamins and magnesium
and the risk of hearing loss in the US general population. Am. J. Clin. Nutr. 2014, 99, 148-
155.
Chole, R. A.; McKenna, M., Pathophysiology of otosclerosis. Otol. Neurotol. 2001, 22, 249-257.
Clarke, S.; Pinaud, F.; Beutel, O.; You, C.; Piehler, J.; Dahan, M., Covalent monofunctionalization
of peptide-coated quantum dots for single-molecule assays. Nano Lett. 2010, 10, 2147-2154.
Clayton, A. E.; Mikulec, A. A.; Mikulec, K. H.; Merchant, S. N.; McKenna, M. J., Association
between osteoporosis and otosclerosis in women. J. Laryngol. Otol. 2004, 118, 617-621.
Coleman, R. E.; McCloskey, E. V., Bisphosphonates in oncology. Bone 2011, 49, 71-76.
Collins, M. T.; Boehm, M., It ANKH necessarily so. J. Clin. Endocrinol. Metab. 2011, 96, 72-74.
Coppens, I., Targeting lipid biosynthesis and salvage in apicomplexan parasites for improved
chemotherapies. Nat. Rev. Microbiol. 2013, 11, 823-835.
Coscia, M.; Quaglino, E.; Iezzi, M.; Curcio, C.; Pantaleoni, F.; Riganti, C.; Holen, I.; Monkkonen,
H.; Boccadoro, M.; Forni, G.; Musiani, P.; Bosia, A.; Cavallo, F.; Massaia, M., Zoledronic acid
repolarizes tumour-associated macrophages and inhibits mammary carcinogenesis by targeting the
mevalonate pathway. J. Cell Mol. Med. 2010, 14, 2803-2815.
Cosman, F.; Eriksen, E. F.; Recknor, C.; Miller, P. D.; Guanabens, N.; Kasperk, C.; Papanastasiou,
P.; Readie, A.; Rao, H.; Gasser, J. A.; Bucci-Rechtweg, C.; Boonen, S., Effects of intravenous
zoledronic acid plus subcutaneous teriparatide [rhPTH(1-34)] in postmenopausal osteoporosis. J.
Bone Miner. Res. 2011, 26, 503-511.
Coxon, F. P.; Benford, H. L.; Russell, R. G.; Rogers, M. J., Protein synthesis is required for caspase
activation and induction of apoptosis by bisphosphonate drugs. Mol. Pharmacol. 1998, 54, 631-
638.
Coxon, F. P.; Ebetino, F. H.; Mules, E. H.; Seabra, M. C.; McKenna, C. E.; Rogers, M. J.,
Phosphonocarboxylate inhibitors of Rab geranylgeranyl transferase disrupt the prenylation and
membrane localization of Rab proteins in osteoclasts in vitro and in vivo. Bone 2005, 37, 349-358.
Coxon, F. P.; Helfrich, M. H.; Larijani, B.; Muzylak, M.; Dunford, J. E.; Marshall, D.; McKinnon,
A. D.; Nesbitt, S. A.; Horton, M. A.; Seabra, M. C.; Ebetino, F. H.; Rogers, M. J., Identification
of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl transferase that specifically
prevents Rab prenylation in osteoclasts and macrophages. J. Biol. Chem. 2001, 276, 48213-48222.
198

Coxon, F. P.; Helfrich, M. H.; Van't Hof, R.; Sebti, S.; Ralston, S. H.; Hamilton, A.; Rogers, M.
J., Protein geranylgeranylation is required for osteoclast formation, function, and survival:
inhibition by bisphosphonates and GGTI-298. J. Bone Miner. Res. 2000, 15, 1467-1476.
Coxon, F. P.; Thompson, K.; Roelofs, A. J.; Ebetino, F. H.; Rogers, M. J., Visualizing mineral
binding and uptake of bisphosphonate by osteoclasts and non-resorbing cells. Bone 2008, 42, 848-
860.
Cremers, S.; Papapoulos, S., Pharmacology of bisphosphonates. Bone 2011, 49, 42-49.
Croucher, P. I.; McDonald, M. M.; Martin, T. J., Bone metastasis: the importance of the
neighbourhood. Nat. Rev. Cancer 2016, 16, 373-386.
Cummings, S. R.; San Martin, J.; McClung, M. R.; Siris, E. S.; Eastell, R.; Reid, I. R.; Delmas, P.;
Zoog, H. B.; Austin, M.; Wang, A.; Kutilek, S.; Adami, S.; Zanchetta, J.; Libanati, C.; Siddhanti,
S.; Christiansen, C.; Trial, F., Denosumab for prevention of fractures in postmenopausal women
with osteoporosis. N. Engl. J. Med. 2009, 361, 756-765.
David, P.; Nguyen, H.; Barbier, A.; Baron, R., The bisphosphonate tiludronate is a potent inhibitor
of the osteoclast vacuolar H(+)-ATPase. J. Bone Miner. Res. 1996, 11, 1498-1507.
Doherty, J. K.; Linthicum, F. H., Spiral ligament and stria vascularis changes in cochlear
otosclerosis: effect on hearing level. Otol. Neurotol. 2004, 25, 457-464.
Dormer, K. J.; Awasthi, V.; Galbraith, W.; Kopke, R. D.; Chen, K.; Wassel, R., Magnetically-
targeted, technetium 99m-labeled nanoparticles to the inner ear. J. Biomed. Nanotechnol. 2008, 4,
174-184.
Duan, X. PhD. Dissertation, Oxford, 2010.
Dumortier, G.; Grossiord, J. L.; Agnely, F.; Chaumeil, J. C., A review of poloxamer 407
pharmaceutical and pharmacological characteristics. Pharm. Res. 2006, 23, 2709-2728.
Dunford, J. E.; Kwaasi, A. A.; Rogers, M. J.; Barnett, B. L.; Ebetino, F. H.; Russell, R. G.;
Oppermann, U.; Kavanagh, K. L., Structure-activity relationships among the nitrogen containing
bisphosphonates in clinical use and other analogues: time-dependent inhibition of human farnesyl
pyrophosphate synthase. J. Med. Chem. 2008, 51, 2187-2195.
Dunford, J. E.; Thompson, K.; Coxon, F. P.; Luckman, S. P.; Hahn, F. M.; Poulter, C. D.; Ebetino,
F. H.; Rogers, M. J., Structure-activity relationships for inhibition of farnesyl diphosphate synthase
in vitro and inhibition of bone resorption in vivo by nitrogen-containing bisphosphonates. J.
Pharmacol. Exp. Ther. 2001, 296, 235-242.
Ealy, M.; Smith, R. J., Otosclerosis. Adv. Otorhinolaryngol. 2011, 70, 122-129.
Ebetino, F. H.; Hogan, A. M.; Sun, S.; Tsoumpra, M. K.; Duan, X.; Triffitt, J. T.; Kwaasi, A. A.;
Dunford, J. E.; Barnett, B. L.; Oppermann, U.; Lundy, M. W.; Boyde, A.; Kashemirov, B. A.;
199

McKenna, C. E.; Russell, R. G., The relationship between the chemistry and biological activity of
the bisphosphonates. Bone 2011, 49, 20-33.
Ebetino, F. H.; Hogan, A. M. L.; Sun, S. T.; Tsoumpra, M. K.; Duan, X. C.; Triffitt, J. T.; Kwaasi,
A. A.; Dunford, J. E.; Barnett, B. L.; Oppermann, U.; Lundy, M. W.; Boyde, A.; Kashemirov, B.
A.; McKenna, C. E.; Russell, R. G. G., The relationship between the chemistry and biological
activity of the bisphosphonates. Bone 2011, 49, 20-33.
El Kechai, N.; Agnely, F.; Mamelle, E.; Nguyen, Y.; Ferrary, E.; Bochot, A., Recent advances in
local drug delivery to the inner ear. Int. J. Pharm. 2015, 494, 83-101.
Fast, D. K.; Felix, R.; Dowse, C.; Neuman, W. F.; Fleisch, H., The effects of diphosphonates on
the growth and glycolysis of connective-tissue cells in culture. Biochem. J. 1978, 172, 97-107.
Felix, R.; Graham, R.; Russell, G.; Fleisch, H., The effect of several diphosphonates on acid
phosphohydrolases and other lysosomal enzymes. Biochim. Biophys. Acta 1976, 429, 429-438.
Fiering, J. O.; Mescher, M. J.; Pararas, E. E.; Borenstein, J. T.; Sewell, W. F.; Kujawa, S. G.;
McKenna, M. J.; Ernest S. Kim Drug delivery apparatus US20150157837A1, 2015.
Figueiredo, J. L.; Passerotti, C. C.; Sponholtz, T.; Nguyen, H. T.; Weissleder, R., A novel method
of imaging calcium urolithiasis using fluorescence. J Urology 2008, 179, 1610-1614.
Finerman, G. A.; Stover, S. L., Heterotopic ossification following hip replacement or spinal cord
injury. Two clinical studies with EHDP. Metab. Bone Dis. Relat. Res. 1981, 3, 337-342.
Fisher, J. E.; Rogers, M. J.; Halasy, J. M.; Luckman, S. P.; Hughes, D. E.; Masarachia, P. J.;
Wesolowski, G.; Russell, R. G.; Rodan, G. A.; Reszka, A. A., Alendronate mechanism of action:
geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast
formation, bone resorption, and kinase activation in vitro. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
133-138.
Fizazi, K.; Carducci, M.; Smith, M.; Damiao, R.; Brown, J.; Karsh, L.; Milecki, P.; Shore, N.;
Rader, M.; Wang, H.; Jiang, Q.; Tadros, S.; Dansey, R.; Goessl, C., Denosumab versus zoledronic
acid for treatment of bone metastases in men with castration-resistant prostate cancer: a
randomised, double-blind study. Lancet 2011, 377, 813-822.
Flanagan, A. M.; Chambers, T. J., Dichloromethylenebisphosphonate (Cl2mbp) Inhibits Bone-
Resorption through Injury to Osteoclasts That Resorb Cl2mbp-Coated Bone. Bone Miner. 1989,
6, 33-43.
Flanagan, A. M.; Chambers, T. J., Dichloromethylenebisphosphonate (Cl2MBP) inhibits bone
resorption through injury to osteoclasts that resorb Cl2MBP-coated bone. Bone Miner. 1989, 6,
33-43.
Fleisch, H.; Bisaz, S., Isolation from urine of pyrophosphate, a calcification inhibitor. Am. J.
Physiol. 1962, 203, 671-675.
200

Fleisch, H.; Russell, R. G.; Francis, M. D., Diphosphonates inhibit hydroxyapatite dissolution in
vitro and bone resorption in tissue culture and in vivo. Science 1969, 165, 1262-1264.
Fleisch, H.; Russell, R. G.; Straumann, F., Effect of pyrophosphate on hydroxyapatite and its
implications in calcium homeostasis. Nature 1966, 212, 901-903.
Fleish, H.; Neuman, W. F., Mechanisms of calcification: role of collagen, polyphosphates, and
phosphatase. Am. J. Physiol. 1961, 200, 1296-1300.
Fletcher, S.; Gunning, P. T., Mild, efficient and rapid O-debenzylation of ortho-substituted phenols
with trifluoroacetic acid. Tetrahedron Lett. 2008, 49, 4817-4819.
Flora, L.; Hassing, G. S.; Cloyd, G. G.; Bevan, J. A.; Parfitt, A. M.; Villanueva, A. R., The long-
term skeletal effects of EHDP in dogs. Metab. Bone Dis. Relat. Res. 1981, 3, 289-300.
Fournier, P. G.; Daubine, F.; Lundy, M. W.; Rogers, M. J.; Ebetino, F. H.; Clezardin, P., Lowering
bone mineral affinity of bisphosphonates as a therapeutic strategy to optimize skeletal tumor
growth inhibition in vivo. Cancer Res. 2008, 68, 8945-8953.
Francis, M. D., The inhibition of calcium hydroxypatite crystal growth by polyphosphonates and
polyphosphates. Calcif. Tissue Res. 1969, 3, 151-162.
Francis, M. D.; Briner, W. W., The effect of phosphonates on dental enamel in vitro and calculus
formation in vivo. Calcif. Tissue Res. 1973, 11, 1-9.
Francis, M. D.; Flora, L.; King, W. R., The effects of disodium ethane-1-hydroxy-1, 1-
diphosphonate on adjuvant induced arthritis in rats. Calcif. Tissue Res. 1972, 9, 109-121.
Francis, M. D.; Gray, J. A.; Griebstein, W. J., The formation and influence of surface phases on
calcium phosphate solids. Adv. Oral Biol. 1968, 3, 83-120.
Francis, M. D.; Russell, R. G.; Fleisch, H., Diphosphonates inhibit formation of calcium phosphate
crystals in vitro and pathological calcification in vivo. Science 1969, 165, 1264-1266.
Francis, M. D.; Valent, D. J., Historical perspectives on the clinical development of
bisphosphonates in the treatment of bone diseases. J. Musculoskelet. Neuronal. Interact. 2007, 7,
2-8.
Frith, J. C.; Monkkonen, J.; Blackburn, G. M.; Russell, R. G.; Rogers, M. J., Clodronate and
liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5'-(beta,
gamma-dichloromethylene) triphosphate, by mammalian cells in vitro. J. Bone Miner. Res. 1997,
12, 1358-1367.
Fumagalli, F.; Racagni, G.; Riva, M. A., Shedding light into the role of BDNF in the
pharmacotherapy of Parkinson's disease. Pharmacogenomics J. 2006, 6, 95-104.
201

Garnero, P.; Shih, W. J.; Gineyts, E.; Karpf, D. B.; Delmas, P. D., Comparison of new biochemical
markers of bone turnover in late postmenopausal osteoporotic women in response to alendronate
treatment. J. Clin. Endocrinol. Metab. 1994, 79, 1693-1700.
Gasser, J. A.; Ingold, P.; Venturiere, A.; Shen, V.; Green, J. R., Long-term protective effects of
zoledronic acid on cancellous and cortical bone in the ovariectomized rat. J. Bone Miner. Res.
2008, 23, 544-551.
Gibbs, S. L., Near infrared fluorescence for image-guided surgery. Quant. Imaging Med. Surg.
2012, 2, 177-187.
Glorieux, F. H.; Bishop, N. J.; Plotkin, H.; Chabot, G.; Lanoue, G.; Travers, R., Cyclic
administration of pamidronate in children with severe osteogenesis imperfecta. N. Engl. J. Med.
1998, 339, 947-952.
Gober, H. J.; Kistowska, M.; Angman, L.; Jeno, P.; Mori, L.; De Libero, G., Human T cell receptor
gammadelta cells recognize endogenous mevalonate metabolites in tumor cells. J. Exp. Med. 2003,
197, 163-168.
Grbic, J. T.; Black, D. M.; Lyles, K. W.; Reid, D. M.; Orwoll, E.; McClung, M.; Bucci-Rechtweg,
C.; Su, G., The incidence of osteonecrosis of the jaw in patients receiving 5 milligrams of
zoledronic acid: data from the health outcomes and reduced incidence with zoledronic acid once
yearly clinical trials program. J. Am. Dent. Assoc. 2010, 141, 1365-1370.
Grbic, J. T.; Landesberg, R.; Lin, S. Q.; Mesenbrink, P.; Reid, I. R.; Leung, P. C.; Casas, N.;
Recknor, C. P.; Hua, Y.; Delmas, P. D.; Eriksen, E. F.; Health, O.; Reduced Incidence with
Zoledronic Acid Once Yearly Pivotal Fracture Trial Research, G., Incidence of osteonecrosis of
the jaw in women with postmenopausal osteoporosis in the health outcomes and reduced incidence
with zoledronic acid once yearly pivotal fracture trial. J. Am. Dent. Assoc. 2008, 139, 32-40.
Green, J. R.; Muller, K.; Jaeggi, K. A., Preclinical pharmacology of CGP 42'446, a new, potent,
heterocyclic bisphosphonate compound. J. Bone Miner. Res. 1994, 9, 745-751.
Gross, D.; Williams, W. S., Streaming potential and the electromechanical response of
physiologically-moist bone. J. Biomech. 1982, 15, 277-295.
Grove, J. E.; Brown, R. J.; Watts, D. J., The intracellular target for the antiresorptive
aminobisphosphonate drugs in Dictyostelium discoideum is the enzyme farnesyl diphosphate
synthase. J. Bone Miner. Res. 2000, 15, 971-981.
Guitton, M.; Puel, J.-L.; Pujol, R.; Ruel, J.; Wang, J. Methods using NMDA receptor antagonists
for the treatment of tinnitus induced by cochlear excitotoxicity. US20150265552A1, 2015.
Guo, Z.; Park, S.; Yoon, J.; Shin, I., Recent progress in the development of near-infrared
fluorescent probes for bioimaging applications. Chem. Soc. Rev. 2014, 43, 16-29.
202

Hahn, H.; Salt, A. N.; Biegner, T.; Kammerer, B.; Delabar, U.; Hartsock, J. J.; Plontke, S. K.,
Dexamethasone levels and base-to-apex concentration gradients in the scala tympani perilymph
after intracochlear delivery in the guinea pig. Otol. Neurotol. 2012, 33, 660-665.
Halasy-Nagy, J. M.; Rodan, G. A.; Reszka, A. A., Inhibition of bone resorption by alendronate
and risedronate does not require osteoclast apoptosis. Bone 2001, 29, 553-559.
Hall, A., Rho GTPases and the actin cytoskeleton. Science 1998, 279, 509-514.
Hamid, M.; Trune, D., Issues, indications, and controversies regarding intratympanic steroid
perfusion. Curr. Opin. Otolaryngol. Head Neck Surg. 2008, 16, 434-440.
Han, D. Y.; Harada, N.; Tomoda, K.; Yamashita, T., Characterization of the calcium influx
induced by depolarization of guinea pig cochlear spiral ganglion cells. ORL J. Otorhinolaryngol.
Relat. Spec. 1994, 56, 125-129.
Harris, S. T.; Watts, N. B.; Genant, H. K.; McKeever, C. D.; Hangartner, T.; Keller, M.; Chesnut,
C. H., 3rd; Brown, J.; Eriksen, E. F.; Hoseyni, M. S.; Axelrod, D. W.; Miller, P. D., Effects of
risedronate treatment on vertebral and nonvertebral fractures in women with postmenopausal
osteoporosis: a randomized controlled trial. Vertebral Efficacy With Risedronate Therapy (VERT)
Study Group. JAMA 1999, 282, 1344-1352.
Hashemi, J.; Shahfarhat, A.; Beheshtian, A., Fibrodysplasia ossificans progressiva: report of a case
and review of articles. Iran J. Radiol. 2011, 8, 113-117.
Heldin, P.; Asplund, T.; Ytterberg, D.; Thelin, S.; Laurent, T. C., Characterization of the molecular
mechanism involved in the activation of hyaluronan synthetase by platelet-derived growth factor
in human mesothelial cells. Biochem. J. 1992, 283 ( Pt 1), 165-170.
Henneman, Z. J.; Nancollas, G. H.; Ebetino, F. H.; Russell, R. G.; Phipps, R. J., Bisphosphonate
binding affinity as assessed by inhibition of carbonated apatite dissolution in vitro. J. Biomed.
Mater. Res. A 2008, 85, 993-1000.
Hiroi-Furuya, E.; Kameda, T.; Hiura, K.; Mano, H.; Miyazawa, K.; Nakamaru, Y.; Watanabe-
Mano, M.; Okuda, N.; Shimada, J.; Yamamoto, Y.; Hakeda, Y.; Kumegawa, M., Etidronate
(EHDP) inhibits osteoclastic-bone resorption, promotes apoptosis and disrupts actin rings in
isolate-mature osteoclasts. Calcif. Tissue Int. 1999, 64, 219-223.
Hokugo, A.; Sun, S. T.; Park, S.; McKenna, C. E.; Nishimura, I., Equilibrium-dependent
bisphosphonate interaction with crystalline bone mineral explains anti-resorptive
pharmacokinetics and prevalence of osteonecrosis of the jaw in rats. Bone 2013, 53, 59-68.
Hoskison, E.; Daniel, M.; Al-Zahid, S.; Shakesheff, K. M.; Bayston, R.; Birchall, J. P., Drug
delivery to the ear. Ther. Deliv. 2013, 4, 115-124.
Hu, A.; Parnes, L. S., Intratympanic steroids for inner ear disorders: a review. Audiol. Neurootol.
2009, 14, 373-382.
203

Hu, L.; Sun, Y.; Wu, Y., Advances in chitosan-based drug delivery vehicles. Nanoscale 2013, 5,
3103-3111.
Hughes, D. E.; MacDonald, B. R.; Russell, R. G.; Gowen, M., Inhibition of osteoclast-like cell
formation by bisphosphonates in long-term cultures of human bone marrow. J. Clin. Invest. 1989,
83, 1930-1935.
Hughes, D. E.; Wright, K. R.; Uy, H. L.; Sasaki, A.; Yoneda, T.; Roodman, G. D.; Mundy, G. R.;
Boyce, B. F., Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J.
Bone Miner. Res. 1995, 10, 1478-1487.
Humandiagram.info Blank Ear Diagram. http://humandiagram.info/how-important-ear-diagram-
labeled-is/blank-ear-diagram/#main (accessed April 26, 2016).
Ibrahim, A.; Scher, N.; Williams, G.; Sridhara, R.; Li, N.; Chen, G.; Leighton, J.; Booth, B.;
Gobburu, J. V.; Rahman, A.; Hsieh, Y.; Wood, R.; Vause, D.; Pazdur, R., Approval summary for
zoledronic acid for treatment of multiple myeloma and cancer bone metastases. Clin. Cancer Res.
2003, 9, 2394-2399.
Igarashi, M.; Ohashi, K.; Ishii, M., Morphometric comparison of endolymphatic and perilymphatic
spaces in human temporal bones. Acta Otolaryngol. 1986, 101, 161-164.
Ito, M.; Chokki, M.; Ogino, Y.; Satomi, Y.; Azuma, Y.; Ohta, T.; Kiyoki, M., Comparison of
cytotoxic effects of bisphosphonates in vitro and in vivo. Calcif. Tissue Int. 1998, 63, 143-147.
Iyer, G.; Pinaud, F.; Xu, J.; Ebenstein, Y.; Li, J.; Chang, J.; Dahan, M.; Weiss, S., Aromatic
aldehyde and hydrazine activated peptide coated quantum dots for easy bioconjugation and live
cell imaging. Bioconjug Chem. 2011, 22, 1006-1011.
Jaffer, F. A.; Libby, P.; Weissleder, R., Optical and multimodality molecular imaging: insights
into atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 2009, 29, 1017-1024.
Jahnke, W.; Henry, C., An in vitro assay to measure targeted drug delivery to bone mineral.
ChemMedChem 2010, 5, 770-776.
Jain, N.; Weinstein, R. S., Giant osteoclasts after long-term bisphosphonate therapy: diagnostic
challenges. Nat. Rev. Rheumatol. 2009, 5, 341-346.
Jang, S. W.; Liu, X.; Yepes, M.; Shepherd, K. R.; Miller, G. W.; Liu, Y.; Wilson, W. D.; Xiao, G.;
Blanchi, B.; Sun, Y. E.; Ye, K., A selective TrkB agonist with potent neurotrophic activities by
7,8-dihydroxyflavone. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 2687-2692.
Janner, M.; Muhlbauer, R. C.; Fleisch, H., Sodium EDTA enhances intestinal absorption of two
bisphosphonates. Calcif. Tissue Int. 1991, 49, 280-283.
Jauhiainen, M.; Monkkonen, H.; Raikkonen, J.; Monkkonen, J.; Auriola, S., Analysis of
endogenous ATP analogs and mevalonate pathway metabolites in cancer cell cultures using liquid
204

chromatography-electrospray ionization mass spectrometry. J. Chromatogr. B Analyt. Technol.
Biomed. Life Sci. 2009, 877, 2967-2975.
Jung, A.; Bisaz, S.; Gebauer, U.; Russell, R. G.; Morgan, D. B.; Fleisch, H., The uptake and
metabolism of (32-P)pyrophosphate by mouse calvaria in vitro. Biochem. J. 1974, 140, 175-183.
Kang, W. S.; Sun, S.; Nguyen, K.; Kashemirov, B.; McKenna, C. E.; Hacking, S. A.; Quesnel, A.
M.; Sewell, W. F.; McKenna, M. J.; Jung, D. H., Non-Ototoxic Local Delivery of Bisphosphonate
to the Mammalian Cochlea. Otol. Neurotol. 2015, 36, 953-960.
Kavanagh, K. L.; Dunford, J. E.; Bunkoczi, G.; Russell, R. G.; Oppermann, U., The crystal
structure of human geranylgeranyl pyrophosphate synthase reveals a novel hexameric arrangement
and inhibitory product binding. J. Biol. Chem. 2006, 281, 22004-22012.
Kavanagh, K. L.; Guo, K.; Dunford, J. E.; Wu, X.; Knapp, S.; Ebetino, F. H.; Rogers, M. J.; Russell,
R. G.; Oppermann, U., The molecular mechanism of nitrogen-containing bisphosphonates as
antiosteoporosis drugs. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 7829-7834.
Keegan, M. E.; McKenna, M. J. Drug-eluting staples prosthesis. US20150320551A1, 2015.
Keller, R. K.; Fliesler, S. J., Mechanism of aminobisphosphonate action: characterization of
alendronate inhibition of the isoprenoid pathway. Biochem. Biophys. Res. Commun. 1999, 266,
560-563.
Khan, A. A.; Morrison, A.; Hanley, D. A.; Felsenberg, D.; McCauley, L. K.; O'Ryan, F.; Reid, I.
R.; Ruggiero, S. L.; Taguchi, A.; Tetradis, S.; Watts, N. B.; Brandi, M. L.; Peters, E.; Guise, T.;
Eastell, R.; Cheung, A. M.; Morin, S. N.; Masri, B.; Cooper, C.; Morgan, S. L.; Obermayer-Pietsch,
B.; Langdahl, B. L.; Al Dabagh, R.; Davison, K. S.; Kendler, D. L.; Sandor, G. K.; Josse, R. G.;
Bhandari, M.; El Rabbany, M.; Pierroz, D. D.; Sulimani, R.; Saunders, D. P.; Brown, J. P.;
Compston, J.; International Task Force on Osteonecrosis of the, J., Diagnosis and management of
osteonecrosis of the jaw: a systematic review and international consensus. J. Bone Miner. Res.
2015, 30, 3-23.
Khan, S. A.; Kanis, J. A.; Vasikaran, S.; Kline, W. F.; Matuszewski, B. K.; McCloskey, E. V.;
Beneton, M. N.; Gertz, B. J.; Sciberras, D. G.; Holland, S. D.; Orgee, J.; Coombes, G. M.; Rogers,
S. R.; Porras, A. G., Elimination and biochemical responses to intravenous alendronate in
postmenopausal osteoporosis. J. Bone Miner. Res. 1997, 12, 1700-1707.
Khetarpal, U.; Schuknecht, H. F., In search of pathologic correlates for hearing loss and vertigo in
Paget's disease. A clinical and histopathologic study of 26 temporal bones. Ann. Otol. Rhinol.
Laryngol. Suppl. 1990, 145, 1-16.
Khoo, X.; Simons, E. J.; Chiang, H. H.; Hickey, J. M.; Sabharwal, V.; Pelton, S. I.; Rosowski, J.
J.; Langer, R.; Kohane, D. S., Formulations for trans-tympanic antibiotic delivery. Biomaterials
2013, 34, 1281-1288.
205

Kim, B.; Han, G.; Toley, B. J.; Kim, C. K.; Rotello, V. M.; Forbes, N. S., Tuning payload delivery
in tumour cylindroids using gold nanoparticles. Nat. Nanotechnol. 2010, 5, 465-472.
Klein, G.; Martin, J. B.; Satre, M., Methylenediphosphonate, a Metabolic Poison in Dictyostelium-
Discoideum - P-31 Nmr Evidence for Accumulation of Adenosine 5'-(Beta,Gamma-
Methylenetriphosphate) and Diadenosine 5',5'''-P1,P4-(P2,P3-Methylenetetraphosphate).
Biochemistry-Us 1988, 27, 1897-1901.
Klickstein, L. CGF166 Atonal Gene Therapy for Hearing Loss & Vestibular Dysfunction: Review
of NIH OBA protocol #1310-1260.
http://osp.od.nih.gov/sites/default/files/1_1260_CGF166_Klickstein.pdf (accessed April 25).
Klouda, L., Thermoresponsive hydrogels in biomedical applications: A seven-year update. Eur. J.
Pharm. Biopharm. 2015, 97, 338-349.
Kogan, G.; Soltes, L.; Stern, R.; Gemeiner, P., Hyaluronic acid: a natural biopolymer with a broad
range of biomedical and industrial applications. Biotechnol. Lett. 2007, 29, 17-25.
Konstantinopoulos, P. A.; Karamouzis, M. V.; Papavassiliou, A. G., Post-translational
modifications and regulation of the RAS superfamily of GTPases as anticancer targets. Nat. Rev.
Drug Discov. 2007, 6, 541-555.
Kopke, R. D.; Wassel, R. A.; Mondalek, F.; Grady, B.; Chen, K.; Liu, J.; Gibson, D.; Dormer, K.
J., Magnetic nanoparticles: inner ear targeted molecule delivery and middle ear implant. Audiol.
Neurootol. 2006, 11, 123-133.
Kozloff, K. M.; Volakis, L. I.; Marini, J. C.; Caird, M. S., Near-infrared fluorescent probe traces
bisphosphonate delivery and retention in vivo. J. Bone Miner. Res. 2010, 25, 1748-1758.
Kroemer, G.; Reed, J. C., Mitochondrial control of cell death. Nat. Med. 2000, 6, 513-519.
Kucuk, B.; Abe, K.; Ushiki, T.; Inuyama, Y.; Fukuda, S.; Ishikawa, K., Microstructures of the
bony modiolus in the human cochlea: a scanning electron microscopic study. J. Electron Microsc.
1991, 40, 193-197.
Kujawa, S. G.; Liberman, M. C., Acceleration of age-related hearing loss by early noise exposure:
evidence of a misspent youth. J. Neurosci. 2006, 26, 2115-2123.
Kujawa, S. G.; Liberman, M. C., Adding insult to injury: cochlear nerve degeneration after
"temporary" noise-induced hearing loss. J. Neurosci. 2009, 29, 14077-14085.
Kwek, E. B.; Goh, S. K.; Koh, J. S.; Png, M. A.; Howe, T. S., An emerging pattern of
subtrochanteric stress fractures: a long-term complication of alendronate therapy? Injury 2008, 39,
224-231.
206

Lajud, S. A.; Han, Z.; Chi, F. L.; Gu, R.; Nagda, D. A.; Bezpalko, O.; Sanyal, S.; Bur, A.; Han, Z.;
O'Malley, B. W.; Li, D., A regulated delivery system for inner ear drug application. J. Control
Release 2013, 166, 268-276.
Lal, S.; Clare, S. E.; Halas, N. J., Nanoshell-enabled photothermal cancer therapy: impending
clinical impact. Acc. Chem. Res. 2008, 41, 1842-1851.
Lalwani, A. K.; Kysar, J. W. System and method to locally deliver therapeutic agent to inner ear.
US20150265824A1, 2015.
Lawson, M. A.; Xia, Z.; Barnett, B. L.; Triffitt, J. T.; Phipps, R. J.; Dunford, J. E.; Locklin, R. M.;
Ebetino, F. H.; Russell, R. G., Differences between bisphosphonates in binding affinities for
hydroxyapatite. J. Biomed. Mater. Res. B Appl. Biomater. 2010, 92, 149-155.
Leder, B. Z.; Tsai, J. N.; Uihlein, A. V.; Burnett-Bowie, S. A.; Zhu, Y.; Foley, K.; Lee, H.; Neer,
R. M., Two years of Denosumab and teriparatide administration in postmenopausal women with
osteoporosis (The DATA Extension Study): a randomized controlled trial. J. Clin. Endocrinol.
Metab. 2014, 99, 1694-1700.
Lehenkari, P. P.; Kellinsalmi, M.; Napankangas, J. P.; Ylitalo, K. V.; Monkkonen, J.; Rogers, M.
J.; Azhayev, A.; Vaananen, H. K.; Hassinen, I. E., Further insight into mechanism of action of
clodronate: inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable, adenine-
containing metabolite. Mol. Pharmacol. 2002, 61, 1255-1262.
Lerner, U. H.; Larsson, A., Effects of four bisphosphonates on bone resorption, lysosomal enzyme
release, protein synthesis and mitotic activities in mouse calvarial bones in vitro. Bone 1987, 8,
179-189.
Leu, C. T.; Luegmayr, E.; Freedman, L. P.; Rodan, G. A.; Reszka, A. A., Relative binding affinities
of bisphosphonates for human bone and relationship to antiresorptive efficacy. Bone 2006, 38,
628-636.
Li, Y.; Manickam, G.; Ghoshal, A.; Subramaniam, P., More efficient palladium catalyst for
hydrogenolysis of benzyl groups. Synth. Commun. 2006, 36, 925-928.
Lichter, J.; Vollrath, B.; Duron, S. G.; Lebel, C.; Piu, F.; Ye, Q.; Dellamary, L. A.; Trammel, A.
M.; Scaife, M. C.; Harris, J. P. Controlled release antimicrobial compositions and methods for the
treatment of otic disorders. US20150150793A1, 2015.
Lichter, J.; Vollrath, B.; Trammel, A. M.; Duron, S. G.; Piu, F.; Dellamary, L. A.; Ye, Q.; Lebel,
C.; Scaife, M. C.; Harris, J. P. Controlled-release ion channel modulator compositions and methods
for the treatment of otic disorders. US20130150410A1, 2015.
Lichter, J.; Vollrath, B.; Trammel, A. M.; Duron, S. G.; Piu, F.; Dellamary, L. A.; Ye, Q.; Lebel,
C.; Scaife, M. C.; Harris, J. P. Controlled release auris sensory cell modulator compositions and
methods for the treatment of otic disorders. US20150313839A1, 2015.
207

Lichter, J.; Vollrath, B.; Trammel, A. M.; Duron, S. G.; Scaife, M. C.; Piu, F.; Harris, J. P.
Sustained release corticosteroid compositions for treatment of otic disorders. US20150313838A1,
2015.
Liktor, B.; Szekanecz, Z.; Batta, T. J.; Sziklai, I.; Karosi, T., Perspectives of pharmacological
treatment in otosclerosis. Eur. Arch. Otorhinolaryngol. 2013, 270, 793-804.
Lin, J. H.; Duggan, D. E.; Chen, I. W.; Ellsworth, R. L., Physiological disposition of alendronate,
a potent anti-osteolytic bisphosphonate, in laboratory animals. Drug Metab. Dispos. 1991, 19, 926-
932.
Ling, Y.; Sahota, G.; Odeh, S.; Chan, J. M.; Araujo, F. G.; Moreno, S. N.; Oldfield, E.,
Bisphosphonate inhibitors of Toxoplasma gondi growth: in vitro, QSAR, and in vivo
investigations. J. Med. Chem. 2005, 48, 3130-3140.
Lipton, A., New therapeutic agents for the treatment of bone diseases. Expert Opin. Biol. Ther.
2005, 5, 817-832.
Logothetis, C. J.; Navone, N. M.; Lin, S. H., Understanding the biology of bone metastases: key
to the effective treatment of prostate cancer. Clin. Cancer Res. 2008, 14, 1599-1602.
Loo, C.; Lin, A.; Hirsch, L.; Lee, M. H.; Barton, J.; Halas, N.; West, J.; Drezek, R., Nanoshell-
enabled photonics-based imaging and therapy of cancer. Technol. Cancer Res. Treat. 2004, 3, 33-
40.
Lopez-Escamez, J. A.; Carey, J.; Chung, W. H.; Goebel, J. A.; Magnusson, M.; Mandala, M.;
Newman-Toker, D. E.; Strupp, M.; Suzuki, M.; Trabalzini, F.; Bisdorff, A., Diagnostic criteria for
Meniere's disease. Consensus document of the Barany Society, the Japan Society for Equilibrium
Research, the European Academy of Otology and Neurotology (EAONO), the American Academy
of Otolaryngology-Head and Neck Surgery (AAO-HNS) and the Korean Balance Society. Acta
Otorrinolaringol. Esp. 2016, 67, 1-7.
Lowik, C. W. G. M.; Vanderpluijm, G.; Vanderweepals, L. J. A.; Vantreslongdegroot, H. B.;
Bijvoet, O. L. M., Migration and Phenotypic Transformation of Osteoclast Precursors into Mature
Osteoclasts - the Effect of a Bisphosphonate. J. Bone Miner. Res. 1988, 3, 185-192.
Luckman, S. P.; Coxon, F. P.; Ebetino, F. H.; Russell, R. G.; Rogers, M. J., Heterocycle-containing
bisphosphonates cause apoptosis and inhibit bone resorption by preventing protein prenylation:
evidence from structure-activity relationships in J774 macrophages. J. Bone Miner. Res. 1998, 13,
1668-1678.
Luckman, S. P.; Hughes, D. E.; Coxon, F. P.; Graham, R.; Russell, G.; Rogers, M. J., Nitrogen-
containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational
prenylation of GTP-binding proteins, including Ras. J. Bone Miner. Res. 1998, 13, 581-589.
208

Maalouf, M. A.; Wiemer, A. J.; Kuder, C. H.; Hohl, R. J.; Wiemer, D. F., Synthesis of fluorescently
tagged isoprenoid bisphosphonates that inhibit protein geranylgeranylation. Bioorg. Med. Chem.
2007, 15, 1959-1966.
Manfredi, M.; Merigo, E.; Guidotti, R.; Meleti, M.; Vescovi, P., Bisphosphonate-related
osteonecrosis of the jaws: a case series of 25 patients affected by osteoporosis. Int. J. Oral
Maxillofac. Surg. 2011, 40, 277-284.
Marma, M. S.; Xia, Z.; Stewart, C.; Coxon, F.; Dunford, J. E.; Baron, R.; Kashemirov, B. A.;
Ebetino, F. H.; Triffitt, J. T.; Russell, R. G.; McKenna, C. E., Synthesis and biological evaluation
of alpha-halogenated bisphosphonate and phosphonocarboxylate analogues of risedronate. J. Med.
Chem. 2007, 50, 5967-5975.
Marshall, A. H.; Fanning, N.; Symons, S.; Shipp, D.; Chen, J. M.; Nedzelski, J. M., Cochlear
implantation in cochlear otosclerosis. Laryngoscope 2005, 115, 1728-1733.
Martin, M. B.; Grimley, J. S.; Lewis, J. C.; Heath, H. T., 3rd; Bailey, B. N.; Kendrick, H.; Yardley,
V.; Caldera, A.; Lira, R.; Urbina, J. A.; Moreno, S. N.; Docampo, R.; Croft, S. L.; Oldfield, E.,
Bisphosphonates inhibit the growth of Trypanosoma brucei, Trypanosoma cruzi, Leishmania
donovani, Toxoplasma gondii, and Plasmodium falciparum: a potential route to chemotherapy. J.
Med. Chem. 2001, 44, 909-916.
Martin, M. B.; Sanders, J. M.; Kendrick, H.; de Luca-Fradley, K.; Lewis, J. C.; Grimley, J. S.; Van
Brussel, E. M.; Olsen, J. R.; Meints, G. A.; Burzynska, A.; Kafarski, P.; Croft, S. L.; Oldfield, E.,
Activity of bisphosphonates against Trypanosoma brucei rhodesiense. J. Med. Chem. 2002, 45,
2904-2914.
Masarachia, P.; Weinreb, M.; Balena, R.; Rodan, G. A., Comparison of the distribution of 3H-
alendronate and 3H-etidronate in rat and mouse bones. Bone 1996, 19, 281-290.
Mashiba, T.; Hirano, T.; Turner, C. H.; Forwood, M. R.; Johnston, C. C.; Burr, D. B., Suppressed
bone turnover by bisphosphonates increases microdamage accumulation and reduces some
biomechanical properties in dog rib. J. Bone Miner. Res. 2000, 15, 613-620.
Mashiba, T.; Mori, S.; Burr, D. B.; Komatsubara, S.; Cao, Y.; Manabe, T.; Norimatsu, H., The
effects of suppressed bone remodeling by bisphosphonates on microdamage accumulation and
degree of mineralization in the cortical bone of dog rib. J. Bone Miner. Metab. 2005, 23 Suppl, 36-
42.
Massa, S. M.; Yang, T.; Xie, Y.; Shi, J.; Bilgen, M.; Joyce, J. N.; Nehama, D.; Rajadas, J.; Longo,
F. M., Small molecule BDNF mimetics activate TrkB signaling and prevent neuronal degeneration
in rodents. J. Clin. Invest. 2010, 120, 1774-1785.
McKenna, C. E.; Kashemirov, B. A.; Bala, J. L. F. Synthesis of drug conjugates via reaction with
epoxide-containing linkers. US20080312440A1, 2008.
209

McKenna, C. E.; Kashemirov, B. A.; Blazewska, K. M.; Mallard-Favier, I.; Stewart, C. A.; Rojas,
J.; Lundy, M. W.; Ebetino, F. H.; Baron, R. A.; Dunford, J. E.; Kirsten, M. L.; Seabra, M. C.; Bala,
J. L.; Marma, M. S.; Rogers, M. J.; Coxon, F. P., Synthesis, chiral high performance liquid
chromatographic resolution and enantiospecific activity of a potent new geranylgeranyl transferase
inhibitor, 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid. J. Med. Chem.
2010, 53, 3454-3464.
McKenna, M. J.; Nguyen-Huynh, A. T.; Kristiansen, A. G., Association of otosclerosis with Sp1
binding site polymorphism in COL1A1 gene: evidence for a shared genetic etiology with
osteoporosis. Otol. Neurotol. 2004, 25, 447-450.
McWilliams, M. L.; Chen, G. D.; Fechter, L. D., Characterization of the ototoxicity of
difluoromethylornithine and its enantiomers. Toxicol. Sci. 2000, 56, 124-132.
Meyer, T. Pharmaceutical compositions for the treatment of inner ear disorders.
US20150265532A1, 2015.
Meyer, T. Methods and composition for treating and preventing tinnitus. US20150306178A1,
2015.
Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu,
A. M.; Gambhir, S. S.; Weiss, S., Quantum dots for live cells, in vivo imaging, and diagnostics.
Science 2005, 307, 538-544.
Mikulec, A. A.; Plontke, S. K.; Hartsock, J. J.; Salt, A. N., Entry of substances into perilymph
through the bone of the otic capsule after intratympanic applications in guinea pigs: implications
for local drug delivery in humans. Otol. Neurotol. 2009, 30, 131-138.
Mitrofan, L. M.; Pelkonen, J.; Monkkonen, J., The level of ATP analog and isopentenyl
pyrophosphate correlates with zoledronic acid-induced apoptosis in cancer cells in vitro. Bone
2009, 45, 1153-1160.
Mondal, S. B.; Gao, S.; Zhu, N.; Liang, R.; Gruev, V.; Achilefu, S., Real-time fluorescence image-
guided oncologic surgery. Adv. Cancer Res. 2014, 124, 171-211.
Monkkonen, H.; Auriola, S.; Lehenkari, P.; Kellinsalmi, M.; Hassinen, I. E.; Vepsalainen, J.;
Monkkonen, J., A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine
nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing
bisphosphonates. Br. J. Haematol. 2006, 147, 437-445.
Monkkonen, H.; Kuokkanen, J.; Holen, I.; Evans, A.; Lefley, D. V.; Jauhiainen, M.; Auriola, S.;
Monkkonen, J., Bisphosphonate-induced ATP analog formation and its effect on inhibition of
cancer cell growth. Anti-Cancer Drugs 2008, 19, 391-399.
Montalvetti, A.; Bailey, B. N.; Martin, M. B.; Severin, G. W.; Oldfield, E.; Docampo, R.,
Bisphosphonates are potent inhibitors of Trypanosoma cruzi farnesyl pyrophosphate synthase. J.
Biol. Chem. 2001, 276, 33930-33937.
210

Montalvetti, A.; Fernandez, A.; Sanders, J. M.; Ghosh, S.; Van Brussel, E.; Oldfield, E.; Docampo,
R., Farnesyl pyrophosphate synthase is an essential enzyme in Trypanosoma brucei. In vitro RNA
interference and in vivo inhibition studies. J. Biol. Chem. 2003, 278, 17075-17083.
Moreno, B.; Bailey, B. N.; Luo, S.; Martin, M. B.; Kuhlenschmidt, M.; Moreno, S. N.; Docampo,
R.; Oldfield, E., (31)P NMR of apicomplexans and the effects of risedronate on Cryptosporidium
parvum growth. Biochem. Biophys. Res. Commun. 2001, 284, 632-637.
Mukherjee, S.; Huang, C.; Guerra, F.; Wang, K.; Oldfield, E., Thermodynamics of
bisphosphonates binding to human bone: a two-site model. J. Am. Chem. Soc. 2009, 131, 8374-
8375.
Mukherjee, S.; Song, Y.; Oldfield, E., NMR investigations of the static and dynamic structures of
bisphosphonates on human bone: a molecular model. J. Am. Chem. Soc. 2008, 130, 1264-1273.
Mukkamala, D.; No, J. H.; Cass, L. M.; Chang, T. K.; Oldfield, E., Bisphosphonate inhibition of
a Plasmodium farnesyl diphosphate synthase and a general method for predicting cell-based
activity from enzyme data. J. Med. Chem. 2008, 51, 7827-7833.
Muller, U.; Barr-Gillespie, P. G., New treatment options for hearing loss. Nat. Rev. Drug Discov.
2015, 14, 346-365.
Murakami, H.; Takahashi, N.; Sasaki, T.; Udagawa, N.; Tanaka, S.; Nakamura, I.; Zhang, D.;
Barbier, A.; Suda, T., A Possible Mechanism of the Specific Action of Bisphosphonates on
Osteoclasts - Tiludronate Preferentially Affects Polarized Osteoclasts Having Ruffled Borders.
Bone 1995, 17, 137-144.
Murakami, H.; Takahashi, N.; Tanaka, S.; Nakamura, I.; Udagawa, N.; Nakajo, S.; Nakaya, K.;
Abe, M.; Yuda, Y.; Konno, F.; Barbier, A.; Suda, T., Tiludronate inhibits protein tyrosine
phosphatase activity in osteoclasts. Bone 1997, 20, 399-404.
Mustafa, D. A.; Kashemirov, B. A.; McKenna, C. E., Microwave-assisted synthesis of nitrogen-
containing 1-hydroxymethylenebisphosphonate drugs. Tetrahedron Lett. 2011, 52, 2285-2287.
Mynatt, R.; Hale, S. A.; Gill, R. M.; Plontke, S. K.; Salt, A. N., Demonstration of a longitudinal
concentration gradient along scala tympani by sequential sampling of perilymph from the cochlear
apex. J. Assoc. Res. Otolaryngol. 2006, 7, 182-193.
Nadol, J. B., Jr., Patterns of neural degeneration in the human cochlea and auditory nerve:
implications for cochlear implantation. Otolaryngol. Head Neck Surg. 1997, 117, 220-228.
Nancollas, G. H.; Tang, R.; Phipps, R. J.; Henneman, Z.; Gulde, S.; Wu, W.; Mangood, A.; Russell,
R. G.; Ebetino, F. H., Novel insights into actions of bisphosphonates on bone: differences in
interactions with hydroxyapatite. Bone 2006, 38, 617-627.
211

Nieves, J. W.; Bilezikian, J. P.; Lane, J. M.; Einhorn, T. A.; Wang, Y.; Steinbuch, M.; Cosman, F.,
Fragility fractures of the hip and femur: incidence and patient characteristics. Osteoporos. Int.
2010, 21, 399-408.
Nishikawa, M.; Akatsu, T.; Katayama, Y.; Yasutomo, Y.; Kado, S.; Kugal, N.; Yamamoto, M.;
Nagata, N., Bisphosphonates act on osteoblastic cells and inhibit osteoclast formation in mouse
marrow cultures. Bone 1996, 18, 9-14.
Nizamov, S.; Willig, K. I.; Sednev, M. V.; Belov, V. N.; Hell, S. W., Phosphorylated 3-
heteroarylcoumarins and their use in fluorescence microscopy and nanoscopy. Chemistry (Easton)
2012, 18, 16339-16348.
Odvina, C. V.; Zerwekh, J. E.; Rao, D. S.; Maalouf, N.; Gottschalk, F. A.; Pak, C. Y., Severely
suppressed bone turnover: a potential complication of alendronate therapy. J. Clin. Endocrinol.
Metab. 2005, 90, 1294-1301.
Ohyama, K.; Salt, A. N.; Thalmann, R., Volume flow rate of perilymph in the guinea-pig cochlea.
Hear. Res. 1988, 35, 119-129.
Oldfield, E., Targeting isoprenoid biosynthesis for drug discovery: bench to bedside. Acc. Chem.
Res. 2010, 43, 1216-1226.
O'Malley, B. W.; Li, D. Chitosan-based matrixes for controlled-release delivery systems for inner
ear drug application. US20130189241A1, 2013.
Opas, E. E.; Rutledge, S. J.; Golub, E.; Stern, A.; Zimolo, Z.; Rodan, G. A.; Schmidt, A.,
Alendronate inhibition of protein-tyrosine-phosphatase-meg1. Biochem. Pharmacol. 1997, 54,
721-727.
Ory, B.; Moriceau, G.; Trichet, V.; Blanchard, F.; Berreur, M.; Redini, F.; Rogers, M.; Heymann,
D., Farnesyl diphosphate synthase is involved in the resistance to zoledronic acid of osteosarcoma
cells. J. Cell Mol. Med. 2008, 12, 928-941.
Ott, S. M., Long-term safety of bisphosphonates. J. Clin. Endocrinol. Metab. 2005, 90, 1897-1899.
Owens, G.; Jackson, R.; Lewiecki, E. M., An integrated approach: bisphosphonate management
for the treatment of osteoporosis. Am. J. Manag. Care 2007, 13 Suppl 11, S290-308; quiz S309-
212.
Paasche, G.; Gibson, P.; Averbeck, T.; Becker, H.; Lenarz, T.; Stover, T., Technical report:
modification of a cochlear implant electrode for drug delivery to the inner ear. Otol. Neurotol.
2003, 24, 222-227.
Papapetrou, P. D., Bisphosphonate-associated adverse events. Hormones (Athens) 2009, 8, 96-110.
Papapoulos, S.; Lippuner, K.; Roux, C.; Lin, C. J.; Kendler, D. L.; Lewiecki, E. M.; Brandi, M. L.;
Czerwinski, E.; Franek, E.; Lakatos, P.; Mautalen, C.; Minisola, S.; Reginster, J. Y.; Jensen, S.;
212

Daizadeh, N. S.; Wang, A.; Gavin, M.; Libanati, C.; Wagman, R. B.; Bone, H. G., The effect of 8
or 5 years of denosumab treatment in postmenopausal women with osteoporosis: results from the
FREEDOM Extension study. Osteoporos. Int. 2015, 26, 2773-2783.
Papapoulos, S.; Lippuner, K.; Roux, C.; Lin, C. J. F.; Kendler, D. L.; Lewiecki, E. M.; Brandi, M.
L.; Czerwinski, E.; Franek, E.; Lakatos, P.; Mautalen, C.; Minisola, S.; Reginster, J. Y.; Jensen,
S.; Daizadeh, N. S.; Wang, A.; Gavin, M.; Libanati, C.; Wagman, R. B.; Bone, H. G., The effect
of 8 or 5 years of denosumab treatment in postmenopausal women with osteoporosis: results from
the FREEDOM Extension study. Osteoporos. Int. 2015, 26, 2773-2783.
Papapoulos, S. E.; Hoekman, K.; Lowik, C. W. G. M.; Vermeij, P.; Bijvoet, O. L. M., Application
of an Invitro Model and a Clinical Protocol in the Assessment of the Potency of a New
Bisphosphonate. J. Bone Miner. Res. 1989, 4, 775-781.
Paparodis, R.; Buehring, B.; Pelley, E. M.; Binkley, N., A case of an unusual subtrochanteric
fracture in a patient receiving denosumab. Endocr. Pract. 2013, 19, e64-68.
Pedrosa, P.; Vinhas, R.; Fernandes, A.; Baptista, P. V., Gold Nanotheranostics: Proof-of-Concept
or Clinical Tool? Nanomaterials 2015, 5, 1853-1879.
Pelorgeas, S.; Martin, J. B.; Satre, M., Cytotoxicity of dichloromethane diphosphonate and of 1-
hydroxyethane-1,1-diphosphonate in the amoebae of the slime mould Dictyostelium discoideum.
A 31P NMR study. Biochem. Pharmacol. 1992, 44, 2157-2163.
Pentikainen, P. J.; Elomaa, I.; Nurmi, A. K.; Karkkainen, S., Pharmacokinetics of clodronate in
patients with metastatic breast cancer. Int. J. Clin. Pharmacol. Ther. Toxicol. 1989, 27, 222-228.
Peters, G.; Lin, J.; Arriaga, M. A.; Nuss, D. W.; Schaitkin, B.; Walvekar, R. R., Middle-ear
endoscopy and trans-tympanic drug delivery using an interventional sialendoscope: feasibility
study in human cadaveric temporal bones. J. Laryngol. Otol. 2010, 124, 1263-1267.
Pinaud, F.; King, D.; Moore, H. P.; Weiss, S., Bioactivation and cell targeting of semiconductor
CdSe/ZnS nanocrystals with phytochelatin-related peptides. J. Am. Chem. Soc. 2004, 126, 6115-
6123.
Piu, F.; Ye, Q.; Dellamary, L.; Lebel, C. Prevention of and recovery from drug-induced ototoxicity
using corticosteroids and JNK inhibitors. US20130045957A1, 2013.
Piu, F.; Ye, Q.; Dellamary, L. A.; Lebel, C. Methods for the treatment of pediatric otic disorders.
US20150290124, 2015.
Plontke, S. K.; Biegner, T.; Kammerer, B.; Delabar, U.; Salt, A. N., Dexamethasone concentration
gradients along scala tympani after application to the round window membrane. Otol. Neurotol.
2008, 29, 401-406.
213

Plontke, S. K.; Mynatt, R.; Gill, R. M.; Borgmann, S.; Salt, A. N., Concentration gradient along
the scala tympani after local application of gentamicin to the round window membrane.
Laryngoscope 2007, 117, 1191-1198.
Plotkin, L. I.; Bivi, N.; Bellido, T., A bisphosphonate that does not affect osteoclasts prevents
osteoblast and osteocyte apoptosis and the loss of bone strength induced by glucocorticoids in
mice. Bone 2011, 49, 122-127.
Plotkin, L. I.; Weinstein, R. S.; Parfitt, A. M.; Roberson, P. K.; Manolagas, S. C.; Bellido, T.,
Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J. Clin. Invest.
1999, 104, 1363-1374.
Poirrier, A. L.; Van den Ackerveken, P.; Kim, T. S.; Vandenbosch, R.; Nguyen, L.; Lefebvre, P.
P.; Malgrange, B., Ototoxic drugs: Difference in sensitivity between mice and guinea pigs. Toxicol.
Lett. 2010, 193, 41-49.
Puljula, E.; Turhanen, P.; Vepsalainen, J.; Monteil, M.; Lecouvey, M.; Weisell, J., Structural
requirements for bisphosphonate binding on hydroxyapatite: NMR study of bisphosphonate partial
esters. ACS Med. Chem. Lett. 2015, 6, 397-401.
Quai, M.; Repetto, C.; Barbaglia, W.; Cereda, E., Fast deprotection of phenoxy benzyl ethers in
transfer hydrogenation assisted by microwave. Tetrahedron Lett. 2007, 48, 1241-1245.
Quesnel, A. M.; Seton, M.; Merchant, S. N.; Halpin, C.; McKenna, M. J., Third-generation
bisphosphonates for treatment of sensorineural hearing loss in otosclerosis. Otol. Neurotol. 2012,
33, 1308-1314.
Raikkonen, J.; Crockett, J. C.; Rogers, M. J.; Monkkonen, H.; Auriola, S.; Monkkonen, J.,
Zoledronic acid induces formation of a pro-apoptotic ATP analogue and isopentenyl
pyrophosphate in osteoclasts in vivo and in MCF-7 cells in vitro. Br. J. Haematol. 2009, 157, 427-
435.
Rama-Lopez, J.; Cervera-Paz, F. J.; Manrique, M., Cochlear implantation of patients with far-
advanced otosclerosis. Otol. Neurotol. 2006, 27, 153-158.
Ramsden, R.; Rotteveel, L.; Proops, D.; Saeed, S.; van Olphen, A.; Mylanus, E., Cochlear
implantation in otosclerotic deafness. Adv. Otorhinolaryngol. 2007, 65, 328-334.
Rasband, W. S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA,
http://imagej.nih.gov/ij/, 1997-2014.
Rask-Andersen, H.; Schrott-Fischer, A.; Pfaller, K.; Glueckert, R., Perilymph/modiolar
communication routes in the human cochlea. Ear Hear. 2006, 27, 457-465.
Rauch, F.; Glorieux, F. H., Osteogenesis imperfecta, current and future medical treatment. Am. J.
Med. Genet. C Semin. Med. Genet. 2005, 139C, 31-37.
214

Reid, D. M.; Devogelaer, J. P.; Saag, K.; Roux, C.; Lau, C. S.; Reginster, J. Y.; Papanastasiou, P.;
Ferreira, A.; Hartl, F.; Fashola, T.; Mesenbrink, P.; Sambrook, P. N.; investigators, H., Zoledronic
acid and risedronate in the prevention and treatment of glucocorticoid-induced osteoporosis
(HORIZON): a multicentre, double-blind, double-dummy, randomised controlled trial. Lancet
2009, 373, 1253-1263.
Reid, I. R., Efficacy, effectiveness and side effects of medications used to prevent fractures. J.
Intern. Med. 2015, 277, 690-706.
Reid, I. R., Short-term and long-term effects of osteoporosis therapies. Nat. Rev. Endocrinol. 2015,
11, 418-428.
Reid, I. R.; Black, D. M.; Eastell, R.; Bucci-Rechtweg, C.; Su, G.; Hue, T. F.; Mesenbrink, P.;
Lyles, K. W.; Boonen, S.; Trial, H. P. F.; Committees, H. R. F. T. S., Reduction in the risk of
clinical fractures after a single dose of zoledronic Acid 5 milligrams. J. Clin. Endocrinol. Metab.
2013, 98, 557-563.
Reid, I. R.; Brown, J. P.; Burckhardt, P.; Horowitz, Z.; Richardson, P.; Trechsel, U.; Widmer, A.;
Devogelaer, J. P.; Kaufman, J. M.; Jaeger, P.; Body, J. J.; Brandi, M. L.; Broell, J.; Di Micco, R.;
Genazzani, A. R.; Felsenberg, D.; Happ, J.; Hooper, M. J.; Ittner, J.; Leb, G.; Mallmin, H.; Murray,
T.; Ortolani, S.; Rubinacci, A.; Saaf, M.; Samsioe, G.; Verbruggen, L.; Meunier, P. J., Intravenous
zoledronic acid in postmenopausal women with low bone mineral density. N. Engl. J. Med. 2002,
346, 653-661.
Reid, I. R.; Gamble, G. D.; Mesenbrink, P.; Lakatos, P.; Black, D. M., Characterization of and risk
factors for the acute-phase response after zoledronic acid. J. Clin. Endocrinol. Metab. 2010, 95,
4380-4387.
Reitsma, P. H.; Bijvoet, O. L.; Verlinden-Ooms, H.; van der Wee-Pals, L. J., Kinetic studies of
bone and mineral metabolism during treatment with (3-amino-1-hydroxypropylidene)-1,1-
bisphosphonate (APD) in rats. Calcif. Tissue Int. 1980, 32, 145-157.
Reszka, A. A.; Halasy-Nagy, J. M.; Masarachia, P. J.; Rodan, G. A., Bisphosphonates act directly
on the osteoclast to induce caspase cleavage of mst1 kinase during apoptosis. A link between
inhibition of the mevalonate pathway and regulation of an apoptosis-promoting kinase. J. Biol.
Chem. 1999, 274, 34967-34973.
Ridley, A. J.; Hall, A., The small GTP-binding protein rho regulates the assembly of focal
adhesions and actin stress fibers in response to growth factors. Cell 1992, 70, 389-399.
Ridley, A. J.; Paterson, H. F.; Johnston, C. L.; Diekmann, D.; Hall, A., The small GTP-binding
protein rac regulates growth factor-induced membrane ruffling. Cell 1992, 70, 401-410.
Rodriguez, N.; Bailey, B. N.; Martin, M. B.; Oldfield, E.; Urbina, J. A.; Docampo, R., Radical
cure of experimental cutaneous leishmaniasis by the bisphosphonate pamidronate. J. Infect. Dis.
2002, 186, 138-140.
215

Roelofs, A. J.; Coxon, F. P.; Ebetino, F. H.; Lundy, M. W.; Henneman, Z. J.; Nancollas, G. H.;
Sun, S.; Blazewska, K. M.; Bala, J. L.; Kashemirov, B. A.; Khalid, A. B.; McKenna, C. E.; Rogers,
M. J., Fluorescent risedronate analogues reveal bisphosphonate uptake by bone marrow monocytes
and localization around osteocytes in vivo. J. Bone Miner. Res. 2010, 25, 606-616.
Roelofs, A. J.; Coxon, F. P.; Ebetino, F. H.; Lundy, M. W.; Henneman, Z. J.; Nancollas, G. H.;
Sun, S. T.; Blazewska, K. M.; Bala, J. L. F.; Kashemirov, B. A.; Khalid, A. B.; McKenna, C. E.;
Rogers, M. J., Fluorescent Risedronate Analogues Reveal Bisphosphonate Uptake by Bone
Marrow Monocytes and Localization Around Osteocytes In Vivo. J. Bone Miner. Res. 2010, 25,
606-616.
Roelofs, A. J.; Jauhiainen, M.; Monkkonen, H.; Rogers, M. J.; Monkkonen, J.; Thompson, K.,
Peripheral blood monocytes are responsible for gammadelta T cell activation induced by
zoledronic acid through accumulation of IPP/DMAPP. Br. J. Haematol. 2009, 144, 245-250.
Roelofs, A. J.; Stewart, C. A.; Sun, S.; Blazewska, K. M.; Kashemirov, B. A.; McKenna, C. E.;
Russell, R. G.; Rogers, M. J.; Lundy, M. W.; Ebetino, F. H.; Coxon, F. P., Influence of bone
affinity on the skeletal distribution of fluorescently labeled bisphosphonates in vivo. J. Bone Miner.
Res. 2012, 27, 835-847.
Roelofs, A. J.; Stewart, C. A.; Sun, S. T.; Blazewska, K. M.; Kashemirov, B. A.; McKenna, C. E.;
Russell, R. G. G.; Rogers, M. J.; Lundy, M. W.; Ebetino, F. H.; Coxon, F. P., Influence of Bone
Affinity on the Skeletal Distribution of Fluorescently Labeled Bisphosphonates In Vivo. J. Bone
Miner. Res. 2012, 27, 835-847.
Roelofs, A. J.; Thompson, K.; Ebetino, F. H.; Rogers, M. J.; Coxon, F. P., Bisphosphonates:
molecular mechanisms of action and effects on bone cells, monocytes and macrophages. Curr.
Pharm. Des. 2010, 16, 2950-2960.
Rogers, M. J.; Brown, R. J.; Hodkin, V.; Blackburn, G. M.; Russell, R. G.; Watts, D. J.,
Bisphosphonates are incorporated into adenine nucleotides by human aminoacyl-tRNA synthetase
enzymes. Biochem. Biophys. Res. Commun. 1996, 224, 863-869.
Rogers, M. J.; Chilton, K. M.; Coxon, F. P.; Lawry, J.; Smith, M. O.; Suri, S.; Russell, R. G.,
Bisphosphonates induce apoptosis in mouse macrophage-like cells in vitro by a nitric oxide-
independent mechanism. J. Bone Miner. Res. 1996, 11, 1482-1491.
Rogers, M. J.; Crockett, J. C.; Coxon, F. P.; Monkkonen, J., Biochemical and molecular
mechanisms of action of bisphosphonates. Bone 2011, 49, 34-41.
Rogers, M. J.; Ji, X.; Russell, R. G.; Blackburn, G. M.; Williamson, M. P.; Bayless, A. V.; Ebetino,
F. H.; Watts, D. J., Incorporation of bisphosphonates into adenine nucleotides by amoebae of the
cellular slime mould Dictyostelium discoideum. Biochem. J. 1994, 303 ( Pt 1), 303-311.
Rogers, M. J.; Russell, R. G.; Blackburn, G. M.; Williamson, M. P.; Watts, D. J., Metabolism of
halogenated bisphosphonates by the cellular slime mould Dictyostelium discoideum. Biochem.
Biophys. Res. Commun. 1992, 189, 414-423.
216

Rogers, M. J.; Xiong, X.; Brown, R. J.; Watts, D. J.; Russell, R. G.; Bayless, A. V.; Ebetino, F. H.,
Structure-activity relationships of new heterocycle-containing bisphosphonates as inhibitors of
bone resorption and as inhibitors of growth of Dictyostelium discoideum amoebae. Mol.
Pharmacol. 1995, 47, 398-402.
Rondeau, J. M.; Bitsch, F.; Bourgier, E.; Geiser, M.; Hemmig, R.; Kroemer, M.; Lehmann, S.;
Ramage, P.; Rieffel, S.; Strauss, A.; Green, J. R.; Jahnke, W., Structural basis for the exceptional
in vivo efficacy of bisphosphonate drugs. ChemMedChem 2006, 1, 267-273.
Roy, S.; Glueckert, R.; Johnston, A. H.; Perrier, T.; Bitsche, M.; Newman, T. A.; Saulnier, P.;
Schrott-Fischer, A., Strategies for drug delivery to the human inner ear by multifunctional
nanoparticles. Nanomedicine 2012, 7, 55-63.
Russell, R. G., Excretion of Inorganic Pyrophosphate in Hypophosphatasia. Lancet 1965, 2, 461-
464.
Russell, R. G., Bisphosphonates: mode of action and pharmacology. Pediatrics 2007, 119 Suppl
2, S150-162.
Russell, R. G., Bisphosphonates: the first 40 years. Bone 2011, 49, 2-19.
Russell, R. G.; Bisaz, S.; Donath, A.; Morgan, D. B.; Fleisch, H., Inorganic pyrophosphate in
plasma in normal persons and in patients with hypophosphatasia, osteogenesis imperfecta, and
other disorders of bone. J. Clin. Invest. 1971, 50, 961-969.
Russell, R. G.; Xia, Z.; Dunford, J. E.; Oppermann, U.; Kwaasi, A.; Hulley, P. A.; Kavanagh, K.
L.; Triffitt, J. T.; Lundy, M. W.; Phipps, R. J.; Barnett, B. L.; Coxon, F. P.; Rogers, M. J.; Watts,
N. B.; Ebetino, F. H., Bisphosphonates: an update on mechanisms of action and how these relate
to clinical efficacy. Ann. N.Y. Acad. Sci. 2007, 1117, 209-257.
Sahni, M.; Guenther, H. L.; Fleisch, H.; Collin, P.; Martin, T. J., Bisphosphonates act on rat bone
resorption through the mediation of osteoblasts. J. Clin. Invest. 1993, 91, 2004-2011.
Salomo, M.; Jurlander, J.; Nielsen, L. B.; Gimsing, P., How myeloma cells escape bisphosphonate-
mediated killing: development of specific resistance with preserved sensitivity to conventional
chemotherapeutics. Br. J. Haematol. 2003, 122, 202-210.
Salt, A. N.; Gill, R. M.; Hartsock, J. J., Perilymph Kinetics of FITC-Dextran Reveals Homeostasis
Dominated by the Cochlear Aqueduct and Cerebrospinal Fluid. J. Assoc. Res. Otolaryngol. 2015,
16, 357-371.
Salt, A. N.; King, E. B.; Hartsock, J. J.; Gill, R. M.; O'Leary, S. J., Marker entry into vestibular
perilymph via the stapes following applications to the round window niche of guinea pigs. Hear.
Res. 2012, 283, 14-23.
217

Salt, A. N.; Ohyama, K.; Thalmann, R., Radial communication between the perilymphatic scalae
of the cochlea. II: Estimation by bolus injection of tracer into the sealed cochlea. Hear. Res. 1991,
56, 37-43.
Sambrook, P., Quarterly intravenous injection of ibandronate to treat osteoporosis in
postmenopausal women. Clin. Interv. Aging 2007, 2, 65-72.
Sanders, J. M.; Ghosh, S.; Chan, J. M.; Meints, G.; Wang, H.; Raker, A. M.; Song, Y.; Colantino,
A.; Burzynska, A.; Kafarski, P.; Morita, C. T.; Oldfield, E., Quantitative structure-activity
relationships for gammadelta T cell activation by bisphosphonates. J. Med. Chem. 2004, 47, 375-
384.
Santini, D.; Caraglia, M.; Vincenzi, B.; Holen, I.; Scarpa, S.; Budillon, A.; Tonini, G., Mechanisms
of disease: Preclinical reports of antineoplastic synergistic action of bisphosphonates. Nat. Clin.
Pract. Oncol. 2006, 3, 325-338.
Santos, F.; McCall, A. A.; Chien, W.; Merchant, S., Otopathology in Osteogenesis Imperfecta.
Otol. Neurotol. 2012, 33, 1562-1566.
Sarwar, A.; Lee, R.; Depireux, D. A.; Shapiro, B., Magnetic Injection of Nanoparticles Into Rat
Inner Ears at a Human Head Working Distance. IEEE Trans. Magn. 2013, 49, 440-452.
Sato, M.; Grasser, W., Effects of Bisphosphonates on Isolated Rat Osteoclasts as Examined by
Reflected Light-Microscopy. J. Bone Miner. Res. 1990, 5, 31-40.
Sato, M.; Grasser, W., Effects of bisphosphonates on isolated rat osteoclasts as examined by
reflected light microscopy. J. Bone Miner. Res. 1990, 5, 31-40.
Sato, M.; Grasser, W.; Endo, N.; Akins, R.; Simmons, H.; Thompson, D. D.; Golub, E.; Rodan, G.
A., Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast
ultrastructure. J. Clin. Invest. 1991, 88, 2095-2105.
Schenk, R.; Merz, W. A.; Muhlbauer, R.; Russell, R. G.; Fleisch, H., Effect of ethane-1-hydroxy-
1,1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl 2 MDP) on the calcification
and resorption of cartilage and bone in the tibial epiphysis and metaphysis of rats. Calcif. Tissue
Res. 1973, 11, 196-214.
Schibler, D.; Russell, R. G.; Fleisch, H., Inhibition by pyrophosphate and polyphosphate of aortic
calcification induced by vitamin D3 in rats. Clin. Sci. 1968, 35, 363-372.
Schiller, J. S.; Lucas, J. W.; Ward, B. W.; Peregoy, J. A., Summary health statistics for U.S. adults:
National Health Interview Survey, 2010. Vital. Health Stat. 2012, 252, 1-207.
Schindowski, K.; Belarbi, K.; Buee, L., Neurotrophic factors in Alzheimer's disease: role of axonal
transport. Genes Brain Behav. 2008, 7 Suppl 1, 43-56.
218

Schipper, M. L.; Iyer, G.; Koh, A. L.; Cheng, Z.; Ebenstein, Y.; Aharoni, A.; Keren, S.; Bentolila,
L. A.; Li, J.; Rao, J.; Chen, X.; Banin, U.; Wu, A. M.; Sinclair, R.; Weiss, S.; Gambhir, S. S.,
Particle size, surface coating, and PEGylation influence the biodistribution of quantum dots in
living mice. Small 2009, 5, 126-134.
Schuknecht, H. F.; Gacek, M. R., Cochlear pathology in presbycusis. Ann. Otol. Rhinol. Laryngol.
1993, 102, 1-16.
Selander, K.; Lehenkari, P.; Vaananen, H. K., The effects of bisphosphonates on the resorption
cycle of isolated osteoclasts. Calcif. Tissue Int. 1994, 55, 368-375.
Selander, K. S.; Monkkonen, J.; Karhukorpi, E. K.; Harkonen, P.; Hannuniemi, R.; Vaananen, H.
K., Characteristics of clodronate-induced apoptosis in osteoclasts and macrophages. Mol.
Pharmacol. 1996, 50, 1127-1138.
Semaan, M. T.; Gehani, N. C.; Tummala, N.; Coughlan, C.; Fares, S. A.; Hsu, D. P.; Murray, G.
S.; Lippy, W. H.; Megerian, C. A., Cochlear implantation outcomes in patients with far advanced
otosclerosis. Am. J. Otolaryngol. 2012, 33, 608-614.
Sensenig, R.; Sapir, Y.; MacDonald, C.; Cohen, S.; Polyak, B., Magnetic nanoparticle-based
approaches to locally target therapy and enhance tissue regeneration in vivo. Nanomedicine 2012,
7, 1425-1442.
Shah, R. B.; Mehra, R.; Chinnaiyan, A. M.; Shen, R.; Ghosh, D.; Zhou, M.; Macvicar, G. R.;
Varambally, S.; Harwood, J.; Bismar, T. A.; Kim, R.; Rubin, M. A.; Pienta, K. J., Androgen-
independent prostate cancer is a heterogeneous group of diseases: lessons from a rapid autopsy
program. Cancer Res. 2004, 64, 9209-9216.
Shapiro, B.; Depireux, D.; Sarwar, A.; Nacev, A.; Preciado, D.; Hausfeld, J., Pre-Clinical
Development of Magnetic Delivery of Therapy to Middle and Inner Ears. ENT Audiol. News 2014,
23, 54-56.
Shapiro, B.; Depireux, D. A.; Dormer, K.; Rutel, I. B. Device and methods for directing agents to
the middle ear and the inner ear including magnetic field. US20140073835A1, 2014.
Shapiro, B.; Kulkarni, S.; Nacev, A.; Sarwar, A.; Preciado, D.; Depireux, D. A., Shaping magnetic
fields to direct therapy to ears and eyes. Annu. Rev. Biomed. Eng. 2014, 16, 455-481.
Shinomori, Y.; Spack, D. S.; Jones, D. D.; Kimura, R. S., Volumetric and dimensional analysis of
the guinea pig inner ear. Ann. Otol. Rhinol. Laryngol. 2001, 110, 91-98.
Sillero, M. A.; de Diego, A.; Silles, E.; Perez-Zuniga, F.; Sillero, A., Synthesis of bisphosphonate
derivatives of ATP by T4 RNA ligase. FEBS Lett. 2006, 580, 5723-5727.
Silverman, S. L.; Landesberg, R., Osteonecrosis of the jaw and the role of bisphosphonates: a
critical review. Am. J. Med. 2009, 122, S33-45.
219

Singer, F. R.; Ritch, P. S.; Lad, T. E.; Ringenberg, Q. S.; Schiller, J. H.; Recker, R. R.; Ryzen, E.,
Treatment of hypercalcemia of malignancy with intravenous etidronate. A controlled, multicenter
study. The Hypercalcemia Study Group. Arch. Intern. Med. 1991, 151, 471-476.
Singh, A. P.; Zhang, Y.; No, J. H.; Docampo, R.; Nussenzweig, V.; Oldfield, E., Lipophilic
bisphosphonates are potent inhibitors of Plasmodium liver-stage growth. Antimicrob. Agents
Chemother. 2010, 54, 2987-2993.
Snider, W. D., Functions of the Neurotrophins during Nervous-System Development - What the
Knockouts Are Teaching Us. Cell 1994, 77, 627-638.
Sockalingam, R.; Freeman, S.; Cherny, T. L.; Sohmer, H., Effect of high-dose cisplatin on auditory
brainstem responses and otoacoustic emissions in laboratory animals. Am. J. Otol. 2000, 21, 521-
527.
Staal, A.; Frith, J. C.; French, M. H.; Swartz, J.; Gungor, T.; Harrity, T. W.; Tamasi, J.; Rogers,
M. J.; Feyen, J. H., The ability of statins to inhibit bone resorption is directly related to their
inhibitory effect on HMG-CoA reductase activity. J. Bone Miner. Res. 2003, 18, 88-96.
Staecker, H.; Gabaizadeh, R.; Federoff, H.; Van De Water, T. R., Brain-derived neurotrophic
factor gene therapy prevents spiral ganglion degeneration after hair cell loss. Otolaryngol. Head
Neck Surg. 1998, 119, 7-13.
Stancu, C.; Sima, A., Statins: mechanism of action and effects. J. Cell Mol. Med. 2001, 5, 378-
387.
Subramanian, S.; Jaffer, F. A.; Tawakol, A., Optical molecular imaging in atherosclerosis. J. Nucl.
Cardiol. 2010, 17, 135-144.
Sun, H.; Chen, F.; Wang, X.; Liu, Z.; Yang, Q.; Zhang, X.; Zhu, J.; Qiang, L.; Guo, Q.; You, Q.,
Studies on gambogic acid (IV): Exploring structure-activity relationship with IkappaB kinase-beta
(IKKbeta). Eur. J. Med. Chem. 2012, 51, 110-123.
Sun, S.; Blazewska, K. M.; Kadina, A. P.; Kashemirov, B. A.; Duan, X.; Triffitt, J. T.; Dunford, J.
E.; Russell, R. G.; Ebetino, F. H.; Roelofs, A. J.; Coxon, F. P.; Lundy, M. W.; McKenna, C. E.,
Fluorescent Bisphosphonate and Carboxyphosphonate Probes: A Versatile Imaging Toolkit for
Applications in Bone Biology and Biomedicine. Bioconjug Chem. 2016, 27, 329-340.
Sutherland, K. A.; Rogers, H. L.; Tosh, D.; Rogers, M. J., RANKL increases the level of Mcl-1 in
osteoclasts and reduces bisphosphonate-induced osteoclast apoptosis in vitro. Arthritis Res. Ther.
2009, 11, R58.
Suva, L. J.; Griffin, R. J.; Makhoul, I., Mechanisms of bone metastases of breast cancer. Endocr.
Relat. Cancer 2009, 16, 703-713.
Suva, L. J.; Washam, C.; Nicholas, R. W.; Griffin, R. J., Bone metastasis: mechanisms and
therapeutic opportunities. Nat. Rev. Endocrinol. 2011, 7, 208-218.
220

Swan, E. E.; Mescher, M. J.; Sewell, W. F.; Tao, S. L.; Borenstein, J. T., Inner ear drug delivery
for auditory applications. Adv. Drug Deliv. Rev. 2008, 60, 1583-1599.
Szabo, C. M.; Matsumura, Y.; Fukura, S.; Martin, M. B.; Sanders, J. M.; Sengupta, S.; Cieslak, J.
A.; Loftus, T. C.; Lea, C. R.; Lee, H. J.; Koohang, A.; Coates, R. M.; Sagami, H.; Oldfield, E.,
Inhibition of geranylgeranyl diphosphate synthase by bisphosphonates and diphosphates: a
potential route to new bone antiresorption and antiparasitic agents. J. Med. Chem. 2002, 45, 2185-
2196.
Tanaka, S., Regulation of bone destruction in rheumatoid arthritis through RANKL-RANK
pathways. World J. Orthop. 2013, 4, 1-6.
Terkeltaub, R. A., Inorganic pyrophosphate generation and disposition in pathophysiology. Am. J.
Physiol. Cell Physiol. 2001, 281, C1-C11.
Thompson, K.; Dunford, J. E.; Ebetino, F. H.; Rogers, M. J., Identification of a bisphosphonate
that inhibits isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Biochem.
Biophys. Res. Commun. 2002, 290, 869-873.
Thompson, K.; Roelofs, A. J.; Jauhiainen, M.; Monkkonen, H.; Monkkonen, J.; Rogers, M. J.,
Activation of gammadelta T cells by bisphosphonates. Adv. Exp. Med. Biol. 2010, 658, 11-20.
Thompson, K.; Rogers, M. J., Statins prevent bisphosphonate-induced gamma,delta-T-cell
proliferation and activation in vitro. J. Bone Miner. Res. 2004, 19, 278-288.
Thompson, K.; Rogers, M. J.; Coxon, F. P.; Crockett, J. C., Cytosolic entry of bisphosphonate
drugs requires acidification of vesicles after fluid-phase endocytosis. Mol. Pharmacol. 2006, 69,
1624-1632.
Timms, A. E.; Zhang, Y.; Russell, R. G.; Brown, M. A., Genetic studies of disorders of calcium
crystal deposition. Rheumatology (Oxford) 2002, 41, 725-729.
Torres Martin de Rosales, R.; Finucane, C.; Mather, S. J.; Blower, P. J., Bifunctional
bisphosphonate complexes for the diagnosis and therapy of bone metastases. Chem. Commun.
(Camb) 2009, 4847-4849.
Trott, O.; Olson, A. J., AutoDock Vina: improving the speed and accuracy of docking with a new
scoring function, efficient optimization, and multithreading. J. Comput. Chem. 2010, 31, 455-461.
Trott, O.; Olson, A. J., Software News and Update AutoDock Vina: Improving the Speed and
Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading.
J. Comput. Chem. 2010, 31, 455-461.
van beek, E.; Lowik, C.; van der Pluijm, G.; Papapoulos, S., The role of geranylgeranylation in
bone resorption and its suppression by bisphosphonates in fetal bone explants in vitro: A clue to
the mechanism of action of nitrogen-containing bisphosphonates. J. Bone Miner. Res. 1999, 14,
722-729.
221

van Beek, E.; Pieterman, E.; Cohen, L.; Lowik, C.; Papapoulos, S., Nitrogen-containing
bisphosphonates inhibit isopentenyl pyrophosphate isomerase/farnesyl pyrophosphate synthase
activity with relative potencies corresponding to their antiresorptive potencies in vitro and in vivo.
Biochem. Biophys. Res. Commun. 1999, 255, 491-494.
Varela, I.; Pereira, S.; Ugalde, A. P.; Navarro, C. L.; Suarez, M. F.; Cau, P.; Cadinanos, J.; Osorio,
F. G.; Foray, N.; Cobo, J.; de Carlos, F.; Levy, N.; Freije, J. M.; Lopez-Otin, C., Combined
treatment with statins and aminobisphosphonates extends longevity in a mouse model of human
premature aging. Nat. Med. 2008, 14, 767-772.
Viana, L. M.; O'Malley, J. T.; Burgess, B. J.; Jones, D. D.; Oliveira, C. A.; Santos, F.; Merchant,
S. N.; Liberman, L. D.; Liberman, M. C., Cochlear neuropathy in human presbycusis: Confocal
analysis of hidden hearing loss in post-mortem tissue. Hear. Res. 2015, 327, 78-88.
Vitte, C.; Fleisch, H.; Guenther, H. L., Bisphosphonates induce osteoblasts to secrete an inhibitor
of osteoclast-mediated resorption. Endocrinology 1996, 137, 2324-2333.
Von Baeyer, H.; Hofmann, K. A., Acetodiphosphoriga sauer. Ber 1897, 30, 1973.
Wan, G.; Gomez-Casati, M. E.; Gigliello, A. R.; Liberman, M. C.; Corfas, G., Neurotrophin-3
regulates ribbon synapse density in the cochlea and induces synapse regeneration after acoustic
trauma. eLife 2014, 3.
Wang, T. S.; Fawwaz, R. A.; Johnson, L. J.; Mojdehi, G. E.; Johnson, P. M., Bone-seeking
properties of Tc-99m carbonyl diphosphonic acid, dihydroxy-methylene diphosphonic acid and
monohydroxy-methylene phosphoinic acid: concise communication. J. Nucl. Med. 1980, 21, 767-
770.
Wang, T. S.; Mojdehi, G. E.; Fawwaz, R. A.; Johnson, P. M., A study of the relationship between
chemical structure and bone localization of Tc-99m diphosphonic acids: concise communication.
J. Nucl. Med. 1979, 20, 1066-1070.
Wang, X.; Dellamary, L.; Fernandez, R.; Harrop, A.; Keithley, E. M.; Harris, J. P.; Ye, Q.; Lichter,
J.; LeBel, C.; Piu, F., Dose-Dependent Sustained Release of Dexamethasone in Inner Ear Cochlear
Fluids Using a Novel Local Delivery Approach. Audiol. Neurootol. 2009, 14.
Wang, X.; Dellamary, L.; Fernandez, R.; Ye, Q.; LeBel, C.; Piu, F., Principles of inner ear
sustained release following intratympanic administration. Laryngoscope 2011, 121, 385-391.
Wang, X.; Fernandez, R.; Dellamary, L.; Harrop, A.; Ye, Q.; Lichter, J.; Lau, D.; Lebel, C.; Piu,
F., Pharmacokinetics of Dexamethasone Solution following Intratympanic Injection in Guinea Pig
and Sheep. Audiol. Neurootol. 2011, 16.
Wang, X.; Fernandez, R.; Tsivkovskaia, N.; Harrop-Jones, A.; Hou, H. J.; Dellamary, L.; Dolan,
D. F.; Altschuler, R. A.; Lebel, C.; Piu, F., OTO-201: Nonclinical Assessment of a Sustained-
Release Ciprofloxacin Hydrogel for the Treatment of Otitis Media. Otol. Neurotol. 2014, 35, 459-
469.
222

Wark, J. D.; Bensen, W.; Recknor, C.; Ryabitseva, O.; Chiodo, J., 3rd; Mesenbrink, P.; de Villiers,
T. J., Treatment with acetaminophen/paracetamol or ibuprofen alleviates post-dose symptoms
related to intravenous infusion with zoledronic acid 5 mg. Osteoporos. Int. 2012, 23, 503-512.
Watts, N. B.; Chines, A.; Olszynski, W. P.; McKeever, C. D.; McClung, M. R.; Zhou, X.; Grauer,
A., Fracture risk remains reduced one year after discontinuation of risedronate. Osteoporos. Int.
2008, 19, 365-372.
Watts, N. B.; Harris, S. T.; Genant, H. K.; Wasnich, R. D.; Miller, P. D.; Jackson, R. D.; Licata,
A. A.; Ross, P.; Woodson, G. C., 3rd; Yanover, M. J.; et al., Intermittent cyclical etidronate
treatment of postmenopausal osteoporosis. N. Engl. J. Med. 1990, 323, 73-79.
Wegner, I.; Verhagen, J. J.; Stegeman, I.; Vincent, R.; Grolman, W., A systematic review of the
effect of piston diameter in stapes surgery for otosclerosis on hearing results. Laryngoscope 2016,
126, 182-190.
Weinstein, R. S.; Roberson, P. K.; Manolagas, S. C., Giant osteoclast formation and long-term oral
bisphosphonate therapy. N. Engl. J. Med. 2009, 360, 53-62.
Wen, D.; Qing, L.; Harrison, G.; Golub, E.; Akintoye, S. O., Anatomic site variability in rat skeletal
uptake and desorption of fluorescently labeled bisphosphonate. Oral. Dis. 2011, 17, 427-432.
Whitaker, M.; Guo, J.; Kehoe, T.; Benson, G., Bisphosphonates for osteoporosis--where do we go
from here? N. Engl. J. Med. 2012, 366, 2048-2051.
Whyte, M. P., Hypophosphatasia - aetiology, nosology, pathogenesis, diagnosis and treatment. Nat.
Rev. Endocrinol. 2016, 12, 233-246.
Widler, L.; Jaeggi, K. A.; Glatt, M.; Muller, K.; Bachmann, R.; Bisping, M.; Born, A. R.; Cortesi,
R.; Guiglia, G.; Jeker, H.; Klein, R.; Ramseier, U.; Schmid, J.; Schreiber, G.; Seltenmeyer, Y.;
Green, J. R., Highly potent geminal bisphosphonates. From pamidronate disodium (Aredia) to
zoledronic acid (Zometa). J. Med. Chem. 2002, 45, 3721-3738.
Yardley, V.; Khan, A. A.; Martin, M. B.; Slifer, T. R.; Araujo, F. G.; Moreno, S. N.; Docampo,
R.; Croft, S. L.; Oldfield, E., In vivo activities of farnesyl pyrophosphate synthase inhibitors
against Leishmania donovani and Toxoplasma gondii. Antimicrob. Agents Chemother. 2002, 46,
929-931.
Ye, Q.; Dellamary, L. A.; Piu, F. Modulation of gel temperature of poloxamer-containing sustained
release formulations. US20120277199A1, 2012.
Yu, X.; Scholler, J.; Foged, N. T., Interaction between effects of parathyroid hormone and
bisphosphonate on regulation of osteoclast activity by the osteoblast-like cell line UMR-106. Bone
1996, 19, 339-345.
Zaheer, A.; Lenkinski, R. E.; Mahmood, A.; Jones, A. G.; Cantley, L. C.; Frangioni, J. V., In vivo
near-infrared fluorescence imaging of osteoblastic activity. Nat. Biotechnol. 2001, 19, 1148-1154.
223

Zerial, M.; Stenmark, H., Rab GTPases in vesicular transport. Curr. Opin. Cell Biol. 1993, 5, 613-
620.
Zhang, D.; Udagawa, N.; Nakamura, I.; Murakami, H.; Saito, S.; Yamasaki, K.; Shibasaki, Y.;
Morii, N.; Narumiya, S.; Takahashi, N.; et al., The small GTP-binding protein, rho p21, is involved
in bone resorption by regulating cytoskeletal organization in osteoclasts. J. Cell Sci. 1995, 108 ( Pt
6), 2285-2292.
Zhang, F. L.; Casey, P. J., Protein prenylation: molecular mechanisms and functional
consequences. Annu. Rev. Biochem. 1996, 65, 241-269.
Zhang, Q.; Badell, I. R.; Schwarz, E. M.; Boulukos, K. E.; Yao, Z.; Boyce, B. F.; Xing, L., Tumor
necrosis factor prevents alendronate-induced osteoclast apoptosis in vivo by stimulating Bcl-xL
expression through Ets-2. Arthritis Rheum. 2005, 52, 2708-2718.
Zimolo, Z.; Wesolowski, G.; Rodan, G. A., Acid extrusion is induced by osteoclast attachment to
bone. Inhibition by alendronate and calcitonin. J. Clin. Invest. 1995, 96, 2277-2283.
Zou, J.; Pyykko, I.; Bjelke, B.; Dastidar, P.; Toppila, E., Communication between the
perilymphatic scalae and spiral ligament visualized by in vivo MRI. Audiol. Neurootol. 2005, 10,
145-152.
Zuccato, C.; Cattaneo, E., Role of brain-derived neurotrophic factor in Huntington's disease. Prog.
Neurobiol. 2007, 81, 294-330.

224

APPENDIX A: Chapter 2 Supporting Data

225

A. 1-
1
H NMR Spectrum of RIS linker (2.2)

A. 2-
31
P NMR Spectrum of RIS linker (2.2)
226

A. 3 - Chromatograph of the Purification of Extended Rislinker using SAX RP HPLC
227

A. 4- ESI-MS of 2.3
N
P
OH
P
H
N
OH
OH
OH
O
OH
O
OH
O
OH
O
Chemical Formula:C
14
H
23
N
2
O
11
P
2
+
ExactMass:457.08
MolecularWeight:457.29
[M-2H]
-

Dimer of 2.3
228

A. 5-
1
H NMR of 2.3

A. 6-
31
P NMR of 2.3
229

A. 7- ESI-MS of 2.6

[M-H]
-

230

A. 8-
1
H NMR of 2.6

A. 9-
31
P NMR of 2.6
231

A. 10-
1
H NMR of 2.8

A. 11-
31
P NMR of 2.8
232

A. 12- ESI-MS of the reaction mixture containing 2.8 and lysine
P
OH
P
OH
OH
O
OH
O
OH
N
H
O
HO
O
ChemicalFormula: C
7
H
15
NO
10
P
2
ExactMass:335.02
MolecularWeight:335.14
[M-H]
-

[M-H]
-

233

A. 13-
1
H NMR of 2.12
234

A. 14- ESI-MS (-) of 2.13
[M-H]
-

[M-H]
-
of 2.14
235

A. 15- LC-MS of 2.13
[M-H]
-

236

A. 16-
1
H NMR of 2.13 after 3X Dowex exchanges

A. 17-
31
P NMR of 2.13 after 3X Dowex exchanges
237

APPENDIX B: Chapter 3 Supporting Data

238

B. 1-
1
H NMR Spectrum of N-Boc-allylamine (3.2)

B. 2-
1
H NMR Spectrum of tert-butyl (oxiran-2-ylmethyl)carbamate (3.3)

239

B. 3-
1
H NMR Spectrum of N-Boc-RIS linker (3.5)

B. 4-
31
P NMR Spectrum of N-Boc-RIS linker (3.5)
240

B. 5-
1
H NMR Spectrum of RIS linker (3.6)

B. 6-
31
P NMR Spectrum of RIS linker (3.6)
241

B. 7- Chromatograms for C18 semi- preparative HPLC purification of the reaction mixture after
size exclusion chromatography of 5(6)-FAM-RIS (3.7a and 3.7b).

B. 8- UV-VIS spectra of 5- and 6-FAM-RIS (3.7a and 3.7b)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
200 240 280 320 360 400 440 480 520 560 600
Absorbance
Wavelength (nm)
UV-VIS Spectra of 5- and 6-FAM-RIS
6-FAM-RIS
5-FAM-RIS
Chrom. 1 0.0 mins. 33.7 mins.
6
5
4
3
2
1
RIS linker
Retention time: 3.0 min
5,6-FAM
Retention time: 12.0 min
6-FAM-RIS
Retention time: 15.0 min
5-FAM-RIS
Retention time: 24.0 min
242

B. 9- Fluorescence spectra of 5- and 6-FAM-RIS (3.7a and 3.7b)

B. 10- Chromatograms for C18 semi- preparative HPLC purification of the reaction mixture of
S-Cy5-RIS (3.7c).

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
400 440 480 520 560 600 640 680 720 760 800
Normalized Intensity
Wavelength (nm)
Fluorescence Spectra of 5- and 6-FAM-RIS
5-FAM-RIS-Emission
5-FAM-RIS-Excitation
6-FAM-RIS-Emission
6-FAM-RIS-Excitation
Chrom. 1 0.0 mins. 17.6 mins.
1
Rislinker
Retention time: 3.0 min
S-Cy5 Dye
Retention time: 15.0min
S-Cy5-RIS
Retention time: 9.0 min
243

B. 11- UV-Vis spectrum of S-Cy5-RIS (3.7c).

B. 12- Fluorescence pectra of S-Cy5-RIS (3.7c).
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
200 240 280 320 360 400 440 480 520 560 600 640 680 720 760 800
Absorbance
Wavelength (nm)
UV-Vis Spectrum of S-Cy5-RIS
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
400 450 500 550 600 650 700 750 800
Normalized Intensity
Wavelength (nm)
Fluorescence Spectrum of S-Cy5-RIS
Emission
Excitation
244

B. 13- Chromatograms for C18 semi- preparative HPLC purification of the reaction mixture of
AF647-RIS. (3.7d).

B. 14-UV-Vis Spectrum of AF647-RIS (3.7d).

0
0.1
0.2
0.3
0.4
0.5
400 430 460 490 520 550 580 610 640 670 700
Absorbance
Wavelength (nm)
UV-Vis Spectrum of AF647-RIS
Rislinker
Retention time: 3.0 min
AF647-RIS
Retention time: 22.5 min
AF647 Dye
Retention time: 21.0min
245

B. 15- Fluorescence Spectra of AF647-RIS (3.7d).

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
400 440 480 520 560 600 640 680 720 760 800
Normalized intensity
Wavelength (nm)
Fluorescence Spectra of AF647-RIS
Emission
Excitation
246

B. 16-
1
H NMR Spectrum of 5-FAM-RIS (3.7a).

B. 17-
31
P NMR Spectrum of 5-FAM-RIS (3.7a).
247

B. 18-
1
H NMR Spectrum of 6-FAM-RIS (3.7b).

B. 19-
31
P NMR Spectrum of 6-FAM-RIS (3.7b).
248

B. 20 -
1
H NMR Spectrum of S-Cy5-RIS (3.7c).

B. 21-
31
P NMR Spectrum of S-Cy5-RIS (3.7c).
pH = 6.51
249

B. 22-
1
H NMR Spectrum of AF647-RIS (3.7d).

B. 23-
31
P NMR Spectrum of AF647-RIS (3.7d).
250

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0
f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
A (s)
1.38
B (s)
2.78
C (s)
3.45
D (s)
5.03
9.00
5.79
1.93
0.72
1.38
2.78
3.45
5.03
Z
X
Y
Y
X = Solvent Residue
Y = Ether
Z = Grease

B. 24-
1
H NMR Spectrum of NHS-NBoc-Beta Alanine (3.11).

B. 25- Chromatograms for C18 preparative HPLC purification of the reaction mixture of N-Boc-
RIS-V1 linker (3.14).

N-Boc-RIS-V1
Retention time: 22.3 min
RIS linker
Retention time: 11.7 min
251

B. 26-
1
H NMR Spectrum of NBoc-RIS-V1 linker (3.14).
B. 27-
31
P NMR Spectrum of NBoc-RIS-V1 linker (3.14).
252

B. 28-
1
H NMR Spectrum of RIS-V1 linker (3.17).

B. 29-
31
P NMR Spectrum of RIS-V1 linker (3.17).
pH = 2.05
253

B. 30- Chromatograms for C18 semi- preparative HPLC purification of the reaction mixture after
size exclusion chromatography of 5(6)-FAM-RIS-V1 (3.20a and 3.20b).

B. 31- UV-Vis spectra of 5 FAM-RIS-V1- and 6-FAM-RIS-V1 (3.20a and 3.20b, respectively)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
250 280 310 340 370 400 430 460 490 520 550
Absorbance
Wavelength (nm)
UV-Vis Spectra of 5- and 6-FAM-RIS-V1
5-FAM-RIS-V1
6-FAM-RIS-V1
Chrom. 1 0.0 mins. 39.3 mins.
5
4
3
2
1
RIS-V1 linker
Retention time: 3.0 min
5,6-FAM
Retention time: 12.0 min
6-FAM-RIS-V1
Retention time: 20.0 min
5-FAM-RIS-V1
Retention time: 30.0 min
254

B. 32- Fluorescence spectra of 5- and 6-FAM-RIS-V1 (3.20a and 3.20b, respectively)

B. 33- HPLC Chromatograph of S-Cy5-RIS-V1 (3.20c)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
400 450 500 550 600 650 700
Normalized Intensity
Wavelength (nm)
Fluorescence Spectra of 5- and 6-FAM-RIS-V1
6-FAM-RIS-V1-Excitation
6-FAM-RIS-V1-Emission
5-FAM-RIS-V1-Excitation
5-FAM-RIS-V1-Emission
Chrom. 1 0.0 mins. 23.2 mins.
1
RIS-V1 linker
Retention time: 3.0 min
S-Cy5 Dye
Retention time: 19.0 min
S-Cy5-RIS-V1
Retention time: 12.0 min
255

B. 34- UV-Vis Spectrum of S-Cy5-RIS-V1 (3.20c)

B. 35- Fluorescence spectra of S-Cy5-RIS-V1 (3.20c)

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
200 240 280 320 360 400 440 480 520 560 600 640 680 720 760 800
Absorbance
Wavelength (nm)
UV-Vis Spectrum of S-Cy5-RIS-V1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
400 450 500 550 600 650 700 750 800
Normalized Intensity
Wavelength (nm)
Fluorescence Spectra of S-Cy5-ExtRis-V1
Emission
Excitation
256

B. 36. Chromatograms for C18 semi- preparative HPLC purification of the reaction mixture of
AF647-RIS-V1. (3.20d)

B. 37- UV-Vis Spectrum of AF647-RIS-V1 (3.20d)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
1.1
200 250 300 350 400 450 500 550 600 650 700 750 800
Absorbance
Wavelength (nm)
UV-Vis Spectrum of AF-Ris-V1
Chrom. 1 0.0 mins. 45.2 mins.
4
3
2
1
RIS-V1 linker
Retention time: 5.4 min
AF647-RIS-V1
Retention time: 29.0 min
AF647-Dye
Retention time: 31.0 min
257

B. 38- Fluorescence spectra of AF647-RIS-V1 (3.20d)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
400 450 500 550 600 650 700 750 800
Normalized Intensity
Wavelength (nm)
Fluorescence Spectra of AF647-RIS-V1
AF647-Ris-V1-Emission
AF647-Ris-V1-Excitation
258

B. 39-
1
H NMR in D2O of 5-FAM-RIS-V1 (3.20a)

B. 40-
31
P NMR in D2O of 5-FAM-RIS-V1 (3.20a)
pH = 6.0
259

Supplemental Figure 9.
1
H NMR in D2O of 6-FAM-RIS-V1 (3.20b)

Supplemental Figure 10.
31
P NMR in D2O of 6-FAM-RIS-V1 (3.20b)
pH = 6.0
260

B. 41-
1
H NMR Spectrum of S-Cy5-RIS-V1 (3.20c)

B. 42-
31
P NMR Spectrum of S-Cy5-RIS-V1 (3.20c)

pH = 5.87
261

B. 43. 1H NMR in D2O of AF647-RIS-V1 (3.20d)

B. 44.
31
P NMR in D2O at pH 6.0 of AF647-RIS-V1 (3.20d)

262

B. 45-
1
H NMR Spectrum of 3.12

Chrom. 1 0.0 mins. 32.4 mins.
8
7
6
5
4
3
2
1
NBoc-RIS-M
Retention time: 15.30 min
RIS linker
Retention time: 7.50 min
263

B. 46- C18 HPLC Chromatograph of 3.15

B. 47-
1
H NMR Spectrum of 3.15

B. 48-
31
P NMR Spectrum of 3.15
pH = 6.67
264

B. 49- 1H NMR Spectrum of 3.18

B. 50-
31
P NMR Spectrum of 3.18
pH = 2.13
265

B. 51- Chromatograms for C18 semi- preparative HPLC purification of the reaction mixture after
size exclusion chromatography of 5(6)-FAM-RIS-M (3.21a and 3.21b).

B. 52- UV-Vis spectra of 5- and 6-FAM-RIS-M (3.21a and 3.21b).
0
0.1
0.2
0.3
0.4
0.5
250 280 310 340 370 400 430 460 490 520 550
Absorbance
Wavelength (nm)
UV-Vis Spectra of 5- and 6-FAM-RIS-M
5-FAMRIS-M
6-FAM-RIS-M
Chrom. 1 0.0 mins. 39.4 mins.
1
6-FAM-RIS-M
Retention time: 23 min
5-FAM-RIS-M
Retention time: 34 min
5-FAM
Retention time: 30 min
6-FAM
Retention time: 15 min
RIS-M
Retention time: 3 min
266

B. 53- Fluorescence spectra of 5- and 6-FAM-RIS-M (3.21a and 3.21b).

B. 54- HPLC Chromatograph of 3.21c

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
400 430 460 490 520 550 580 610 640 670 700
Normalized Intensity
Wavelength (nm)
Fluorescence Spectra of 5- and 6-FAM-RIS-M
5-FAM-RIS-M-Emission
5-FAM-RIS-M-Excitation
6-FAM-RIS-M-Emission
6-FAM-RIS-M-Excitation
Chrom. 1 0.0 mins. 27.0 mins.
1
RIS-M
Retention time: 3.0 min
S-Cy5 Dye
Retention time: 15.0 min
S-Cy5-RIS-M
Retention time: 23 min
267

B. 55- UV-Vis Spectrum of 3.21c

B. 56- Fluorescence spectra of S-Cy5-ExtRIS-M 3.21c

-0.05
0.05
0.15
0.25
0.35
0.45
0.55
0.65
200 240 280 320 360 400 440 480 520 560 600 640 680 720 760 800
Absorbance
Wavelength (nm)
UV-Vis Spectrum of S-Cy5-RIS-M
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
400 450 500 550 600 650 700 750 800
Normalized Intensity
Wavelength (nm)
Fluorescence Spectra of S-Cy-ExtRis-M
Excitation
Emission
268

B. 57- HPLC Chromatograph of 3.21d

B. 58- UV-Vis Spectrum of 3.21d

-0.05
0.05
0.15
0.25
0.35
0.45
0.55
0.65
200 240 280 320 360 400 440 480 520 560 600 640 680 720 760 800
Absorbance
Wavelength (nm)
UV-Vis Spectrum of AF647-RIS-M
Chrom. 1 0.0 mins. 17.5 mins.
1
RIS-M
Retention time: 3.0 min
AF647-RIS-M
Retention time: 23 min
AF647 Dye
Retention time: 15.0 min
269

Supplemental Figure 1- Fluorescence spectra of AF647-ExtRIS-M (3.21d)

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
400 450 500 550 600 650 700 750 800
Normalized Intensity
Wavelength (nm)
Fluorescence Spectra of AF647-ExtRis-M
Emission
Excitation
270

B. 59-
1
H NMR Spectrum of 3.21a

B. 60-
31
P NMR Spectrum of 3.21a

pH = 5.08
271

B. 61- - 1H NMR Spectrum of 3.21b

B. 62-
31
P NMR Spectrum of 3.21b
pH = 5.90
272

B. 63-
1
H NMR Spectrum of 3.21c

B. 64-
31
P NMR Spectrum of 3.21c

pH = 5.85
273

B. 65-
1
H NMR Spectrum of 3.21d

B. 66-
31
P NMR Spectrum of 3.21d
pH = 6.30
274

B. 67-
1
H NMR Spectrum of 3.13

B. 68- HPLC chromatograph of the purification of NBoc-RIS-V2 (3.16)
Chrom. 1 0.0 mins. 28.3 mins.
4
3
2
1
NBoc-RIS-V2
Retention time: 27.0 min
RIS linker
Retention time: 7.50 min
275

B. 69-
1
H NMR Spectrum of 3.16

B. 70-
31
P NMR Spectrum of 3.16

N
P
HO
P
H
N
OH
OH
OH
O
OH
O
OH
O
O
4
NHBoc
pH = 5.86
276

B. 71-
1
H NMR Spectrum of 3.19

B. 72-
31
P NMR Spectrum of 3.19

pH = 8.3
277

B. 73- Chromatograms for C18 semi- preparative HPLC purification of the reaction mixture after
size exclusion chromatography of 5(6)-FAM-RIS-V2 (3.22a and 3.22b).

B. 74- UV-Vis spectra of 5- and 6-FAM-RIS-V2 (3.22a and 3.22b).
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
200 230 260 290 320 350 380 410 440 470 500 530
Absorbance
Wavelength (nm)
UV-VIS Spectra of 5- and 6-FAM-RIS-V2
5-FAM-ExtRis-V2
6-FAM-ExtRis-V2
Chrom. 1 0.0 mins. 70.8 mins.
1
4
3
2
RIS-linker-V2
Retention time: 5.20 min
5-FAM-RIS-V2
Retention time: 44.0 min
6-FAM-RIS-V2
Retention time: 33.9 min
5,6-FAM
Retention time: 12.0 and
27.0 min, respectively
278

B. 75- Fluorescence spectra of 5- and 6-FAM-RIS-V2 (3.22a and 3.22b).

B. 76- HPLC Chromatograph of S-Cy5-RIS-V2 (3.22c).
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
400 440 480 520 560 600 640 680 720 760 800
Normalized Intensity
Wavelength (nm)
Fluorescence Spectra of 5- and 6-FAM-RIS-V2
5-FAM-RIS-V2-Emission
5-FAM-RIS-V2-Excitation
6-FAM-RIS-V2-Emission
6-FAM-RIS-V2-Excitation
RIS-V2
Retention time: 3.0 min
Chrom. 1 0.0 mins. 27.3 mins.
1
S-Cy5-RIS-V2
Retention time: 23 min
S-Cy5 Dye
Retention time: 15.0 min
279

B. 77- UV-Vis spectrum of S-Cy5-RIS-V2 (3.22c)

B. 78- Fluorescence spectra of S-Cy5-RIS-V2 (3.22c)

-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
200 250 300 350 400 450 500 550 600 650 700 750 800
Absorbance
Wavelength (nm)
UV-Vis Spectrum of S-Cy5-RIS-V2
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
400 450 500 550 600 650 700 750 800
Normalized Intensity
Wavelength (nm)
Fluorescence Spectra of S-Cy5-RIS-V2
Emission
Excitation
280

B. 79- Chromatograms for semi- preparative HPLC purification of the reaction mixture of
AF647-RIS-V2 (3.22d)

B. 80- UV-Vis spectrum of AF647-RIS-V2 (3.22d)
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
200 250 300 350 400 450 500 550 600 650 700 750 800
Absorbance
Wavelength (nm)
UV-Vis Spectrum of AF-Ris-V2
RIS-V2 linker
Retention time: 5.4 min
AF647-RIS-V2
Retention time: 16.7 min
AF647 Dye
Retention time: 17.3. min
281

B. 81- Fluorescence spectra of AF647-RIS-V2 (3.22d)

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
400 450 500 550 600 650 700 750 800
Normalized Intensity
Wavelength (nm)
Fluorescence Spectra of AF647-RIS-V2
AF647-Ris-V2-Emission
AF647-Ris-V2-Excitation
282

B. 82-
1
H NMR in D2O of 5-FAM-RIS-V2 (3.22a)

B. 83-
31
P NMR in D2O of 5-FAM-RIS-V2 (3.22a)
pH = 6.0
283

B. 84-
1
H NMR in D2O of 6-FAM-RIS-V2 (3.22b)

B. 85-
31
P NMR in D2O of 6-FAM-RIS-V2 (3.22b)
pH = 6.3
284

B. 86-
1
H NMR Spectrum of 3.22c

B. 87-
31
P NMR Spectrum of 3.22c

285

B. 88-
1
H NMR in D2O of AF647-RIS-V2 (3.22d)
B. 89-
31
P NMR in D2O of AF647-RIS-V2 (3.22d)
pH = 6.3
286

B. 90- ESI-MS of 5-FAM-RIS (3.7a)
[M-2H]
-

[M-2H + Na]
-

287

B. 91- ESI-MS of 6-FAM-RIS (3.7b)
[M-2H]
-

[M-2H + Na]
-

288

B. 92- LC-MS of S-Cy5-RIS (3.7c)
[M-3H]
-

[M-4H]
2-

289

B. 93- ESI-MS of AF647-RIS (3.7d)
[M-5H]
3-

[M-5H + Na]
3-

290

B. 94- LC-MS of 5-FAM-RIS-V1 (3.20a)
[M-2H]
-

291

B. 95- HRMS of 6-FAM-RIS-V1 (3.20b)
292

B. 96- LC-MS of S-Cy5-RIS-V1 (3.20c)
[M-3H]
2-

[M-3H]
-

293

B. 97- ESI-MS of AF647-RIS-V1 (3.20d)
N
O
H
C
H
C
H
C
H
C
C
H
N
SO3H
HO
3
S
HO
3
S
SO3H
NH
O
HN
HO
N
P
P
OH
O
O
HO
OH
OH
OH
Chemical Formula: C
49
H
69
N
5
O
22
P
2
S
4
2+
Exact Mass: 1269.28
Molecular Weight: 1270.29
294

B. 98- LC-MS of 5-FAM-RIS-M (3.21a)
[M-2H]
-

295

B. 99- LC-MS of 6-FAM-RIS-M (3.21b)
[M-2H]
-

296

B. 100- LC-MS of S-Cy5-RIS-M (3.21c)
[M-3H]
2-

[M-3H]
2-
+ Acetate
297

B. 101- LC-MS of AF647-RIS-M (3.21d)
[M-3H]
2-

[M-3H + Na]
2-

[M-3H + 2Na]
2-

298

B. 102- HRMS of 5-FAM-RIS-V2 (3.22a)
299

B. 103- ESI-MS of 6-FAM-RIS-V2 (3.22b)
300

B. 104- LC-MS of S-Cy5-RIS-V2 (3.22c)
[M-2H]
2-

[M-2H]
-

301

B. 105- ESI-MS of AF647-RIS-V2 (3.22d)

[M-5H + Na]
2-

[M-5H]
3-

[M-5H + Na]
2-

[M-4H]
2-

302

APPENDIX C: Chapter 4 Supporting Data

303

Time
(h)
Abs at
493 nm
ε (M
-
1
cm
-1
)
V (µl) Amoun
t (µmol)
Total
Amount
(µmol)
Left Binding %
Binding
0 0.7766 73000 500 0.01064 0.01064 1 0 0
0.25 0.6775 73000 500 0.00928 0.01064 0.872 0.128 12.8
0.5 0.6412 73000 450 0.00791 0.00957 0.826 0.174 17.4
0.75 0.6273 73000 400 0.00687 0.00851 0.808 0.192 19.2
1 0.604 73000 350 0.00579 0.00745 0.778 0.222 22.2
1.25 0.6012 73000 300 0.00494 0.00638 0.774 0.226 22.6
1.5 0.5673 73000 250 0.00389 0.00532 0.730 0.270 27.0
24 0.2167 73000 200 0.00119 0.00426 0.279 0.721 72.1

C. 1- Preliminary data on the HAP binding of 5-FAM-RIS using method 1 at C ~ 20 µM for 1.5
h
Time
(h)
Abs at
493 nm
ε (M
-
1
cm
-1
)
V (µl) Amount
(µmol)
Total
Amount
(µmol)
Left Binding %
Binding
0 0.7904 73000 500 0.01083 0.01083 1.000 0.000 0
0.25 0.6883 73000 500 0.00943 0.01083 0.871 0.129 12.9
0.5 0.6559 73000 450 0.00809 0.00974 0.830 0.170 17.0
0.75 0.6395 73000 400 0.00701 0.00866 0.809 0.191 19.1
1 0.6293 73000 350 0.00603 0.00758 0.796 0.204 20.4
1.25 0.6221 73000 300 0.00511 0.00650 0.787 0.213 21.3
1.5 0.5908 73000 250 0.00405 0.00541 0.747 0.253 25.3
24 0.2628 73000 200 0.00144 0.00433 0.332 0.668 66.8

C. 2- Preliminary data on the HAP binding of 5-FAM-ZOL using method 1 at C ~ 20 µM for 1.5
h
Time
(h)
Abs @
493
nm
ε (M
-
1
cm
-1
)
Volum
e (µL)
Amount
(µmol)
Total
amount
(µmol)
Left Binding %
Binding
0 0.7653 73000 500 0.01048 0.01048 1.0000 0.0000 0.00
2 0.5305 73000 500 0.00727 0.01048 0.6932 0.3068 30.68
4 0.5181 73000 450 0.00639 0.00944 0.6770 0.3230 32.30
6 0.4895 73000 400 0.00536 0.00839 0.6396 0.3604 36.04
8 0.4688 73000 350 0.00450 0.00734 0.6126 0.3874 38.74
10 0.4517 73000 300 0.00371 0.00629 0.5902 0.4098 40.98
24 0.469 73000 250 0.00321 0.00524 0.6128 0.3872 38.72
27 0.4587 73000 200 0.00251 0.00419 0.5994 0.4006 40.06

C. 3 - Preliminary data on the HAP binding of 5-FAM-RIS using method 1 at C ~ 20 µM for 27
h
304

Time
(h)
Abs @
493
nm
ε (M
-
1
cm
-1
)
Volum
e (µL)
Amount
(µmol)
Total
amount
(µmol)
Left Binding %
Binding
0 0.7653 73000 500 0.01048 0.01048 1.0000 0.0000 0.00
2 0.5305 73000 500 0.00727 0.01048 0.6932 0.3068 30.68
4 0.5181 73000 450 0.00639 0.00944 0.6770 0.3230 32.30
6 0.4895 73000 400 0.00536 0.00839 0.6396 0.3604 36.04
8 0.4688 73000 350 0.00450 0.00734 0.6126 0.3874 38.74
10 0.4517 73000 300 0.00371 0.00629 0.5902 0.4098 40.98
24 0.469 73000 250 0.00321 0.00524 0.6128 0.3872 38.72
27 0.4587 73000 200 0.00251 0.00419 0.5994 0.4006 40.06

C. 4- Preliminary data on the HAP binding of 5-FAM-ZOL using method 1 at C ~ 20 µM for 27
h
Time
(h)
Abs @
493
nm
ε (M
-
1
cm
-1
)
Volum
e (µL)
Amount
(µmol)
Total
amount
(µmol)
Left Binding %
Binding
0 0.3707 73000 500 0.0051 0.0051 1.0000 0.000 0.0
2 0.2656 73000 500 0.0036 0.0051 0.7165 0.284 28.4
4 0.2348 73000 450 0.0029 0.0046 0.6334 0.367 36.7
6 0.2086 73000 400 0.0023 0.0041 0.5627 0.437 43.7
8 0.1869 73000 350 0.0018 0.0036 0.5042 0.496 49.6
10 0.1885 73000 300 0.0015 0.0030 0.5085 0.492 49.2
24 0.1448 73000 250 0.0010 0.0025 0.3906 0.609 60.9
27 0.1414 73000 200 0.0008 0.0020 0.3814 0.619 61.9

C. 5- Preliminary data on the HAP binding of 5-FAM-RIS using method 1 at C ~ 10 µM for 27 h

Time
(h)
Abs @
493
nm
ε (M
-
1
cm
-1
)
Volum
e (µL)
Amount
(µmol)
Total
amount
(µmol)
Left Binding %
Binding
0 0.3357 73000 500 0.00460 0.00460 1.000 0.000 0.00
2 0.2171 73000 500 0.00297 0.00460 0.647 0.353 35.33
4 0.1928 73000 450 0.00238 0.00414 0.574 0.426 42.57
6 0.1789 73000 400 0.00196 0.00368 0.533 0.467 46.71
8 0.1724 73000 350 0.00165 0.00322 0.514 0.486 48.64
10 0.1659 73000 300 0.00136 0.00276 0.494 0.506 50.58
24 0.1338 73000 250 0.00092 0.00230 0.399 0.601 60.14
27 0.1339 73000 200 0.00073 0.00184 0.399 0.601 60.11

C. 6- Preliminary data on the HAP binding of 5-FAM-ZOL using method 1 at C ~ 10 µM for 27
h
305

Time
(h)
Abs @
493
nm
ε (M
-
1
cm
-1
)
Volum
e (µL)
Amount
(µmol)
Total
amount
(µmol)
Left Binding %
Binding
0 0.1746 73000 500 0.002392 0.002392 1.000 0.000 0.000
2 0.0972 73000 500 0.001332 0.002392 0.557 0.443 44.3
4 0.0841 73000 450 0.001037 0.002153 0.482 0.518 51.8
6 0.0775 73000 400 0.000849 0.001913 0.444 0.556 55.6
8 0.0696 73000 350 0.000667 0.001674 0.399 0.601 60.1
10 0.0599 73000 300 0.000572 0.001435 0.399 0.601 60.1
24 0.042 73000 250 0.000410 0.001196 0.343 0.657 65.7
27 0.0387 73000 200 0.000230 0.000957 0.241 0.759 75.9

C. 7- Preliminary data on the HAP binding of 5-FAM-RIS using method 1 at C ~ 5 µM for 27 h

Time
(h)
Abs @
493
nm
ε (M
-
1
cm
-1
)
Volum
e (µL)
Amount
(µmol)
Total
amount
(µmol)
Left Binding %
Binding
0 0.1976 73000 500 0.002707 0.002707 1 0 0
2 0.1342 73000 500 0.001838 0.002707 0.6791 0.3209 32.09
4 0.1079 73000 450 0.001330 0.002436 0.5461 0.4539 45.39
6 0.0939 73000 400 0.001029 0.002165 0.4752 0.5248 52.48
8 0.0877 73000 350 0.000841 0.001895 0.4438 0.5562 55.62
10 0.0766 73000 300 0.000630 0.001624 0.3877 0.6123 61.23
24 0.0372 73000 250 0.000255 0.001353 0.1883 0.8117 81.17
27 0.0394 73000 200 0.000216 0.001083 0.1994 0.8006 80.06

C. 8- Preliminary data on the HAP binding of 5-FAM-ZOL using method 1 at C ~ 5 µM for 27 h
Time Trial 1 Trial 2 Trial 3 Avg. Ratio (Measured / Theoritical V)
0 500 500 500 500 1.00
20 435 435 435 435 0.97
40 375 380 375 376.7 0.94
60 325 320 315 320 0.91
80 265 270 260 265 0.88
100 210 200 200 203.3 0.81
120 145 150 140 145 0.73
140 95 85 85 88.3 0.59

C. 9- Volume calibration of sample at each time point using method 1
306

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 15.67 13.84 15.42
Diameter (Mm) 5.0 5.0 5.1
Thickness (Mm) 0.3 0.2 0.2
Total Surface Area 44.0 42.4 44.0

Time
(Min)
Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 27.27 0.620 28.89 0.682 31.20 0.708 0.670 0.045 0.026
40 41.03 0.933 41.07 0.969 41.55 0.943 0.949 0.018 0.011
60 50.31 1.144 51.51 1.215 51.20 1.163 1.174 0.037 0.021
80 56.22 1.279 51.78 1.222 59.06 1.341 1.280 0.060 0.035
100 62.27 1.416 53.12 1.253 67.72 1.538 1.402 0.143 0.082
120 68.24 1.552 61.83 1.459 74.24 1.686 1.566 0.114 0.066
140 71.98 1.637 68.11 1.607 75.76 1.720 1.655 0.059 0.034

C. 10- Data table on the HAP binding of 5-FAM-RIS using method 1

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 11.72 14.2 12.64
Diameter (Mm) 4.95 5.1 5.1
Thickness (Mm) 0.2 0.2 0.2
Total Surface Area 41.6 44.0 44.0

Time
(Min)
Trial 1 Trial 1
Norm
Trial 2 Trial 2
Norm
Trial 3 Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 26.22 0.631 17.71 0.402 4.33 0.098 0.377 0.267 0.154
40 22.88 0.550 23.29 0.529 16.20 0.368 0.482 0.100 0.058
60 28.90 0.695 14.06 0.319 19.97 0.454 0.489 0.190 0.110
80 31.37 0.755 35.15 0.798 23.25 0.528 0.694 0.145 0.084
100 31.57 0.759 44.52 1.011 28.56 0.649 0.806 0.186 0.107
120 39.80 0.957 51.62 1.172 41.62 0.945 1.025 0.128 0.074
140 46.02 1.107 57.82 1.313 41.55 0.943 1.121 0.185 0.107

C. 11- Data table on the HAP binding of 5-FAM-RIS using method 2

307

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 7.670 11.670 7.390
Diameter (Mm) 4.850 4.900 5.000
Thickness (Mm) 0.100 0.200 0.100
Total Surface Area 38.5 40.8 40.8

Time
(Min)
Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 20.84 0.474 16.25 0.383 14.29 0.325 0.394 0.075 0.044
40 24.34 0.554 28.33 0.668 27.03 0.614 0.612 0.057 0.033
60 30.94 0.704 37.87 0.893 33.22 0.754 0.784 0.098 0.057
80 38.09 0.866 42.33 0.999 38.81 0.881 0.915 0.072 0.042
100 43.62 0.992 45.49 1.073 46.00 1.045 1.037 0.041 0.024
120 48.66 1.107 51.04 1.204 49.49 1.124 1.145 0.052 0.030
140 71.00 1.615 58.07 1.370 55.94 1.270 1.418 0.177 0.102

C. 12- Data table on the HAP binding of 6-FAM-RIS using method 1

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 10.490 11.750 6.570
Diameter (Mm) 4.900 5.100 5.000
Thickness (Mm) 0.200 0.200 0.100
Total Surface Area 40.8 44.0 40.8

Time
(Min)
Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 27.15 0.666 24.72 0.561 22.13 0.542 0.590 0.067 0.038
40 44.01 1.079 38.69 0.879 30.98 0.759 0.906 0.162 0.093
60 52.52 1.288 46.48 1.055 38.47 0.942 1.095 0.176 0.102
80 56.46 1.385 58.45 1.327 45.95 1.126 1.279 0.136 0.079
100 58.05 1.424 63.75 1.448 49.29 1.207 1.360 0.132 0.076
120 62.54 1.534 66.00 1.499 56.36 1.381 1.471 0.080 0.046
140 66.95 1.642 68.93 1.565 64.97 1.592 1.600 0.039 0.022

C. 13- Data table on the HAP binding of 5-FAM-ZOL using method 1

308

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 16.080 7.420 8.580
Diameter (Mm) 5.100 4.900 4.900
Thickness (Mm) 0.200 0.100 0.100
Total Surface Area 44.0 39.2 39.2

Time
(Min)
Trial 1 Trial 1
Norm
Trial 2 Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 0.60 0.014 2.62 0.067 5.73 0.146 0.075 0.067 0.038
40 1.35 0.031 1.73 0.044 1.59 0.040 0.038 0.007 0.004
60 -1.56 -0.035 2.96 0.076 -0.14 -0.003 0.012 0.057 0.033
80 5.12 0.116 4.03 0.103 3.38 0.086 0.102 0.015 0.009
100 6.94 0.158 8.24 0.210 8.55 0.218 0.195 0.033 0.019
120 12.21 0.277 8.19 0.209 14.31 0.365 0.284 0.078 0.045
140 15.96 0.362 16.33 0.416 15.87 0.404 0.394 0.028 0.016

C. 14- Data table on the HAP binding of AF647-RISPC using method 1

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 16.970 10.760 22.060
Diameter (Mm) 5.000 5.100 5.100
Thickness (Mm) 0.200 0.100 0.350
Total Surface Area 42.4 42.4 46.4

Time
(Min)
Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 26.99 0.637 27.47 0.647 40.40 0.870 0.718 0.132 0.076
40 44.32 1.045 36.51 0.860 53.93 1.161 1.022 0.152 0.088
60 50.45 1.190 49.17 1.159 63.16 1.360 1.236 0.108 0.063
80 68.10 1.606 58.65 1.382 70.89 1.526 1.505 0.114 0.066
100 55.66 1.313 69.57 1.639 72.93 1.570 1.508 0.172 0.099
120 69.36 1.636 79.04 1.862 76.42 1.646 1.715 0.128 0.074
140 74.57 1.759 83.59 1.970 77.53 1.669 1.799 0.154 0.089

C. 15- Data table on the HAP binding of ROX-RISPC using method 1

309

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 13.980 12.580 15.200
Diameter (Mm) 4.950 5.100 5.000
Thickness (Mm) 0.200 0.200 0.200
Total Surface Area 41.6 44.0 42.4

Time
(Min)
Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 73.19 1.760 58.11 1.319 67.33 1.588 1.556 0.222 0.128
40 86.64 2.084 78.27 1.777 88.77 2.094 1.985 0.180 0.104
60 92.48 2.224 88.01 1.999 95.38 2.250 2.158 0.138 0.080
80 93.66 2.253 91.03 2.067 94.79 2.236 2.185 0.103 0.059
100 94.51 2.273 79.80 1.812 90.44 2.134 2.073 0.236 0.136
120 89.22 2.146 90.41 2.053 95.69 2.257 2.152 0.102 0.059
140 95.88 2.306 96.32 2.187 87.80 2.071 2.188 0.117 0.068

C. 16- Data table on the HAP binding of ROX-RIS using method 1

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 8.960 8.370 7.160
Diameter (Mm) 4.800 4.900 4.950
Thickness (Mm) 0.100 0.100 0.100
Total Surface Area 37.7 39.2 40.0

Time
(Min)
Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
4
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 17.11 0.454 13.12 0.334 9.64 0.241 0.343 0.107 0.062
40 17.78 0.472 21.11 0.538 13.34 0.333 0.448 0.104 0.060
60 25.55 0.678 18.11 0.461 16.93 0.423 0.521 0.138 0.079
80 23.93 0.635 28.20 0.719 22.90 0.572 0.642 0.074 0.042
100 32.40 0.860 34.84 0.888 27.38 0.684 0.811 0.111 0.064
120 33.06 0.877 42.39 1.080 35.29 0.882 0.946 0.116 0.067
140 40.33 1.070 48.43 1.234 41.69 1.042 1.115 0.104 0.060

C. 17- Data table on the HAP binding of AF647-RIS using method 1

310

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 8.790 8.100 8.520
Diameter (Mm) 5.000 5.100 4.800
Thickness (Mm) 0.100 0.100 0.100
Total Surface Area 40.8 42.4 37.7

Time
(Min)
Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 42.97 1.053 32.05 0.755 32.60 0.865 0.891 0.150 0.087
40 60.34 1.478 59.11 1.393 41.33 1.097 1.323 0.200 0.116
60 65.48 1.604 58.30 1.374 53.40 1.417 1.465 0.122 0.071
80 76.74 1.880 70.73 1.667 63.98 1.698 1.748 0.115 0.066
100 92.17 2.258 76.50 1.803 71.10 1.887 1.983 0.242 0.140
120 89.86 2.201 82.45 1.943 76.72 2.036 2.060 0.131 0.076
140 91.36 2.238 90.50 2.133 83.49 2.216 2.195 0.056 0.032

C. 18- Data table on the HAP binding of S-Cy5-RIS using method 1

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 20.960 10.220 12.160
Diameter (Mm) 4.950 4.900 5.000
Thickness (Mm) 0.300 0.200 0.200
Total Surface Area 43.1 40.8 42.4

Time
(Min)
Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 25.74 0.597 25.58 0.627 22.93 0.541 0.588 0.044 0.025
40 46.67 1.082 36.77 0.902 38.70 0.913 0.966 0.101 0.058
60 51.59 1.196 44.89 1.101 45.84 1.081 1.126 0.061 0.035
80 58.14 1.348 42.87 1.052 47.24 1.114 1.171 0.156 0.090
100 58.65 1.360 50.51 1.239 51.77 1.221 1.273 0.075 0.044
120 67.49 1.565 54.11 1.327 57.02 1.345 1.412 0.132 0.076

C. 19- Data table on the HAP binding of 5-FAM-RIS-V1 using method 1

311

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 13.290 13.330 15.690
Diameter (Mm) 5.000 5.100 5.000
Thickness (Mm) 0.200 0.200 0.300
Total Surface Area 42.4 44.0 44.0

Time
(Min)
Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 12.67 0.299 23.53 0.534 15.59 0.355 0.396 0.123 0.071
40 29.27 0.691 33.61 0.763 32.33 0.736 0.730 0.037 0.021
60 29.25 0.690 38.92 0.884 36.51 0.831 0.801 0.100 0.058
80 35.92 0.847 41.69 0.947 41.92 0.954 0.916 0.059 0.034
100 41.08 0.969 49.81 1.131 47.55 1.082 1.061 0.083 0.048
120 45.32 1.069 54.25 1.232 63.96 1.455 1.252 0.194 0.112

C. 20- Data table on the HAP binding of 6-FAM-RIS-V1 using method 1

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 9.460 10.280 7.580
Diameter (Mm) 4.900 4.800 5.050
Thickness (Mm) 0.200 0.200 0.100
Total Surface Area 40.8 39.2 41.6

Time
(Min)
Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 11.15 0.273 4.74 0.121 3.68 0.088 0.161 0.099 0.057
40 14.27 0.350 9.07 0.231 9.77 0.235 0.272 0.067 0.039
60 13.89 0.341 11.27 0.288 8.94 0.215 0.281 0.063 0.037
80 17.79 0.436 13.06 0.333 17.21 0.413 0.394 0.054 0.031
100 22.27 0.546 17.20 0.439 20.13 0.484 0.490 0.054 0.031
120 27.79 0.682 26.31 0.671 23.78 0.571 0.641 0.061 0.035
140 36.31 0.891 30.50 0.778 29.49 0.708 0.792 0.092 0.053

C. 21- Data table on the HAP binding of AF647-RIS-V1 using method 1

312

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 8.560 8.220 9.020
Diameter (Mm) 4.900 4.800 4.900
Thickness (Mm) 0.100 0.100 0.200
Total Surface Area 39.2 37.7 40.8

Time
(Min)
Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 23.52 0.599 21.54 0.572 24.37 0.598 0.590 0.016 0.009
40 36.84 0.939 40.45 1.074 36.98 0.907 0.973 0.088 0.051
60 46.94 1.196 43.60 1.157 49.08 1.204 1.186 0.025 0.014
80 53.55 1.365 50.97 1.353 55.50 1.361 1.360 0.006 0.004
100 60.17 1.534 58.73 1.559 65.62 1.610 1.567 0.039 0.022
120 70.53 1.798 62.80 1.667 72.13 1.769 1.744 0.069 0.040
140 80.28 2.046 74.11 1.967 79.50 1.950 1.988 0.051 0.030

C. 22- Data table on the HAP binding of S-Cy5-RIS-V1 using method 1

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 10.830 7.880 13.490
Diameter (Mm) 5.000 4.900 5.000
Thickness (Mm) 0.200 0.100 0.200
Total Surface Area 42.4 39.2 42.4

Time
(Min)
Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 26.78 0.632 29.72 0.758 7.63 0.180 0.523 0.304 0.175
40 39.50 0.932 45.95 1.171 30.18 0.712 0.938 0.230 0.133
60 41.86 0.988 53.16 1.355 37.34 0.881 1.074 0.249 0.144
80 45.56 1.075 61.16 1.559 38.73 0.914 1.182 0.336 0.194
100 55.03 1.298 39.49 1.007 46.68 1.101 1.135 0.149 0.086
120 60.42 1.425 69.82 1.780 55.28 1.304 1.503 0.247 0.143
140 67.20 1.585 76.87 1.959 61.37 1.448 1.664 0.265 0.153

C. 23- Data table on the HAP binding of 5-FAM-RIS-M using method 1
313

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 10.830 10.170 11.040
Diameter (Mm) 5.000 5.000 4.900
Thickness (Mm) 0.200 0.200 0.200
Total Surface Area 42.4 42.4 40.8

Time
(Min)
Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 29.33 0.692 24.75 0.584 28.35 0.695 0.657 0.063 0.037
40 42.34 0.999 34.22 0.807 40.48 0.993 0.933 0.109 0.063
60 44.49 1.049 41.08 0.969 42.20 1.035 1.018 0.043 0.025
80 44.75 1.056 43.55 1.027 45.70 1.121 1.068 0.048 0.028
100 55.51 1.310 56.27 1.327 53.55 1.313 1.317 0.009 0.005
120 63.77 1.504 58.73 1.386 60.63 1.487 1.459 0.064 0.037
140 70.64 1.666 60.06 1.417 68.13 1.671 1.585 0.145 0.084

C. 24- Data table on the HAP binding of 6-FAM-RIS-M using method 1

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 9.370 8.970 6.700
Diameter (Mm) 4.750 4.850 4.700
Thickness (Mm) 0.100 0.100 0.100
Total Surface Area 36.9 38.5 36.2

Time (Min) Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
4
Trial 3
Norm
Avg. Std
Dev
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000
20 16.67 0.451 14.91 0.388 10.08 0.279 0.373 0.087
40 16.68 0.452 12.25 0.319 10.49 0.290 0.354 0.086
60 20.68 0.560 20.81 0.541 13.77 0.381 0.494 0.098
80 19.82 0.537 25.24 0.656 7.37 0.204 0.466 0.235
100 27.93 0.757 31.43 0.817 23.75 0.657 0.744 0.081
120 35.44 0.960 29.52 0.768 31.03 0.858 0.862 0.096
140 39.22 1.062 43.95 1.143 39.30 1.087 1.097 0.041

C. 25- Data table on the HAP binding of AF647-RIS-M using method 1
314

Trial Trial 4 Trial 2 Trial 3
Mass (Mg) 6.630 8.100 8.520
Diameter (Mm) 4.800 5.100 4.800
Thickness (Mm) 0.100 0.100 0.100
Total Surface Area 37.7 42.4 37.7

Time
(Min)
Trial 4 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 32.96 0.875 40.06 0.944 34.30 0.910 0.910 0.035 0.020
40 35.35 0.938 41.84 0.986 52.45 1.392 1.105 0.249 0.144
60 42.47 1.127 55.36 1.305 59.18 1.571 1.334 0.223 0.129
80 63.56 1.687 67.35 1.587 68.40 1.815 1.696 0.114 0.066
100 64.37 1.708 76.55 1.804 75.04 1.992 1.835 0.144 0.083
120 63.93 1.697 85.37 2.012 80.15 2.127 1.945 0.223 0.129
140 77.62 2.060 91.34 2.152 84.70 2.248 2.153 0.094 0.054

C. 26- Data table on the HAP binding of S-Cy5-RIS-M using method 1

Trial Trial 1 Trial 5 Trial 3
Mass (Mg) 16.370 11.580 11.380
Diameter (Mm) 5.000 4.900 4.950
Thickness (Mm) 0.200 0.200 0.200
Total Surface Area 42.4 40.8 41.6

Time
(Min)
Trial 1 Trial 1
Norm
Trial
5
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 26.53 0.626 18.74 0.460 25.73 0.619 0.568 0.094 0.054
40 35.34 0.834 28.17 0.691 32.64 0.785 0.770 0.073 0.042
60 38.43 0.907 38.48 0.944 39.70 0.955 0.935 0.025 0.015
80 44.52 1.050 44.81 1.099 42.91 1.032 1.061 0.035 0.020
100 55.56 1.311 49.73 1.220 46.15 1.110 1.214 0.100 0.058
120 62.85 1.483 59.23 1.453 49.15 1.182 1.373 0.166 0.096
140 71.20 1.680 67.77 1.662 63.13 1.518 1.620 0.088 0.051

C. 27- Data table on the HAP binding of 5-FAM-RIS-V2 using method
315

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 11.660 7.050 10.040
Diameter (Mm) 5.000 4.800 4.900
Thickness (Mm) 0.200 0.100 0.200
Total Surface Area 42.4 37.7 40.8

Time
(Min)
Trial 1 Trial 1
Norm
Trial
4
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 17.42 0.411 21.23 0.563 12.40 0.304 0.426 0.130 0.075
40 22.66 0.534 24.91 0.661 20.79 0.510 0.568 0.081 0.047
60 23.86 0.563 28.69 0.761 23.49 0.576 0.633 0.111 0.064
80 31.12 0.734 35.98 0.955 27.51 0.675 0.788 0.148 0.085
100 37.42 0.883 33.87 0.899 28.59 0.701 0.828 0.110 0.063
120 40.67 0.960 39.42 1.046 36.97 0.907 0.971 0.070 0.041
140 48.04 1.133 94.17 2.499 43.54 1.068 1.567 0.808 0.467

C. 28- Data table on the HAP binding of 6-FAM-RIS-V2 using method 1

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 8.510 7.050 10.590
Diameter (Mm) 5.000 4.900 4.800
Thickness (Mm) 0.100 0.100 0.100
Total Surface Area 40.8 39.2 37.7
Density/Area 0.208 0.180 0.281

Time
(Min)
Trial 1 Trial 1
Norm
Trial 2 Trial 3
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 -4.63 -0.114 4.86 0.124 -5.22 -0.139 -0.043 0.145 0.084
40 15.39 0.377 9.41 0.240 -2.80 -0.074 0.181 0.231 0.134
60 9.81 0.240 13.04 0.332 4.77 0.126 0.233 0.103 0.060
80 5.35 0.131 13.00 0.331 7.30 0.194 0.219 0.103 0.059
100 16.90 0.414 15.26 0.389 10.11 0.268 0.357 0.078 0.045
120 23.13 0.567 19.74 0.503 19.12 0.507 0.526 0.035 0.020
140 30.79 0.754 33.36 0.850 28.72 0.762 0.789 0.053 0.031

C. 29- Data table on the HAP binding of AF647-RIS-V2 using method 1
316

Trial Trial 1 Trial 2 Trial 3
Mass (Mg) 8.110 8.380 9.590
Diameter (Mm) 4.900 4.900 4.900
Thickness (Mm) 0.100 0.100 0.100
Total Surface Area 39.2 39.2 39.2

Time
(Min)
Trial 1 Trial 1
Norm
Trial
2
Trial 2
Norm
Trial
3
Trial 3
Norm
Avg. Std
Dev
Std
Error
0 0.00 0.000 0.00 0.000 0.00 0.000 0.000 0.000 0.000
20 25.57 0.652 21.88 0.558 29.80 0.759 0.656 0.101 0.058
40 36.57 0.932 34.40 0.877 34.91 0.890 0.899 0.029 0.017
60 51.77 1.319 61.69 1.572 37.92 0.966 1.286 0.304 0.176
80 41.34 1.054 52.47 1.337 34.40 0.877 1.089 0.232 0.134
100 56.76 1.447 57.35 1.462 49.37 1.258 1.389 0.113 0.065
120 51.56 1.314 65.09 1.659 58.53 1.492 1.488 0.172 0.100
140 91.93 2.343 70.39 1.794 59.84 1.525 1.887 0.417 0.241

C. 30- Data table on the HAP binding of S-Cy5-RIS-V2 using method 1

317

Method
Compounds
HAP Column ± Error HAP Discs ± Error
ROX-RIS 1.554 0.001046 1.478 0.1305
S-Cy5-RIS N/A N/A 1.414 0.1298
S-Cy5-RIS-M N/A N/A 1.308 0.0971
ZOL 1.287 0.000645 N/A N/A
S-Cy5-RIS-V1 N/A N/A 1.117 0.0676
5(6)-RhR-RIS 1.085 0.000875 N/A N/A
RIS 1.064 0.000479 N/A N/A
MIN 1.064 0.000479 N/A N/A
5(6)-FAM-RIS 1.000 0.0009 1.000 0.0832
800CW-ZOL 0.996 0.000531 N/A N/A
S-Cy5-RIS-V2 N/A N/A 0.990 0.0746
5-FAM-RIS-V1 N/A N/A 0.908 0.0618
6-FAM-RIS-M N/A N/A 0.939 0.0553
6-FAM-ZOL 0.877 0.000418 N/A N/A
5-FAM-RIS-V2 N/A N/A 0.865 0.0656
6-FAM-MIN 0.845 0.001336 N/A N/A
5-FAM-ZOL 0.844 0.00038 0.969 0.0788
ROX-RISPC 0.825 0.000937 1.075 0.0949
5-FAM-RIS-M V N/A 0.810 0.0776
AF647-ZOL 0.796 0.000462 N/A N/A
5-FAM-MIN 0.788 0.000829 N/A N/A
AF647-RIS 0.787 0.000418 0.578 0.0568
6-FAM-RIS-V1 N/A N/A 0.756 0.0561
6-FAM-RIS N/A N/A 0.739 0.0466
5(6)-RhR-RISPC 0.722 0.000766 N/A N/A
RIS 0.617 0.000359 N/A N/A
6-FAM-RIS-V2 N/A N/A 0.590 0.0570
MINPC 0.585 0.000263 N/A N/A
5-FAM-MINPC 0.559 0.000826 N/A N/A
5(6)-FAM-RISPC 0.550 0.000429 N/A N/A
AF647-RIS-M N/A N/A 0.530 0.0457
5(6)-FAM-dRIS 0.527 0.000298 N/A N/A
6-FAM-MINPC 0.524 0.000408 N/A N/A
RISPC 0.500 0.000325 N/A N/A
AF647-RISPC 0.489 0.00022 0.139 0.0158
AF647-RIS-V1 N/A N/A 0.349 0.0302
AF647-RIS-V2 N/A N/A 0.255 0.0354
C. 31- Summary of the relative HAP binding of dye-BP conjugates using two different methods
318

APPENDIX D: Chapter 6 Supporting Data
319

D. 1-
31
P NMR of reaction mixture after 23 h

D. 2- HPLC chromatograph of the purification of ZOL linker
Chrom. 1 0.0 mins. 45.0 mins.
4
3
2
1
Dialkylation N- and O- ZOL
linker
Retention time: 7.0 min
ZOL linker
Retention time: 11.0 min
320

D. 3-
1
H NMR of ZOL linker
D. 4-
31
P NMR of 6.4
321

Chrom. 1 0.0 mins. 38.8 mins.
6
5
4
3
2
1

D. 5- HPLC chromatograph of the purification of 5,6-FAM-ZOL

Zol linker (6.4)
Retention time: 7.0 min
6-FAM-ZOL
Retention time: 16.0 min
5-FAM-ZOL
Retention time: 28.3 min
322

D. 6-
1
H NMR of 5-FAM-ZOL
D. 7-
31
H NMR of 5-FAM-ZOL
P OH
P
HO
N
H
O HO O
N
N
HO
O
O
OH
OH
O
HO
HO
O
323

D. 8- UV-VIS spectrum of 5-FAMZOL

D. 9- Fluorescence spectrum of 5-FAM-ZOL
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
200 250 300 350 400 450 500 550 600
Absorbance
Wavelength (nm)
UV-Vis Spectrum OF 5-FAM-ZOL
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
400 450 500 550 600 650 700
Normalized Intensity
Wavelength (nm)
Fluorescence Spectra of 5-FAM-ZOL
Emission OF 5-FAM-ZOL
Excitation of 5-FAM-ZOL
324

D. 10-
1
H NMR spectrum of 6-FAMZOL
D. 11 -
31
P NMR spectrum of 6-FAMZOL
325

D. 12-UV-VIS spectrum of 6-FAMZOL

D. 13-Fluorescence spectrum of 6-FAMZOL

0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
190 220 250 280 310 340 370 400 430 460 490 520 550
Absorbance
Wavelength (nm)
UV-Vis Spectrum of 6-FAMZol
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
300 340 380 420 460 500 540 580 620 660 700
Normalized Intensity
Wavelength (nm)
Fluorescence Spectrum of 6-FAMZol
Emission
Excitation
326

D. 14- Mass spectrum (ESI-MS, negative mode) of 6.5
P OH
P
HO
N
H
O HO O
N
N
HO
O
O
OH
OH
O
HO
HO
O
Chemical Formula: C
29
H
28
N
3
O
14
P
2
+
Exact Mass: 704.10
Molecular Weight: 704.50
[M-2H]
2-

327

D. 15- Mass spectrum (ESI-MS, negative mode) of 6.6
[M-2H]
2-

328

D. 16-
31
P NMR spectra to confirm in vitro release of RIS from its formulation with P407

Concentration (M) Average absorbance at 206 nm Standard Error
6.77717E-05 0.2964 0.003
8.47146E-05 0.3612 0.005
0.000135543 0.5911 0.001
0.000169429 0.7177 0.005
0.000203315 0.8593 0.004
0.000254144 1.0719 0.006
0.000271087 1.1565 0.002
0.000304973 1.2929 0.003
0.000338858 1.4249 0.004

D. 17- Data for the calibration curve of ZOL

Phosphate
from PBS
buffer
Standard
(Phosphoric
Acid)
Standard
(Phosphoric
Acid)
Standard
(Phosphoric
Acid)
Phosphate
from PBS
buffer
Phosphate
from PBS
buffer
329

Time (h) 0 1 2 3 4 5 6
Trial 1 0.061 1.124 1.491 1.410 1.446 1.448 1.352
Trial 2 0.039 1.102 1.417 1.329 1.337 1.358 1.277
Trial 3 0.050 1.113 1.454 1.370 1.392 1.403 1.315
Average 0.150 1.213 1.554 1.470 1.491 1.503 1.414
Normalized 0.096 0.781 1.000 0.946 0.960 0.967 0.910
D. 18- Data for the release of 5 mM ZOL from 17% w/w P407 using 12 mm diameter membrane.
The data was presented as cumulative release of ZOL over time.

Time (h) 0 1 2 3 4 5 6
Trial 1 0.043 0.812 0.993 0.980 0.953 0.928 0.875
Trial 2 0.035 0.810 0.992 0.979 0.953 0.929 0.888
Trial 3 0.010 0.759 0.935 0.922 0.897 0.874 0.876
Average 0.095 0.878 1.062 1.049 1.023 0.998 0.976
Normalized 0.089 0.827 1.000 0.988 0.963 0.940 0.919
D. 19- Data for the release of 5 mM RIS from 17% w/w P407 using 12 mm diameter membrane.
The data was presented as cumulative release of RIS over time.

Time (h) 0 1 2 3 4 5 6
Trial 1 0.056 0.844 1.029 1.012 0.982 0.956 0.934
Trial 2 0.057 0.809 0.986 0.972 0.942 0.917 0.892
Trial 3 0.051 0.891 1.088 1.063 1.033 1.007 0.982
Average 0.110 0.904 1.090 1.071 1.041 1.016 0.992
Normalized 0.053 0.820 1.000 0.982 0.953 0.928 0.905
D. 20- Data for the release of 5 mM RIS with magnesium stearate from 17% w/w P407 using 12
mm diameter membrane. The data was presented as cumulative release of RIS over time.

Time (h) 0 1 2 3 4 5 6
Trial 1 0.006 0.789 1.031 1.045 1.017 0.986 0.961
Trial 2 0.011 0.856 1.092 1.101 1.059 1.031 1.004
Trial 3 0.008 0.823 1.062 1.073 1.038 1.009 0.983
Average 0.119 0.934 1.173 1.184 1.149 1.120 1.094
Normalized 0.008 0.767 0.990 1.000 0.968 0.940 0.916
D. 21- Data for the release of 5 mM RIS as tetrabutyl ammonium salt (2equivalents) from 17%
w/w P407 using 12 mm diameter membrane. The data was presented as cumulative release of RIS
over time.

330

Time (h) 0 1 2 3 4 5 6
Trial 1 0.315 0.776 1.300 1.366 1.412 1.357 1.300
Trial 2 0.302 0.550 1.123 1.317 1.292 1.261 1.211
Trial 3 0.274 0.462 0.988 1.262 1.320 1.248 1.197
Average 0.297 0.596 1.137 1.315 1.341 1.288 1.236
Normalized 0.222 0.444 0.848 0.981 1.000 0.961 0.922
D. 22- Data for the release of methylprenisolone hemisuccinate (MPS) from 17% w/w P407 using
6.5 mm diameter membrane. The data was presented as normalized cumulative release of MPS
over time.

Time (h) 0 1 2 3 4 5 6
Trial 1 0.012 0.464 0.588 0.634 0.639 0.630 0.621
Trial 2 0.014 0.422 0.612 0.630 0.625 0.620 0.613
Trial 3 0.031 0.502 0.675 0.690 0.686 0.678 0.669
Average 0.019 0.463 0.625 0.651 0.650 0.643 0.635
Normalized 0.029 0.710 0.959 1.000 0.998 0.987 0.974
D. 23- Data for the release of 2% w/w methylprenisolone hemisuccinate (MPS) from 17% w/w
P407 using 12 mm diameter membrane. The data was presented as normalized cumulative release
of MPS over time.

Time (h) 0 1 2 3 4 5 6
Trial 1 0.007 0.282 0.506 0.629 0.725 0.727 0.801
Trial 2 0.008 0.215 0.469 0.591 0.652 0.719 0.795
Trial 3 0.007 0.190 0.443 0.532 0.607 0.707 0.760
Average 0.007 0.229 0.473 0.584 0.661 0.717 0.785
D. 24- Data for the release of 2% w/w methylprenisolone hemisuccinate (MPS) from 25% w/w
P407 using 6.5 mm diameter membrane. The data was presented as cumulative release of MPS
over time.

Time (h) 0 1 2 3 4 5 6
Trial 1 0.108 0.666 1.555 1.494 1.471 1.324 1.326
Trial 2 0.084 0.736 1.735 1.575 1.525 1.456 1.426
Trial 3 0.109 0.655 1.529 1.496 1.485 1.382 1.294
Average 0.100 0.686 1.606 1.521 1.494 1.387 1.348
Normalized 0.062 0.427 1.000 0.947 0.930 0.864 0.839
D. 25- Data for the release of 5 mM RIS without P407 using 6.5 mm diameter membrane. The
data was presented as normalized cumulative release of RIS over time.

331

Time (h) 0 1 2 3 4 5 6
Trial 1 0.120 0.778 1.835 1.856 1.715 1.475 1.472
Trial 2 0.079 0.629 1.574 1.655 1.583 1.507 1.337
Trial 3 0.422 0.780 1.678 1.651 1.623 1.490 1.416
Average 0.252 0.760 1.740 1.713 1.635 1.508 1.419
Normalized 0.145 0.437 1.000 0.984 0.940 0.867 0.815
D. 26- Data for the release of 5 mM RIS with 17% w/w P407 using 6.5 mm diameter membrane.
The data was presented as normalized cumulative release of RIS over time.

Time (h) 0 1 2 3 4 5 6
Trial 1 0.001 0.241 0.670 0.815 0.862 0.884 0.871
Trial 2 0.018 0.195 0.564 0.771 0.877 0.910 0.914
Trial 3 0.018 0.163 0.526 0.751 0.791 0.799 0.798
Average 0.012 0.200 0.587 0.779 0.843 0.864 0.861
Normalized 0.014 0.231 0.679 0.901 0.975 1.000 0.996
D. 27- Data for the release of 5 mM RIS with 25% w/w P407 using 6.5 mm diameter membrane.
The data was presented as normalized cumulative release of RIS over time.

Time (h) 0 1 2 3 4 5 6
Trial 1 0.010 0.324 0.975 1.291 1.500 1.696 1.672
Trial 2 0.006 0.302 0.933 1.273 1.416 1.529 1.579
Trial 3 0.013 0.309 0.835 1.136 1.432 1.486 1.524
Average 0.010 0.312 0.914 1.233 1.450 1.571 1.592
Normalized 0.006 0.196 0.575 0.775 0.911 0.987 1.000
D. 28- Data for the release of 5 mM ZOL with 25% w/w P407 using 6.5 mm diameter membrane.
The data was presented as normalized cumulative release of ZOL over time.

Time (h) 0 1 2 3 4 5 6
Trial 1 0.012 0.717 0.827 0.831 0.821 0.809 0.797
Trial 2 0.036 0.604 0.666 0.667 0.658 0.655 0.645
Trial 3 0.017 0.581 0.683 0.688 0.678 0.671 0.663
Average 0.066 0.678 0.770 0.773 0.764 0.756 0.746
D. 29- Data for the release of 5 mM 5-FAM-ZOL with 17% w/w P407 using 12 mm diameter
membrane. The data was presented as normalized cumulative release of 5-FAM-ZOL over time.

332

Time (h) 0 1 2 3 4 5 6
Trial 1 0.017 0.658 0.717 0.715 0.709 0.698 0.689
Trial 2 0.027 0.631 0.693 0.694 0.684 0.674 0.667
Trial 3 0.020 0.667 0.725 0.723 0.715 0.705 0.697
Average 0.065 0.696 0.756 0.754 0.746 0.737 0.728
D. 30- Data for the release of 5 mM 6-FAM-ZOL with 17% w/w P407 using 12 mm diameter
membrane. The data was presented as normalized cumulative release of 6-FAM-ZOL over time.

Time (h) 0 1 2 3 4 5 6
Trial 1 0.107 0.328 0.513 0.532 0.517 0.509 0.500
Trial 2 0.120 0.297 0.491 0.528 0.510 0.508 0.473
Trial 3 0.101 0.273 0.440 0.466 0.462 0.453 0.441
Average 0.109 0.299 0.482 0.508 0.496 0.490 0.471
Normalized 0.215 0.588 0.947 1.000 0.977 0.964 0.927
D. 31- Data for the release of 5 mM 5-FAM-ZOL with 17% w/w P407 using 6.5 mm diameter
membrane. The data was presented as normalized cumulative release of 5-FAM-ZOL over time.

Time (h) 0 1 2 3 4 5 6
Trial 1 0.000021 0.151 0.297 0.408 0.485 0.523 0.541
Trial 2 0.00235 0.167 0.301 0.411 0.469 0.508 0.528
Trial 3 0.00846 0.150 0.271 0.378 0.453 0.496 0.512
Average 0.00361 0.156 0.290 0.399 0.469 0.509 0.527
Normalized 0.00685 0.296 0.549 0.757 0.890 0.966 1.000
D. 32- Data for the release of 5 mM 5-FAM-ZOL with 25% w/w P407 using 6.5 mm diameter
membrane. The data was presented as normalized cumulative release of 5-FAM-ZOL over time.

333

APPENDIX E: Chapter 7 Supporting Data
334

E. 1-
1
H NMR Spectrum of 7.1
335

E. 2- LC-MS of 7.1
m/z
Dimer of 7.1
[M-H]
-

336

E. 3- Flash Column Chromatograph of the Purification of 7.3
BnO OBn
O
OBn
337

E. 4-
1
H NMR Spectrum of 7.3

E. 5-
13
C NMR Spectrum of 7.3
BnO OBn
O
OBn
BnO OBn
O
OBn
338

E. 6-
1
H NMR Spectrum of 7.5a
E. 7-
13
C NMR Spectrum of 7.5a
339

E. 8- Flash Column Chromatograph of the Purification of 7.5b
340

E. 9-
1
H NMR Spectrum of 7.5b
E. 10-
13
C NMR Spectrum of 7.5b
OBn
BnO OBn
O
O
OH
OBn
BnO OBn
O
O
OH
341

E. 11- Flash Column Chromatograph of the Purification of 7.6
OBn
BnO OH
O
O
OH
342

E. 12-
1
H NMR Spectrum of 7.6
E. 13-
13
C NMR Spectrum of 7.6
OBn
BnO OH
O
O
OH
OBn
BnO OH
O
O
OH
343

E. 14-
1
H NMR Spectrum of 7.7
E. 15-
13
C NMR Spectrum of 7.7
344

E. 16-
1
H NMR Spectrum of 7.8

345

E. 17- LC-MS of 7.8

[M-H]
-

346

E. 18 - Flash Column Chromatograph of the Purification of 7.10
347

E. 19-
1
H NMR Spectrum of 7.10

E. 20-
1
H NMR Spectrum of 7.11
348

E. 21- Flash Column Chromatograph of the Purification of 7.11
349

E. 22- LC-MS Analysis of 7.11
7.11 +TFA
Dimer of 7.11
Dimer of
7.11 + TFA
[M-H]
-

350

E. 23-
1
H NMR Spectrum of 7.12

E. 24-
1
H NMR Spectrum of 7.13
351

E. 25-
31
P NMR Spectrum of 7.13

E. 26-
1
H NMR Spectrum of 7.16
352

E. 27-
1
H NMR Spectrum of 7.17
E. 28-
31
P NMR Spectrum of 7.17
pH = 8.3
353

E. 29-
1
H NMR Spectrum of 7.19

E. 30-
31
P NMR Spectrum of 7.19

354

E. 31- Mass Spectrum of 7.19
7.19
N
P
HO
P
H
N
OH
OH
OH
O
OH
O
OH
O
O
4
N
H
O
O
OH
O
OH
Chemical Formula: C
37
H
48
N
3
O
18
P
2
+
Exact Mass: 884.24
MolecularWeight: 884.74
[M-2H]
-

355

E. 32- HPLC chromatograph for 7.19
E. 33-
1
H NMR Spectrum of 7.20
Chrom. 1 0.0 mins. 25.1 mins.
1
OH
HO O
O
O
O
N
O
O
7.26
t retention: 7.5 min
7.25
t retention: 12.8 min
7.19
t retention: 21.2 min
356

E. 34-
1
H NMR Spectrum of 7.20
[M-H]
-

357

E. 35-
1
H NMR Spectrum of 7.22

E. 36-
1
H NMR Spectrum of 7.23
358

Supplemental Figure 1 -
1
H NMR Spectrum of 7.24

359

E. 37- Mass spectrum of 7.24
7.24 + Na
360

E. 38-
1
H NMR Spectrum of 7.25
O
O
4
NH
O
O
O
O
OH
OH
N
O
O
361

E. 39- Mass spectrum of 7.25
7.25 + Na
362

E. 40-
1
H NMR Spectrum of 7.26
E. 41-
31
P NMR Spectrum of 7.26

Abstract (if available)

Abstract Bisphosphonates are a commonly prescribed class of medications currently used to treat postmenopausal osteoporosis, Paget’s disease, bone metastatic cancers, and other bone-related diseases. They are stable analogues of pyrophosphates and belong to a class of compounds that contains a P-C-P backbone. The presence of the two phosphonate groups enables the compounds to chelate calcium, which is found in bone mineral. This chelating ability of bisphosphonates to calcium is responsible for their specific uptake by the bone, where the drugs inhibit osteoclast mediated bone resorption. ❧ The unique high bone binding affinity of bisphosphonates is an attractive feature that could be exploited for targeted delivery of other drugs to bone. This idea was further explored in the work presented in this thesis for various applications, including targeted delivery of therapeutics to the bone and bone imaging. Two categories of projects are covered in this thesis: 1) non-ear related studies and 2) ear-related studies. ❧ The first chapter briefly summarizes history of the development of bisphosphonate drugs, their mechanism of action, clinical applications, and side effects. The three subsequent chapters focus on work related to the synthesis and characterization of various nitrogen-containing bisphosphonate analogues used in targeted drug delivery and bone imaging. Topics covered in these chapters are 1) the synthesis of a novel N-containing bisphosphonate drug possessing an activated carboxyl moiety for targeted delivery of peptide-coated gold nanoparticles to bone metastatic niches

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Studies of bisphosphate-conjugated fluorescent imaging compounds and 8-oxo-dGTP derivatives

PDF

Fluorescent imaging probes of nitrogen-containing bone active drugs: design, synthesis and applications

PDF

Design and synthesis of bisphosphonate analogs as inhibitors of farnesyl pyrophosphate synthase

PDF

I. Microwave-assisted synthesis of phosphonic acids; II. Design and synthesis of polymerase β lyase domain inhibitors

PDF

Synthesis of fluorescent conjugates of risedronate and related analogues for bone imaging

PDF

Nucleophilic fluorination of bisphosphonates and its application in PET imaging

PDF

Deoxyribonucleoside triphosphate analogues for inhibition of therapeutically important enzymes

PDF

Chemical investigations in drug discovery and drug delivery

PDF

Optimization of nanomedicine based drug delivery systems for the treatment of solid tumors

PDF

Synthetic studies of phosphonate derivatives

PDF

Synthetic studies of chemical probes for i) DNA, ii) RNA polymerases and iii) tropomyosin receptor kinase

PDF

Multivalent smart elastin-like polypeptide therapeutics with drug delivery and biosensing applications.

PDF

Cationic cell penetrating peptides: characterization of transport properties in epithelial cells and their utilization as delivery systems for protein and peptide drugs

PDF

Nucleoside phosphonate prodrugs: synthesis and antiviral activity

PDF

Syntheses of a series of fluorous amphiphiles and modulation of the relaxivity of gadolinium(III)-based contrast agents

PDF

Ruthenium catalyzed hydrogen-borrowing amine alkylation reactions

PDF

Synthesis, structural analysis and in vitro antiviral activities of novel cyclic and acyclic (S)-HPMPA and (S)-HPMPC tyrosinamide prodrugs

PDF

Analytical investigation of the proteasome inhibitor Bortezomib and the total synthesis of specialized pro-resolving lipid mediators

PDF

The use of non-covalent interactions for the modification of nanoparticle surface and MRI contrast agent properties

PDF

Leveraging covalent modification for diverse applications in antiviral discovery and kinase mechanism deconvolution

Asset Metadata

Creator Kim, Nguyen L. T. (author)

Core Title Utilization of bisphosphonate drugs in fluorescent imaging and targeted drug delivery

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 11/15/2016

Defense Date 10/18/2016

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag bisphosphonate drugs,bone imaging,inner ear disorders,OAI-PMH Harvest,targeted drug delivery

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor McKenna, Charles E. (committee chair), Armani, Andrea (committee member), Williams, Travis J. (committee member)

Creator Email kimlnguy@usc.edu,ktvlpsfrn@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-321414

Unique identifier UC11213620

Identifier etd-KimNguyenL-4924.pdf (filename),usctheses-c40-321414 (legacy record id)

Legacy Identifier etd-KimNguyenL-4924.pdf

Dmrecord 321414

Document Type Dissertation

Format application/pdf (imt)

Rights Kim, Nguyen L. T.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA