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Uptake and transport of desmopressin and its lipidized derivatives in Caco-2 cells

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Uptake and transport of desmopressin and its lipidized derivatives in Caco-2 cells

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UPTAKE AND TRANSPORT OF DESMOPRESSIN AND ITS
LIPIDIZED DERIVATIVES IN CACO-2 CELLS
by
Yuan-chen Wu
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
August 2001
Copyright 2001 Yuan-chen Wu
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UMI Number: 1409610
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UMI
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UNIVERSITY OF SOUTHERN CALIFORNIA
The Graduate School
University Park
LOS ANGELES, CALIFORNIA 900894695
This thesis, w ritten b y
U nder th e direction o f h s u r . . . . Thesis
C om m ittee, and approved b y a ll its members,
has been presented to and accepted b y The
Graduate School, in partial fu lfillm en t o f
requirem ents fo r the degree o f
YlMKl-CHEN u lu
Master of Science
Dean o f Graduate Studies
D a te August 7, 2001
THESIS COMMITTEE
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ACKNOWLEDGMENTS
At first, I would like to express my appreciation to Dr. Wei-Chiang
Shen for his academic guidance and support during my graduate study in
USC. I am very grateful to have Dr. Shen as my supervisor.
I would like to specially thank Dr.Curtis Okamoto and Dr. David
Ann for being my committee members and giving advice in my thesis.
In addition, I would like to thank Daisy Shen for her technical
support and valuable assistance in Lab work and normal life.
I would also like to thank Dr. Jeff Wang for his help of preparation
of Desmopressin conjuagtes, discussions and companionship. Tinten Lim,
Cindy Xia, Li Wang, Adam Widera, Karin Belousson, Dr. Jun Wu,
Magarita Shen, Jennica Zaro for their friendship and supports in every
fields.
Finally, I would like to thank my parents and sister for their love
and support.
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TABLE OF CONTENTS
ACKNOWLEDGMENTS..........................................................ii
LIST OF FIGURES.................................................................... vi
ABSTRACT...............................................................................viii
INTRODUCTION........................................................................1
Advantages and limitations of peptide drugs.......................................................... 1
Overcome the limitations of peptide drugs.............................................................2
Background o f Desmopressin (DDAVP)................................................................ 3
Desmopressin: mechanism of action....................................................................... 5
Formation of reversible lipidized DDAVP........................................................... 12
1. Synthesis of L-cysteinyl 2-pyridyl disulfide (CPD).................................13
2. Synthesis of Pal-CPD.................................................................................14
3. Synthesis of DPP........................................................................................14
Caco-2 in vitro m odel............................................................................................ 15
MATERIALS AND METHODS______________________ 18
Chemicals................................................................................................................ 18
Cell line....................................................................................................................18
iii
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Routine cell culture for Caco-2 cells..................................................................... 19
Seeding cells to plates............................................................................................20
Iodination of proteins.............................................................................................21
Uptake study of cells..............................................................................................23
Uptake study of Caco-2 cells (Palmitic Acid Effect)..........................................24
Uptake study of Caco-2 cells (DTT, Palmitic Acid Effect)................................ 25
Pierce BCA protein assay.......................................................................................26
Transport study across Caco-2 cells...................................................................... 27
RESULTS__________________________________________ 29
Iodination............................................................................................................... 30
Cellular uptake.......................................................................................................30
Possible mechanism of increasing cellular uptake...............................................37
Transport................................................................................................................ 47
DISCUSSION.............................................................................50
1 .Can lipidization of peptide drugs increase cellular uptake?............................ 50
2. Does the charge of the linker effect cellular uptake?...................................... 51
3. Does cellular uptake of DPP increase viaconjugation with palmitic acid?.... 51
4. Is the increase of cellular uptake mediated by passive diffusion or active
transport?............................................................................................................ 53
iv
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5. Does better cellular uptake indicate better transport in Caco-2 cell model? .54
CONCLUSIONS____________________________________ 57
REFERENCES_____________________________________ 60
BIBLIOGRAPHY...................................................................... 68
V
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LIST OF FIGURES
FIGURE PAGE
Figure 1 . Structure of desmopressin and the possible cleavage site are 7
digested
Figure 2. Structure of desmopressin and DPP 8
Figure 3. Structure of DPP 9
Figure 4. Structure of DPO 10
Figure 5. Structure of DPOA 11
Figure 6. DPPsysthesis 17
Figure 7. DDAVP was radiolabled with 1 2 5 1 31
Figure 8. DPP was radiolabled w ith1 2 5 1 32
Figure 9. DPO was radiolabled w ith1 2 5 1 33
Figure 10. DPOA was radiolabled with 1 2 5 1 34
Figure 11. Uptake study of lipidized conjugates 35
Figure 12. Cellular uptake (dose response) 38
Figure 13. 1 2 5 1 -DDAVP cell uptake (different medium) 39
Figure 14. 1 2 5 1 -DPP cell uptake (different medium) 40
vi
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Figure 15. I2SI -DDAVP cell uptake with different concentrations
of palmitic acid 41
Figure 16. 1 2 5 1 -DPP cell uptake with different concentrations
of palmitic acid 42
Figure 17. 1 2 5 1 -DDAVP cell uptake (DTT, Palmitic acid effect) 43
Figure 18. 1 2 5 1 -DPP cell uptake (DTT, Palmitic acid effect) 44
Figure 19. Transport study o f 1 2 5 1 -DDAVP, 1 2 5 1 -D P P ,1 2 5 1 -DPO,
1 2 5 1 -DPOA 46
Figure 20. Membrane associated uptake of 1251 -D D A V P,1 2 5 1 -DPP,
12 5 1 -D P O ,1251 -DPOA 47
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Yuan-chen (Daphne) Wu Wei-Chiang S hen, Ph.D
ABSTRACT
UPTAKE AND TRANSPORT OF DESMOPRESSIN AND ITS
LIPIDIZED DERIVATIVES IN CACO-2 CELLS
The most serious limitations in oral administration of desmopressin
is its poor permeability across epithelium of the gastrointestinal tract due to
the large molecular weight, high hydrophilicity, and the susceptibility to
enzymatic or chemical degradation. Fatty acids with different chain lengths
and charges were conjugated with parent desmopressin by reversible
lipidization and were tested in cultured Caco-2 cells as an in vitro model
for their absorption and transport. We found that (a) the increase of cellular
uptake of conjugated desmopressin is mediated by the lipophilicity of the
fatty acid moiety in the conjugate, (b) conjugates with positively charged
fatty acids further enhance the increase of cellular uptake, and (c) Fatty
acid-desmopressin conjugates have a higher affinity towards cell
monolayers in vitro, but they may bind other serum proteins in vivo,
resulting in an increase of plasma half-life after I.V. administration.
viii
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INTRODUCTION
Advantages and limitations of peptide drugs
There are many advantages of using peptides and proteins as
therapeutic drugs for the treatment of human diseases (1,2), because they
are highly potent and specific, and metabolites of them are nontoxic amino
acids. Besides this, we can prepare high purity of human peptides or
proteins in very large quantities by using recombinant DNA technologies.
Protein and peptide base drugs become a very ideal approach for
therapeutic use. But not many therapeutic peptides and proteins drugs for
human diseases are now available in the market. The most severe problem
of them is due to limitations on the delivery of peptide and protein drugs.
So the effective delivery of therapeutic peptides and proteins to their site of
action become a big issue have to overcome in designing a peptide or
protein base drug.
The most serious limitation in protein and peptide drug delivery is
the poor permeability across biological barriers. Due to large molecular
weight and high hydrophilicity of peptides and proteins, it is so hard to
transport through the lipid bilayers of the membrane (2). In addition to the
1
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permeability problem, there are still other problems need to think about,
including the short plasma half-life caused by enzymatic or chemical
degradation and also have problems in formulations, such as they are hard
to encapsulate in certain types of drug carrier system such as liposome and
microspheres (3).
Especially for the oral route of administration of protein and peptide
drugs is a more difficult challenging task. In general, absorption o f most
protein across the intestinal barrier is limited by the physicochemical
properties and characteristics of the cells forming the intestinal barrier. The
most important task is to avoid the enzymatic barriers and overcome the
hydrophilicity that limit of those compounds from reaching the target site.
Overcome the limitations of peptide drugs
Many methods have been tested to solve some of these problems.
One simple approach to overcome these problems is lipidization of the
peptide by conjugating with one or several lipid moieties to increase the
lipophilicity. The method of lipidization is to from an amide bond to link
the carboxyl group of a lipid molecule and an amino group of a peptide.
Such as thyrotropin-releasing hormone (4), insulin (5), and inhibitors of the
2
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KJV protease (6), they use fatty acids and phospholipids as lipid moieties to
modify peptides. And also reported that by lipidization of drugs increases
gastrointestinal absorption (7), enhance transport rate across blood-brain
barrier (8,9), decrease enzymatic degradation (8,10), and prolong plasma
half-life (11,12) of peptide or protein drugs can actually achieved.
However, in practice, there are still many hindrance involved in
peptide and protein lipidization. Such as, the solubilities of a peptide and a
lipid molecule are generally incompatible to each other (13). The peptide is
too hydrophilic, but on the other hand lipid molecule is too lipophilic. They
will limit with water solubility and decrease efficacy of lipidized
conjugates when compared with the parent peptide.
Background of Desmopressin (DDAVP)
Desmopressin is a synthetic antidiuretic hormone that can be
administered orally, intranasally, or parenterally. Desmopressin is a
structural analog of naturally occurring arginine vasopressin (AVP), also
called antidiuretic hormone, or ADH, in which the terminal amino group is
removed and the amino acid residue Arg 8 is replaced by D-Arg.
3
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Desmopressin is more potent and much longer acting than vasopressin and
it was approved by the FDA in 1978.
DDAVP acts to maintain serum osmolarity within a physiologically
acceptable range. And it is used to prevent or control polyuria, polydipsia,
and dehydration in patients with central diabetes insipidus. But it is not
effective for nephrogenic diabetes insipidus. The drug is also control
polydipsia and polyuria that occur following pituitary surgery or trauma,
and to manage primary nocturnal enuresis (14-18). Desmopressin is also
used for the treatment of bleeding episodes in patients with hemophilia A
or Type I von Willbrand’s disease (19). It also approved that has the ability
to improve human memory functions (20). Even though the stability of
DDAVP is increased due to the replacement of the a-amino group to the D-
Arginine, the half-lives of DDAVP in patients are still short (7.8 and
75.5min for the fast and slow phase, respectively) and still require daily to
obtain good effects (19).
Bioavailabilities of DDAVP by administered orally and intranasally
are only 1% and 2-10%, respectively (21-23). Low lipophilicity of
desmopressin (24-6) together with enzymatic degradation at the site of
4
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administration (27-29) contribute to the poor bioavailability of the
therapeutic agent. When administered via orally desmopressin is degraded
by the proteolytic enzyme a-chymotrypsin (26). a-chymotrypsin catalyzes
the cleavage of peptide bonds in which the reactive carbonyl group belongs
to the L-amino acids tryptophan, tyrosine, phenylalanine and to a lesser
extent leucine and methionine (30). The enzyme also catalyzes the
hydrolysis of amides and esters o f aromatic amino acids (30). The substrate
specificity o f a-chymotrypsin therefore cleavage of the Tyr-Phe and /or
Phe-Gln bond in desmopressin might occurred (Fig. 1). Concluding the
disadvantages above, Desmopressin is stable in normal plasma in vitro, but
it was degraded extensively by lysosome in the proximal renal tubule. An
analogue overcome proteolytic degradation before and after absorption
with longer duration of action, minimize the number of injections, reduce
the medical costs, and improve patient compliance should be discovered.
Desmopressin: mechanism o f action
When patients with central diabetes insipidus were administered,
desmopressin exerts antidiuretic effects similar to those of vasopressin. It
5
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increases the resorption of water at the level of the renal collecting duct,
thereby reducing urinary flow and increasing urine osmolarity. Serum
concentration of potassium, sodium and creatinine do not change following
the administration of DDAVP, and urinary excretion o f potassium and
sodium also keeps the same.
But the difference between them is that DDAVP does not induce the
release of adrenocorticotropic hormone or increase plasma cortisol
concentrations, and desmopressin has slight structural differences that
reduce its vasopressive activity and contractile action on visceral smooth
muscle. DDAVP increases plasma factor VIII and plasminogen activator to
a greater extent than equivalent weights of vasopressin.
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Mpa-Ty^-Phe^Gln-Asn-Cys-Pro-D-Arg-Gly-NH2
Desmopressin (DDAVP) Cleavage site
Phe-Gln-Asn-Cys-Pro-D-Arg-GIy-NH2
S-S-Mpa-Tyr
Cleavage site
Gln-Asn-Cys-Pro-D-Arg-Gly-NH2
S-S-Mpa-Tyr-Phe
Cleavage site
Phenylalanine + Gln-Asn-Cys-Pro-D-Arg-Gly-NH2
S-S-Mpa-Tyr
Figure 1. Structure of desmopressin and the possible cleavage site are digested by
a-chymotrypsin. In addition, the proposed degradation of DDAVP into two
intermediates and further degradation of these leading to the formation of
phenylalanine are shown.
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L
O 0 O o b i l O O 0
i -CH2 C H -C — NHj—O I -C -N H —CH-c ]-N H — < t H - C -N — r -< i-N K -C H -C -N H —CHj- C — HHj
6 C H , & fl {n
in , i h it
A
O ' 'NH,
OH di=N H
ta,
Mpi Tyr Pt* Gin Ain Cyi Pro D-A/J GfyNHj
Desmopressin (DDAVP)
M.W= 1069.2
Pal Pal
ys Cys
^CH2CH2CO-Tyr-Phe-Gln-Asn-(^ys-Pro-D-Arg-Gly-NH2
DPP
Figure 2. Structure of desmopressin (DDAVP and DPP. Pal-Cys: N-palmitoyl-
cysteine which is linked to the peptide backbone via a disulfide bond.
8
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^13^31
0 =
0=
Fatty Acid
(Palmitic add)
N H
I
H C —C00H
I
s
•4
N H
Linker
H C—COOH
S
4-~
(cysteine)
HjC— C-
Desmopressin
Tyr-Ptie-Gln-Asn-Cys-Pro-O-Arg-Gly-N^
Figure 3. Structure of DPP
9
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Ct His
0=
C7 H 1 5
0=
Fatty Acid
(Octanoic acid)
N H
H C —C O O H
I
<*2
s
H-
N H
H C —C O O H
T 2
S
4
Linker
(Cysteine)
H 2 C—C-
| Desmopressin
Tyr-Phe-Gln-Asn-Cys-Pro-O-Arg-Gly-N^
Figure 4. Structure of DPO
10
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NH^O
HC
M ^ O
HC
CH.
Aliphatic amine
(Octylamine)
Pg g n r y ^ y g g g jn
H p—C—n — T yrfte£lr> A sn{^sA D D 4rg< ^
Figure 5. Structure of DPOA
1 1
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Formation of reversible lipidized DDAVP
Recently, our laboratory has developed a novel reversible method for
peptide lipidization (31). We use N-palmitoyl cysteinyl 2-pyridyl disulfide
(Pal-CPD) as a lipidization reagent, it is both water soluble and thiol-
reactive. The conjugate with palmitic acid is formed by a reversible thiol-
disulfide exchange reaction between the reagent and either thioconsistant or
thiomodified peptide drug. The existence of a disulfide bond in the
molecule of DDAVP makes it become a good candidate for reversible
lipidization using Pal-CPD.
There are several advantages of reversible lipidization method using
of the disulfide linkage. Firstly, the reversible, biodegradable bond of the
disulfide linkage in the conjugate should allow for the regeneration of the
active peptides and proteins after cellular reduction (32). Secondly, recent
data also suggested that the transcytosis of a protein-macromolecule
conjugate across cell barrier could be achieved only if a reducible,
reversible linkage is used in the conjugation of the protein (33). Taking
DDAVP as an example, the disulfide ring structure is reported to be
essential for the anti-diuretic activity (34). But when we injected DPP, a
12
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derivative of DDAVP without a disulfide ring, subcutaneously displayed a
higher potency than the parent DDAVP. This means a regeneration o f the
disulfide ring structure must occur after the administration to the animal
(35). A reversibly lipidized dipalmitoyl desmopressin (DPP) was
synthesized by the method below and a 250-fold increase of anti-diuretic
potency in Brattleboro rats, which carry hereditary disease of hypothalamic
diabetes insipidus, was found by subcutaneously injection (35). Structure of
Desmopressin and its conjugates are in Fig. 2 to Fig.5. The synthesis of
fatty acid conjugates was briefly stated below, taking DPP as an example.
Procedure is also shown in Fig. 6.
1 . Synthesis of L-cysteinyl 2-pyridyl disulfide (CPD)
L-Cysteine and 2.2-dithiopyridine were 1:1 mixed in ethanol, and
put in 25 °C to react for 18 hrs. In order to remove any precipitate the
solution was centrifuged, and CPD in the supernatant was crystallized by
the addition of cold benzene. The final product after recrystallized in
benzene was a white solid with an m.p. (with decomposition) of 158°C.
The molar yield of the product using this procedure was approximately
60%.
13
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2. Synthesis of Pal-CPD
Triethylamine was added to a solution of CPD in of
dimethylformamide (DMF) and the suspension was reacted with the N-
hydroxysuccinimide ester of palmitic acid in DMF at 35°C for 24 hr, the
suspension start to turned clear during this time. This solution was diluted
with 40 ml of ice-cold water and the Pal-CPD and palmitic acid containing
precipitate was isolated by centrifugation. Pal-CPD was separated from
palmitic acid by suspension of the precipitate in water at PH 7.0, which
dissolved Pal-CPD, but not palmitic acid. Pal-CPD was purified by
repeating precipitation at PH 3 and was characterized by using NMR and
TLC techniques.
3. Synthesis of DPP
DDAVP was dissolved in PH 7.4 PBS and treated with of 0.1M DTT
at 37°C. A small aliquot of the reaction mixture was spotted on a TLC plate
and was developed twice using the organic layer of a mixture of n-butanol:
water: acetic acid (4:5:1) to monitor the reaction by using thin-layer
chromatography (TLC). The reduction of the disulfide bond in DDAVP
14
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was completed within 30 min, Then the reduced DDAVP solution was
mixed with Pal-CPD solution (lOmM, pH7.6) for 30 min at 25°C and,
subsequently, using IN HC1 to acidify to pH 3. The precipitate formed in
the acidified reaction mixture, which consisted of the DPP and the other
reagent, was isolated by using centrifugation (11,220 x g /20 min) and re-
dissolved in 1 ml of dimethylformamide (DMF). DPP was purified by
using a Sephadex® G-15 column (40ml) and then eluted with DMF. DPP-
containing fraction at the void volumn of the column were identified by
TLC analysis (DPP: RF=0.24) and pooled. After the removal of the solvent
under vacuum, purified DPP was obtained.
Caco-2 in vitro model
It is very important to have an appropriate in vitro model to screen
the lipidized polypeptides before determining if there is a significant
difference in the uptake and transport of the fatty acid conjugate of
desmopressin derivatives and parent DDAVP. In order to find out a good
candidate of lipidized DDAVP to administer orally, Caco-2 cell line was
chosen to mimic the intestinal epithelium of GI tract (36,37). Caco-2 cell
15
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line is human colorectal carcinoma cells. There are several advantages of
using Caco-2 cell culture model: (a) because it can serve as a rapid in vitro
screening tool for drug absorption studies, reducing the cost of animal
model, (b) It can provide information at a cellular level on the absorption,
metabolism and transport of drug molecules across intestinal mucosa, (c) It
provides information on the possible toxicity caused by therapeutic agents
on GI tract, (d) It does not suffer from the interspecies differences in the
morphological and physiological characteristics of the intestinal cells
because it is human origin (38). (e) In culture, Caco-2 cells undergo
spontaneous enterocytic differentiation and exhibit epithelial characteristics
(39,36,40). After the cells reach confluence as a monolayer on a
microporous membrane, a brush border and microvilli (41) is developed on
the apical surface with enzymes normally found in the human intestinal
tract, such as aminopeptidase, alkaline phosphatase and sucrase (39). (f) In
addition to the presence of enzymes, the cells also have tight junctions to
investigate what’s the mechanism of peptide transport. Therefore, it is
generally believed that Caco-2 cells are the most suitable model for human
intestinal epithelial cells because of the good correlation between transport
across Caco-2 cells and GI absorption (42).
16
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,) HS-C-g-NH, + 0 K S - S ^ Q — / " Y - s - S - C - g - N H ,
^ COOH — N — '— N ^ COOH
(a) (b) (C)
2) (c) + r ^ , - 0 — W — (c h ^ C H j — $ \ _ s_ s _ c _ Q _ y —1 1 — (o y^C H j
\=N ^ ioSH
(d) (e)
CX X DH
3 ) (e) + DDAVP— SH ► D D A V P -S S C -C -N -jpC C H j^C H a
^ O
(f)
Figure 6. Protein-palmitic acid conjugation using Pal-CPD (e) is synthesized in two
steps: 1) and 2). A protein containing a sulfhydryl group can then be
reacted with Pal-CPD (e) in an aqueous buffer to yield the fatty acid-
derivative conjugate (f), (a) L-cysteine, (b) 2,2-dithiopyridine, (c) CPD,
(d) N-hydroxysuccinimide ester of palmitic acid and (e) Pal-CPD.
17
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MATERIALS AND METHODS
Chemicals
Desmopressin (DDAVP) was purchased from Bachem Biotech
(Torrance, CA). Palmitic acid and Sephadex G-10 gel were obtained from
Sigma (St. Louis, MO). Chemicals and reagents were obtained from Sigma
Chemical Company unless otherwise stated. Fetal bovine serum, trypsin-
EDTA, penicillin/streptomycin, powdered cultured culture media and other
cell culture products were purchased from Gibco-BRL.
Reversible lipidization fatty acid conjugates of DDAVP,such as
DPP, DPO and DPOA were prepared by Dr. Jeff Wang, a research
associate in our laboratory.
Cell line
Human colon carcinoma cell line (Caco-2) was deposit of our
laboratory, Caco-2 originally from the American Type Culture Collection
(ATCC), (Rockvill, MD) and the method of maintaining the cells is a
modification of one used by Pinto, et al. (39). A further and more detailed
18
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description of this spontaneous cell line is provided in the following
sections.
Routine cell culture for Caco-2 cells
Caco-2 cell stocks were seeded at 1.0 X 106 cells for 25 cm2 flask and
maintained in Dulbecco’s Modified Eagle’s Minimum Essential Medium
(D-MEM) (Gibco, Grand Island, NY) supplemented with 10% Fetal bovine
serum (FBS) (Gibco, Grand Island, NY), 1% nonessentiai amino acids
(Gibco, Grand Island, NY), 1% L-glutamine (Gibco, Grand Island, NY)
and penicillin/streptomycin (Gibco, Grand Island, NY) at a final
concentration of about 50 pg /ml, incubated at 37 □ in an atmosphere of 5%
CO2 and 90% humidity. The medium was changed every other two days
and the cells were grown until reaching confluence. At confluence, the
culture medium was removed and the stock was detached from the culture
flask by trypsinization (0.25 ml trypsin/EDTA) for 7 min at 37°C, then a
single cell suspension was achieved by drawing up the suspension by
pipetting and releasing the contents of the pipette against the inside wall of
the culture flask therapy “crushing” or separating the cell aggregates. To
assure single cell suspension the flask was checking by light microscopy.
19
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The cell number was then counted in a Coulter Model counter (Coulter
Electronics, Hialeah, FL) and appropriate dilutions made for seeding new
stock flasks or cluster well plates for experiments.
Seeding cells to plates
For uptake studies, 6-well cluster plates with a well diameter of 35
mm and a growth area of 9.5 cm2 were used (Costar, Coming, NY) and cell
cultures initiated by seeding 1.5 X 105 cells in 2ml per well. For 12-well
cluster plates with a well diameter of 22.6 mm and a growth area of 4 cm2
were used (Costar, Coming, NY) and cell cultures initiated by seeding 4 X
104 cells/1 ml/well. The uptake study was performed on the day when the
cells reached confluence.
For transport studies, Costar’s 24mm transwells with a porous cell
culture insert containing a 0.4|im nucleopore polycarbonate membrane
(Costar, Coming, NY) were used with cells seeded at a density of 40-60 X
103 cells/cm2 in the insert with the same medium that is used for
maintaining cells in culture. The FBS-containing D-MEM, 1.5 ml in the
insert (apical surface) and 2.5 ml in the well (basal surface), was changed
20
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every other day. Confluence usually occurred approximately six to seven
days after passage. After the TEER of the Caco-2 monolayer was reaching
approximately 270 Q • cm2 , which is sufficient to be used for the
determination of drug transport.
Iodination of proteins
Radioiodination o f desmopressin (DDAVP) and its conjugates were
carried out using the chloramine-T method (43). 0.5 mg of DDAVP, DPP,
DPO or DPOA was dissolved in 500pl DMF (N, N, Dimethyl Formamide),
then adding 50 mCi 1 2 5 I, the solution was mixed well. A Chloramine-T
solution (for oxidation) was fresh prepeared by dissolving 4 mg of
Chloramine-T in 0.5 ml of phosphate-buffered saline (PBS). After
complete dissolution, 0.05 ml of the Chloramine-T solution was added to
the protein solution with mixing. The solution was allowed to react 10 min,
with gentle mixing every 2-min. A solution o f sodium metabisulfite
solution (for reduction) was fresh prepared by dissolving 2.4 mg in 0.5 ml
of PBS. An aliquot of 0.05 ml was added to the reaction mixture and the
solution was mix well. The solution was allowed to react for 10 min, with
21
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gentle mixing every two-min. A potassium iodine solution was prepared
by dissolving 10 mg in 1 ml of water. An aliquot of 0.2-ml KI was added to
the reaction mixture and the solution was mixed well. For the native
DDAVP as well as the fatty acid conjugate, the l2 5 I label residues on the
tyrosine residues within the DDAVP and the fatty acid is radiolabeled. The
iodinated protein conjugate was purified with a Sephadex G-10 (sigma,St.
Louis, MO) column (10 ml) by eluting with DMF until 15 ml were
collected in test tubes containing 1 ml each. Each fraction was analyzed in
a gamma counter to determine which fraction contained the radiolabeled
protein, which usually eluted from fraction 4-5, before free iodine eluted
(from fraction 9-10). After collecting elute fractions, the column was
disposed. The protein-containing fractions were isolated and stored in -
80°C and ready to do experiments.
The specific activity was determined by dividing the total
radioactivity of the iodinated protein solution by the amount of protein in
solution and multiple by 0.5 mg. The specific activity was expressed in
terms of mg. The procedure was carried out for the native Desmopressin
and for the fatty acid conjugates.
22
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Uptake study of cells
The amount o f radiolabeled polypeptide associated with fillly
confluent monolayer cell (about one to one-half week old Caco-2 cells was
determined in 6-well cluster plates (Costar, Cambridge, MA). Prior to the
experiment, the 10% FBS-containing DMEM medium was washed with 1.5
ml serum-free DMEM culture medium 3 times. Then treatment medium
consisted of 15 jig/ml of 1 2 5 I-labeled protein, either as DDAVP, DPO,
DPOA, DPP, in serum free medium 2.0 ml of the treatment medium was
used to treat the Caco-2. After one hour, the treatment medium was
removed by gently pipetting and the cells were washed three times with 2
ml ice-cold PBS. The cells were incubated for ten minutes at 37°C in 0.25
ml of 0.25% trypsin/EDTA to detach the cells from the surface of the plate.
The cell suspensions were then transferred to test tubes and centrifuged at
2000 rpm for a 15 min. The cell pellets were washed three times with fresh
2 ml ice-cold PBS by resuspending and centrifuging. The final cell pellets
were counted in a gamma counter to quantify the amount of cell-associated
radioactivity. For all the conjugate and the native DDAVP, this was done in
triplicate or more. During the one-hour time course of the experiment, the
23
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cells were maintained at 37°C in an atmosphere of 5% C 02 and 90%
humidity. After counting with a gamma counter to quantify the amount of
cell-associated radioactivity, the final pellets were dissolved in 250 pi IN
NaOH, then were put in -80°C freezer, and were ready to do Pierce protein
assay.
Uptake study of Caco-2 cells (Palmitic Acid Effect)
The amount of radiolabeled polypeptide associating with fully
confluent cell (about one to one-half week old Caco-2 cells was determined
in 12-well cluster plates (Costar, Cambridge, MA). Prior to the experiment,
the 10% FBS-containing DMEM medium was washed with 1 ml serum-
free DMEM culture medium 3 times. Then treatment medium consisted of
10 (ig/ml of 1 2 5 I-labeled protein, either as DDAVP, DPP and different
concentrations of palmitic acid, 0, 50, 100, 200 pg/ml in 10% FBS
containing-DMEM medium 1.0 ml of the treatment medium was used to
treat the Caco-2. After one hour, the treatment medium was removed by
gently pipetting and the cells were washed three times with 1 ml ice-cold
PBS. The cells were incubated for ten minutes at 37°C in 0.2 ml o f 0.25%
24
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trypsin/EDTA to detach the cells from the surface of the plate. The cell
suspensions were then transferred to test tubes and centrifuged at 2000 rpm
15 minutes. The cell pellets were washed three times with fresh 1 ml ice-
cold PBS by resuspending and centrifuging.
The final cell pellets were counted in a gamma counter to quantify
the amount of cell-associated radioactivity. For all the conjugate and the
native DP, this was done in triplicate or more. During the one-hour time
course of the experiment, the cells were maintained at 37°C in an
atmosphere of 5% CO2 and 90% humidity. After counting with a gamma
counter to quantify the amount of cell-associated radioactivity, the final
pellets were dissolved in 200 pi IN NaOH, then were put in -80°C freezer,
ready to do Pierce protein assay.
Uptake study of Caco-2 cells (DTT, Palmitic Acid Effect)
All procedure are the same as section above except the treatment
medium consisted of 2 or 8 pg/ml of l25I-labeled protein; either as DDAVP
or DPP with 50 mM DTT (1,4-Dithiothereitol) or 100 pg/ml of palmitic
25
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acid in 10% FBS containing-DMEM medium were pre-incubate for 30
minutes in 37°C incubator.
Pierce BCA protein assay
The BCA-protein Reaction scheme:
1) Protein (Peptide bounds) + C u 2 + -> tetradentate-Cu + 1 complex
2) Cu + l +2 Bicinchonic Acid (BCA) -> BCA-Cu + l complex
(purple colored, read at A562)
The cell pellet was already dissolved in IN NaOH, after mixing the
content of each well with a Pasteur pipette, a 5 pi aliquot o f the sample, add
20 pi IX PBS, in duplicate, was placed in a 96-well plate for Pierce assay.
Briefly, the Microtiter Plate Protocol for the Pierce Micro BCA Protein
Assay Kit is as follows: the working reagent was prepared by combining 50
parts BCA reagent A (containing sodium carbonate, sodium bicarbonate
and sodium tartrate in 0.1M NaOH), 1 parts BCA reagent B (containing 4%
cupric sulfate). Then 200 pi was added to each microtiter plate well, which
already contained a 25 pi of a standard, blank or unknown. The samples
were mixed by gently shaking for 30 seconds then covered and incubated at
26
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37°C for 30 minutes. The absorbance was then read at 562 nm with a
microtiter plate reader.
To obtain a protein concentration, a standard curve was also
prepared each time by plotting the blank corrected absorbance at 562 nm vs.
the protein concentration. Using different concentrations of bovine serum
albumin (BSA) to be a standard curve 2.0 mg/ml in a solution of 0.9%
saline and 0.05% sodium azide was serious diluted to 400, 300,200,100,80,
60, 40, 20 (pg/ml). A 25pi aliquot of the BSA standard, in duplicate, was
placed in a 96-well plate for the Pierce assay, then adding working reagent
as method mentioned above.
Transport study across Caco-2 cells
The amount of transcytosis of DDAVP, DPO, DPOA and DPP
across the Caco-2 cell monolayer was determined. The TEER was checked
periodically during the time course of the experiment, because leaky
cellular junctions would allow larger molecules to pass through and would
give an inaccurate representation of the transcytotic capability of the test
models.
27
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Prior to the experiment, the 10% FBS-containing DMEM medium
was rinsed once with serum-free DMEM culture medium. After one-hour
of pre-incubation in 37°C, 1 hr, the 1 mg/ml bovine serum albumin (BSA)
containing DMEM medium was replaced with treatment medium consisted
of 20 (ig/ml of 1 2 5 I-labeIed protein, either as DDAVP, DPO, DPOA and
DPP in 1 mg/ml BSA containing DMEM medium. An aliquot o f 1.5 ml the
treatment medium was placed on the apical surface, while 2.5 ml of serum-
free medium was placed in the basal compartment. Basal medium was
completely removed at 0.5, 1, 2, 3 and 4 hr and was replaced with fresh 1
mg/ml BSA containing DMEM medium. The TEER was measured at 0 and
4 hr. At 4 hr, the medium was removed from both the apical and basal
compartments (to avoid contamination, the basal medium was removed
first) and the cells were washed three times on both sides with ice-cold PBS.
After the rinse, the transwell membranes were removed from the inserts of
transwells and counted in a gamma counter to determine the amount of
radiolabeled protein associated with the cells. The basal medium from each
time point was treated with 0.8 ml 60% trichloroacetic acid (TCA) and
incubate for 15-30 min in 4 °C. After centrifugation at 3000 rpm for 15 min,
28
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the pellet was counted in a gamma counter. During the 4-hr time course of
the experiment, the cells were maintained at 37°C in an atmosphere of 5%
C 02 and 90% humidity incubator except for the time to replace the basal
medium.
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RESULTS
Iodination
DDAVP and its conjugates, DPP, DPO, and DPOA were
radiolabeled with 1 2 5 I . The iodinated proteins were purified with a
sephadex G-10 column by eluting with DMF until 15ml were collected in
test tubes containing 1ml each. Each fraction was analyzed in gamma
counter to determine which fractions contained the highest radiolabled
protein, which usually eluted from fraction 4-5, in front of the free iodine
eluted. The protein-containing fractions with highest count were pooled and
ready to be used for experiments. The results of each compound are shown
in Fig.7 to Fig. 10.
Cellular uptake
Caco-2 cells were incubated with 1 2 5 I-DDAVP, I2 5 I-DPP, 1 2 5 I-DPO or
I2 5 I-DPOA for one hour, and the cell-associated radioactivity, and hence
polypeptide, was measured. As shown in Fig. 11, the cell -associated
uptake of DPP is 7-fold higher than DDAVP. The cell -associated uptake
30
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of eight-carbon chain fatty acid conjugate, i.e. DPO, is less than DDAVP.
However, the uptake of the eight-carbon chain fatty acid conjugate with a
cationic spacer, i.e. DPO A, was 5-fold higher than that of DPO.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Number of fractions Counts (com)
1 54
2 79
3 65
4 110878
5 976379
6 193686
7 116245
8 108740
9 149083
10 156589
1 1 148980
12 142266
13 128540
14 72643
15 11138
125,
l-DDAVP
E
a
tn
c
3
O
o
1200000
1000000
800000
600000
400000
200000
0
-200000
N um ber of fractions
Figure 7. DDAVP was radiolabeled with I2 5 I, the counts of each fraction per 5 ml
was listed above. The fraction 5 with highest DDAVP containing tube was
collected.
32
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DPP
Number of fractions Counts fcpm)
1 55
2 70
3 61
4 86550
5 1285768
6 721410
7 455327
8 495199
9 530501
10 409817
11 71449
12 7580
13 4693
14 3910
15 3304
125I-D P P
1400000
1200000
^ 1000000
a
o,
« T
800000
600000
g 400000
O 200000
-200000 20
Number of fractions
Figure 8. DPP was radiolabeled with 1 2 5 I, the counts of each fraction per 5 ml was
listed above. The fraction 5 with highest DPP containing tube was collected.
33
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DPO
Number of fractions Counts (cpm)
1 54
2 73
3 81
4 1196765
5 923790
6 292011
7 499351
8 614929
9 592157
10 511370
11 348590
12 173531
13 33982
14 8473
15 6025
E
a .
0)
c
3
O
o
1400000
1200000
1000000
800000
600000
400000
200000
0
-200000
1 2 5 i-d p o
Number of fractions
Figure 9. DPO was radiolabeled with I2 S I, the counts of each fraction per 5 ml was
listed above. The fraction 4 with highest DPO containing tube was
collected.
34
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DPOA
Number of fractions Counts (cpm)
1 60
2 60
3 58
4 146787
5 473492
6 236660
7 399722
8 670662
9 873779
10 818207
11 710570
12 259353
13 8146
14 3183
15 2183
j 1000000
IE* 800000
& 600000
400000
§ 200000
-200000 6
Num ber of fractions
Figure 10. DPOA was radiolabeled with l2 5 I, the counts of each fraction per 5 ml was
listed above. The fraction 5 with highest DPOA containing tube was
collected.
35
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Cell uptake (ngijig cell protein)
Cellular Uptake
800
DDAVP D P O D PO A D P P
Proteins
Figure II. Amount of cell-associated 15 jig/ml DDAVP and its conjugates in Caco-2
cells in culture. Cells were incubated with l2 5 I-DDAVP, 1 2 S I-DPP, l2 5 I-
DPO and l2 5 I-DPOA for one hour, rinsed and counted in a gamma counter.
Values are represented as ng /jig Caco-2 cell protein and with standard
deviations indicated as error bars (n=3).
36
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Possible mechanism of increasing cellular uptake
The possible mechanism of increasing cell uptake of DPP was
investigated. There are two possible pathways to transport protein drugs
across cell membranes, one is passive diffusion and the other one is
receptor-mediated, via active transporter, such as fatty acid protein. Since
non-receptor-mediated pathway does not require a membrane receptor, the
uptake is concentrations dependent and non-saturable.
Caco-2 cells were incubated with increasing concentrations of either
i2 5 I-DDAVP or l2 5 I-DPP for one hour, and the cell-associated radioactivity
was measured. The data was shown in Fig. 12 indicating the uptake of both
DDAVP and DPP are not saturable.
The cell uptake of 1 2 5 I -DDAVP, either as the native polypeptide or
as DPP was studied in Caco-2 cell monolayers in the presence and absence
of fetal bovine serum (FBS). The results were presented in Fig. 13 and
Fig. 14, When the uptake studies were carried out in serum -free medium,
DPP demonstrated high affinity towards cell monolayers and was
transported into cells 4-fold higher than DDAVP. The addition of serum to
the medium greatly reduced the uptake of DPP into cell, reducing the
37
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uptake by 77% in the presence of 10% FBS. However, the addition of
palmitic acid to the medium at 50 pg/ml did not show any significant effect
on the uptake of DDAVP an d . The data is shown in Fig. 13 and 14.
When the 1 2 5 I-DDAVP or 1 2 5 I-DPP was incubated with increasing
concentration palmitic acid in 10% FBS-containing DMEM with Caco-2
cells for one hour, no significant difference was observed in the cell-
associated radioactivity (Fig. 15 and Fig. 17). On the other hard, when
palmitic acid conjugate, 1 2 5 I-DPP was added to the medium, the cell -
associated radioactivity was slightly increased with raising concentration of
palmitic acid and also the cell uptake of DPP with and without palmitic
acid in the medium. (Fig. 16 and Fig. 18).
To demonstrate that the higher uptake of DPP was mediated by the
palmitic acid moiety in the conjugate, DPP was reduced with DTT and the
cell uptake in serum-free medium are shown in Fig. 18. The DTT treatment
causes the reduction of disulfide linkages in DPP and the detachment of the
palmitic acid moiety from the conjugate. As presented in Fig. 18, the
reduction with DTT resulted in a drastic decrease in the uptake of DPP into
cells about 65.4%. On the other hand, the uptake of unmodified DDAVP
38
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into cells was also influenced by the DTT treatment, albeit it was less than
that of DPP (Fig. 17).
39
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Cell uptake (ngi|ig cell protein)
Cell uptake (dose response)
2S0
200
ISO
100
76.64
i.52
0 5 10 15 20 25
Drug dOM (ng/ml)
Figure 12. Caco-2 Cells were incubated with different concentrations o f 1 2 5 I-DDAVP
and 1 2 5 I-DPP, such as 0.5, 1, 2, 5, 10, 20 pg/ml, for one hour, rinsed and
counted in a gamma counter. Each point is represented as ng /jag Caco-2
cell protein and with standard deviations indicated as error bars (n=4).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1 2 5 l-ODAVP cell uptake(different medium)
DDAVP(S) DDAVP(F) DDAVP(P)
DDAVP+Otff*r*nt Medium
Figurel3. The cell monolayers were incubated with l2 5 I-DDAVP at 10 fig/ml in
different medium for 60 minutes at 37 °C, rinsed and counted in a gamma
counter. DDAVP (S) represents 10 pg/ml DDAVP in serum free DMEM
medium, DDAVP (F) represents 10 pg/ml DDAVP in 10% FBS
containing-DMEM medium, and DDAVP (P) represents 10 pg/ml
DDAVP in DMEM medium with 50pg/ml palmitic acid. Each point is
represented as ng /pg Caco-2 cell protein and with standard deviations
indicated as error bars (n=3).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1 2 S l-OPP call uptakafdiffarant medium)
DPP(S) DPP(P) DPP(F)
DPP+DHforant Medium
Figure 14. The cell monolayers were incubated with l2 5 I-DPP at 10 |ig/ml in different
medium for 60 minutes at 37 °C and rinsed and counted in a gamma
counter. DPP (S) represents 10 pg/ml DPP in serum free DMEM medium,
DPP (F) represents 10 pg/ml DPP in 10% FBS containing-DMEM
medium, and DPP (P) represents 10 pig/ml DPP in DMEM medium with
50 ng/ml palmitic acid. Each point is represented as ng /|ig Caco-2 cell
protein and with standard deviations indicated as error bars (n=3).
42
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'“ l-O D A V P 0 * 1 1 uptake with different concentrations of palmitic acid
D D A V P(S) D O A V P(F ) D D A V P(50) DDAVP(IOO) D D A V P (200)
DDAVP +Palmitic Acid Concmtrattonfcg/ml)
Figure 15. The cell monolayers were incubated with l2 5 I-DDAVP at 10 |ig/ml in
different medium for 60 minutes at 37 °C and rinsed and counted in a
gamma counter. DDAVP (S) represents 10 pg/ml DDAVP in serum free
DMEM medium, DDAVP (F) represents 10 pg/ml DDAVP in 10% FBS
containing-DMEM medium, DDAVP (50) represents 10 pg/ml DDAVP in
10% FBS containing-DMEM medium with 50pg /ml palmitic acid.
DDAVP (100) represents 10 pg/ml DDAVP in 10% FBS containing-
DMEM medium with lOOpg /ml palmitic acid. DDAVP (200) represents
10 pg/ml DDAVP in 10% FBS containing-DMEM medium with 200pg
/ml palmitic acid. Each point is represented as ng /pg Caco-2 cell protein
and with standard deviations indicated as error bars (n=3).
43
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Cell uptake (ng/pg cell protein)
1 J 5 H)PP Cell uptake with different concentrations of palmitic acid
250
200
150
100
50
0
OPP(S) DPP(F) DPP(50) DPP(100) DPP(200)
DPP ♦ Palmitic Acid Concentration (pg/ml)
Figure 16. The cell monolayers were incubated with l2 5 I-DPP at 10 (ig/ml in different
medium for 60 minutes at 37 °C and rinsed and counted in a gamma
counter. DPP (S) represents 10 pg/ml DPP in serum free DMEM medium,
DPP (F) represents 10 pg/ml DPP in 10% FBS containing-DMEM
medium, DPP (50) represents 10 pg/ml DPP in 10% FBS containing-
DMEM medium with 50pg /ml palmitic acid. DPP (100) represents 10
pg/ml DPP in 10% FBS containing-DMEM medium with lOOpg /ml
palmitic acid. DPP (200) represents 10 pg/ml DPP in 10% FBS
containing-DMEM medium with 200pg /ml palmitic acid. Each point is
represented as ng /pg Caco-2 cell protein and with standard deviations
indicated as error bars (n=3).
44
208.17
68.55 7 3 J0 7*34
i l l
- 40.23
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Cell uptake (ngftg cell protein)
1 2 S I-DDAVP cell uptake (DTT, Palmitic Acid effect)
40
35
X
25
X
15
10
5
0
Figure 17. The cell monolayers were incubated with 1 2 5 I-DDAVP at 2 fig/ml in
different medium for 60 minutes at 37 °C and rinsed and counted in a
gamma counter. DDAVP (S) represents 2 pg/ml DDAVP in serum free
DMEM medium, DDAVP (D) represents 2 pg/ml DDAVP in serum free
DMEM medium with 50mM DTT, DDAVP (F) represents 2 pg/ml
DDAVP in 10% FBS containing-DMEM medium, DDAVP (F+P)
represents 2 pg/ml DDAVP in 10% FBS containing-DMEM medium
with lOOpg /ml palmitic acid. Each point is represented as ng /pg Caco-2
cell protein and with standard deviations indicated as error bars (n=3).
0DAVP(S) DDAVPfD) 00AVP(F)
ODAVP+diffarant medium
DDAVP(F*P)
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1 2 5 I-DPP call uptake (DTT, Palmitic Acid effect)
DPP(S) DPP(D) DPP(F)
DPP*diffiarant medium
DPP(F«P)
Figure 18. The cell monolayers were incubated with 1 2 3 I-DPP at 8 pg/ml in different
medium for 60 minutes at 37 °C and rinsed and counted in a gamma
counter. DPP (S) represents 8 pg/ml DPP in serum free DMEM medium,
DPP (D) represents 8 pg/ml DPP in serum free DMEM medium with
50mM DTT, DPP (F) represents 8 pg/ml DPP in 10% FBS containing-
DMEM medium, DPP (F+P) represents 8 pg/ml DPP in 10% FBS
containing-DMEM medium with lOOpg /ml palmitic acid. Each point is
represented as ng /pg Caco-2 cell protein and with standard deviations
indicated as error bars (n=3).
46
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Transport
To determine if the increase in cellular uptake of the conjugates
would result in a similar increase of the transport across Caco-2 cells, the
transcytosis of 1 2 5 I-DDAVP, 1 2 5 I-DPP, l2 5 I-DPO and I 2 5 I-DPOA in
transwells was determined.
Figure 19 shows the analysis of the 4 hrs basal medium of four
proteins and Figure 20 represents the membrane of Caco-2 associated cell
uptake.
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D rug Transport Percentage (%)
Transport of 1 2 5 I-DDAVP, 1 2 5 l -DPO, i2 5 l-OPOA, 1 2 5 I-DPP
1.6
1.4
1.3
1.0
'DDAVP
DPO
OPOA
DPP 0.7
0.6
0.4
02
0 1.5 0.5 2 3.5 1 2 5 3 4 4.5
Time (hour)
Figure 19. Caco-2 Cells were incubated with 1 2 5 I-DDAVP, 1 2 5 I-DPP, l2 S I-DPO or l2 5 I-
DPOA for four hours in transwells. Basal medium was collected at 0.5, 1,
2, 3, and 4 hr, and counted in a gamma counter. The transcytosis of I2 5 I-
DDAVP, l2 5 I-DPP, l2 5 I-DPO and I2 5 I-DPOA in transwells was determined.
Values are represented as the percentage of the total radioactivity-
transport through Caco-2 cells and with standard deviations indicated as
error bars (n=3).
48
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Caco-2 mambrana aaaocata uptaka (%)
Membrane associated uptake
D D A V P D P O D PO A D P P
Drugs
Figure 20. Caco-2 Cells were incubated with 1 Z 5 I-DDAVP, 1 2 5 I-DPP, l2 5 I-DPO and
1 2 5 I-DPOA for four hours in transwells, cell membrane was collected,
washed, and counted in a gamma counter. The membrane associated
uptake of I2 5 I-DDAVP, l2 5 I-DPP, ,2 5 I-DPO and ,2 5 I-DPOA was
determined. Results are presented as the percentage of radioactivity cell-
associated and with standard deviations indicated as error bars (n=3).
49
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DISCUSSION
Hydrophobic ligands, such as fatty acids, have also been used as
transport ligands in vitro (44) and in vivo (45, 46), and conceivably,
because of their high membrane affinity and low toxicity, may become one
of the most useful carrier ligands for conjugation to peptides and proteins.
There are several questions that can be dressed in my report.
l.Can lipidization of peptide drugs increase cellular uptake?
Initially, the conjugates containing different length of carbon chain
of fatty acid per DDAVP molecule were considered. It was intended to
show that the chain length of fatty acids would make a difference in the
conjugate’s ability to associate with the cell surface as a first step towards
internalization and transcellular transport. Indeed, it was demonstrated that
as the carbon chain length of fatty acids increased, the amount of
radioactivity associated with Caco-2 cells also increased (Figure 11). As
the chain length of the fatty acid was increased, the lipophilic nature of the
conjugate was increased, and leading to a greater attraction to the lipid
bilayer of the cells, and hence the cellular uptake was increased.
50
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2. Does the charge of the linker effect cellular uptake?
As shown in figure 11, with an identical carbon chain length of fatty
acid, the cellular uptake o f DPO A is 6.5-fold higher than that of DPO. The
only difference between DPOA and DPO is the charge, when the DPO and
DPOA are dissolved in DMEM medium, they will carry negative charges
and positive charges respectively. The H atom of DPO is dissociated from
carboxylic acid and becomes negatively charged. The amine group of
DPOA is protonated and DPOA becomes positively charged. It is likely
that, since the cell membrane is negatively charged, the positively charged
DPOA would have a higher binding affinity to the cells.
3. Does cellular uptake of DPP increase via conjugation with
palmitic acid?
When the uptake studies were carried out in serum -free medium,
DPP demonstrated a high affinity towards cell monolayers in vitro and was
transported into cells by a 4-fold higher level when compared to native
DDAVP. The addition of serum to the medium greatly reduced the uptake
of DPP into cell.
51
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On the other hand, as shown in Fig. 15 and Fig. 17, the cell -
associated uptake of DDAVP did not statistically significantly increase
with raising concentrations of palmitic acid and also the cellular uptake of
DDAVP with and without palmitic acid didn’t have significant difference.
But for the palmitic acid conjugate, DPP, the cell -associated uptake of
DPP have a little bit increase with raising concentrations of palmitic acid
and also the cellular uptake of DPP with and without palmitic acid showed
a 42% difference, (Fig. 16 and Fig. 18). Results can be explained by the
affinity of DPP towards not just cell membrane, but also other components
in serum. In particular, binding to fatty acid binding sites of serum protein
may contribute to the increasing circulation time in the plasma.
As presented in Fig. 18, the reduction with DTT resulted in a drastic
decrease in the uptake of DPP into cells. On the other hand, the uptake of
DDAVP into cells was not markedly influenced by the DTT treatment
(Fig. 17), albeit, a small decrease was observed. It is likely that when
DDAVP is treated with DTT, the original ring structure of DDAVP will
open to become a linear form and makes it harder to transport across
membrane. The conclusion of above is that the DPP can increase
52
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circulation time via conjugation with palmitic acid, and that can bind to
fatty acid binding site of serum protein.
4. Is the increase of cellular uptake mediated by passive
diffusion or active transport?
There are two possible pathways to internalize protein drugs across
the intestinal epithelium, one is non-receptor-mediated (i.e. passive
diffusion) and the other one is carrier-mediated active transport. For the
passive diffusion, the uptake of extracellular compartments can occur
without the involvement o f a membrane receptor. This type of endocytosis,
which is derived from constant cell membrane internalization and turnover,
is usually an unsaturable pathway. On the other hand, many carriers in the
intestinal epithelium have been discovered and may have evolved for
efficient absorption of nutrients as evidenced by the fact that some o f the
nutritionally important molecules, such as amino acids, dipeptides and
glucose are transported across the intestinal epithelium by a carrier-
mediated process.
53
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As shown in data, both DDAVP and DPP are not saturable with
increasing concentration of protein. So we concluded that the increase o f
cellular uptake of DPP is mediated by nonspecific absorptive endocytosis
or passive diffusion.
5. Does better cellular uptake indicate better transport in Caco-
2 cell model?
When we compare the results of Fig.l 1 and Fig. 19, the answer of
the above question is probably no. There is a widely held belief that
lipophilic uncharged molecule can easily permeate the cell membrane, and
thus are well absorbed from the GI tract. However, it is becoming clear that
the relationship between the physicochemical properties of molecules and
their ability to transverse across the GI epithelium is quite complex. The
part of complexity in the relationship is due to the highly complex and
dynamic barrier posed by the GI epithelium. As for the passive diffusion,
there are two ways can cross this barrier, (a) by paracellular pathway which
is traveling through the intercellular space, or (b) by transcellular pathway
which is traveling across the epithelial cells (38). The presence of tight
54
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junctions (TJs) around the cells acts to restrict passive diffusion across the
paracellular route. Although active routes of transport have been clearly
characterized (47), it still difficult to delineate that a molecule permeates a
cell monolayer by the paracellular or transcellular route (48). Hydrophobic
molecules are considered to transverse the cell monolayers predominantly
by the transcellular route and hydrophilic molecules predominantly by the
paracellular route (49). By the result we got from above, we know both
DDAVP and DPP are transported via passive diffusion, and also it has been
suggested that the highly hydrophilic desmopressin is largely transported
through Caco-2 cells via paracellular route (50,51).
When we conjugated palmitic acid with DDAVP, the product DPP
become very lipophilic, and maybe the transverse pathway are from
paracellular to transcellular. The properties of the molecule and the
characteristics of the cells that form the barrier dictate whether the
molecule crosses the barrier through or between the cells. For most drugs,
transmembrane pathway is dominant since these substances are generally
hydrophobic and prefer to partition into a lipid environment rather than to
diffuse through the aqueous paracellular pathway. For DPP, maybe
55
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because it is too lipophilic and hard to get out from the lipid bilayer, so the
transport rate is not as good as cellular uptake. Most of the DPP are just
accummulation inside the membrane, membrane associated uptake after 4
hours transport of DPP is higher than DDAVP also confirmed this
speculation (Fig. 20).
But for the DPO and DPOA, the lipophilicity is not as high as DPP;
they might still cross the cell membrane via paracellular pathway. Whereas
increased molecular size should decrease the paracellular permeability
(52,53). However, DPOA is little higher in uptake in Caco-2 cells than
DDAVP, but not in transport study. This difference due to the positive
charge of DPOA which cause higher binding affinity to cell membrane and
consequently, becomes difficult to associate from the membrane. This
might explain the results, that all the cellular uptake and transport rate and
Caco-2 membrane associate uptake after 4 hour transport were not higher
than those of the parent DDAVP (Fig. 11,19,20).
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CONCLUSIONS
Reversible lipidization of peptide drugs introducing many
advantages compare to conventional lipidization methods. The lipidized
products have good water solubility and are appropriate for various
formulations. Reversibly lipidized peptides can also keep bioactivity of
parent drug, because they can regenerate the native peptides in a living
system. By conjugating with a lipophilic fatty acid moieties, the cellular
uptake of peptide drug can be increased. Furthermore, a significantly
prolonged pharmacological activity, as in the case of DPP, can be achieved
due to prolonged plasma half-life by a unique chug biodistribution and a
release kinetics of lipidized peptides (35). Consequently, it is possible to
reduce the numbers of injections and lower the cost of achieving the
desirable therapeutic effect in patients. In addition, the metabolites of
lipidized peptides, DPP, are palmitic acid and cysteine, which are non-toxic
of the living system. These by-products are unlikely to cause any serious
toxicity when compared with other drug carrier. Those advantages also
confirmed in other reports (2,31, and 54) from our laboratory.
57
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Cultured Caco-2 cell model is really a good screening tool for
finding out a good candidate for oral dosage forms of drugs. They have
brush border, microvilli, enzymatic system and tight junctions to mimic the
epithelial characteristics of the GI tract. In addition, they are very easy to
culture and can test a lot of samples at the same time and hence saving a lot
of money compare to in vivo animal models.
In summary, we conclude that: (a) fatty acid-conjugated proteins
have a higher affinity towards cell monolayers in vitro, but also bind other
serum proteins factors, such as serum fatty acid binding site, and can
increase circulation time after administration, (b) Positively charged fatty
acid conjugate, DPOA, help to increase the cellular uptake. (C) The uptake
of fatty acid conjugated polypeptides is mediated by the lipophilicity of the
fatty acid moiety in the conjugate, (d) The biological activity of the peptide
or protein may be retained in the fatty acid conjugate; and (e) Caco-2 cell
model is a good screening tool for discovering a new formulation of
desmopressin. For IV administration, the uptake study is enough. However,
for oral administration, we have to take both cellular uptake and transport
study for consideration. Because passive diffusion across the cellular
58
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barrier was limited kinetically by the equilibrium between protein-bound
drug and free drug partitioned into cell membrane, and also the rate-
limiting dissociation of drug from the cell membrane to target site.
In the future, maybe the chain length between 8-16 carbons of new
fatty acid conjugates of DDAVP can be investigated. A good candidate can
be found to have better lipophilicity affinity to cell membrane, and also
have better dissociation ability to transverse through epithelial cell of GI
tract, and to leave it to the target site. A good oral dosage form of
desmopressin can be made in the future by screening a good candidate in
Caco-2 cell model system.
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(1996).
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BIBLIOGRAPHY
Frokjar,S., Christrup, L and Krogsgaard-Larsen P. (eds). Peptide and Protein Drug
Delivery, Munksgaard, Copengagen, 199S.
Hess, G. P. Chymotrysin-chemical properties and catalysis. In: Boyer, P i), (ed) The
Enzymes, vol III. Academic Press. New York, 1994.
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Louis, MI, 1999.
Lee, V.H.L. (ed). Peptide and Protein Drug Delivery, Marcel Dekkerjnc , New
York, 1990.
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Creator Wu, Yuan-chen (author)

Core Title Uptake and transport of desmopressin and its lipidized derivatives in Caco-2 cells

Degree Master of Science

Degree Program Pharmaceutical Sciences

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

Tag biology, cell,chemistry, pharmaceutical,Health Sciences, Pharmacology,OAI-PMH Harvest

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