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University of Southern California Dissertations and Theses
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Alteration of the in vitro and in vivo processing of a polypeptide, BBI, through conjugation with palmitic acid
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Alteration of the in vitro and in vivo processing of a polypeptide, BBI, through conjugation with palmitic acid
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INFORMATION TO USERS
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ALTERATION OF THE IN VITRO AND jN VIVO
PROCESSING OF A POLYPEPTIDE, BBI, THROUGH
CONJUGATION WITH PALMITIC ACID
by
Laura Roxann Honeycutt
A Dissertation 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)
December 1996
Copyright 1996 Laura Roxann Honeycutt
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UMI Number: 1383529
UMI Microform 1383529
Copyright 1997, by UMI Company. All rights reserved.
This microform edition is protected against unauthorized
copying under Title 17, United States Code.
UMI
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFORNIA 90007
This thesis, written by
Haneycu££*_Laur.a„RQxariii_______________
under the direction of h.fi r. Thesis Committee,
and approved by a ll its members, has been pre
sented to and accepted by the Dean of The
Graduate School, in partial fulfillm ent of the
requirements for the degree of
M a s £ f i r . . Q l . S . Q l e . n . Q . e ________________
D ia n
THESIS C O M M IT T E E
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To Wilson Meng
For his never-ending loving support
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ACKNOWLEDGMENTS
I would like to express my sincere appreciation to Dr. Wei-Chiang Shen
for his guidance and support during my graduate work. I am grateful for
the opportunity to have had him as a mentor.
I would also like to thank my committee members, Dr. Vincent H. L.
Lee and Dr. Robert Koda for their input and advice in my work and thesis.
Additionally, I would like to thank Daisy Shen for her valuable help
and technical support in the lab throughout the years.
My friends and colleagues also deserve recognition for their
encouragement and friendship. In good times and bad, you were there. It
never went unnoticed.
A special, special thanks to Wilson Meng. Much more than a friend,
and sometimes more than I desen/e. I want to thank you for being there
for me. You encouraged me in my academic endeavors and stood by my
side. Thank you for your emotional support and love.
Last and certainly not least, I thank God for carrying me through
the tough times. Without his presence, this would not have been possible.
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TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGMENTS iii
LIST OF FIGURES vi
LIST OF TABLES viii
INTRODUCTION
Barriers to oral route of administration 1
Background on BBI 2
Routes of administration other than the oral route 3
Derivitization of peptides 5
Caco-2 in vitro model 6
MATERIALS AND METHODS
Synthesis and radiolabeling of palmitic acid-conjugated BBI 7
Tissue culture technique 1 1
Uptake / affinity for cells 12
Transport across Caco-2 cells 13
Uptake / cellular processing in Caco-2 cells 14
Pharmacokinetic study 15
Analysis of whole-blood radioactivity composition 16
Determination of pharmacokinetic parameters 17
The in vitro and in vivo binding of BBI and Pal(3)-BBI
to albumin, serum and plasma 17
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RESULTS
Cellular uptake and transport 1 g
Pharmacokinetics 22
Evaluation of binding 31
DISCUSSION 46
CONCLUSIONS 52
REFERENCES 56
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vi
List of Figures
Figure Description Page
1. Amount of cell-associated BBI in Caco-2 cells
in culture 20
2. Transport of 1 2 5 I-BBI and 1 2 5 l-Pal(2)-BBI across
filter-grown Caco-2 cells 21
3. Blood analysis on a Sephadex G-50 (20 ml)
column to determine the amount of intact protein
in each time point 23
4. Curve fit of concentration (mg) versus time (min) for
1 2 5 I-BBI ( • ) and 1 2 5 l-Pal(3)-BBI (■) 26
5. Total radioactivity in blood as percent of the total
injected dose following iv administration of
1 2 S I-BBI (O) and 1 2 5 l-Pal-BBI (• ) in mice 28
6. Total radioactivity in kidneys as percent of the
total injected dose following iv administration of
1 2 5 I-BBI (O) and 1 2 5 l-Pal-BBI ( • ) in mice 29
7. Total radioactivity in the liver as percent of the
total injected dose following iv administration of
1 2 5 I-BBI (O) and 1 2 5 l-Pal-BBI (• ) in mice 30
8. Total radioactivity in the Gl (stomach, small and
large intestines and colon were combined) tract
as percent of the total injected dose following iv
administration of 1 2 5 I-BBI (O) and 1 2 5 l-Pal-BBI ( • )
in mice 32
9. In vitro bindina of 1 2 5 I-BBI to albumin 34
10. In vitro bindina of 1 2 5 l-Pal(31-BBI to albumin 35
11. In vitro bindina of 1 2 5 I-BBI to fetal bovine serum 36
12. In vitro bindina of 1 2 5 l-Pal(31-BBI to fetal bovine
serum 37
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vii
13. In vitro binding of 1 2 5 I-BBI to serum isolated from
a mouse 38
14. In vitro binding of 1 2 5 l-Pal(3)-BBI to serum isolated
from a mouse 39
15. In vivo binding of 1 2 5 l-Pa!(3)-BBI to serum proteins
in a mouse that was injected with the conjugate by
the iv route via the tail vein 40
16. Amount of 1 2 5 I-BBI and 1 2 5 l-Pal(3)-BBI that
associates with the extracellular matrix (trypsin-
removable) and the cellular matrix (cell-associated)
of Caco-2 cells after 24 hours of exposure 42
17. 1 2 5 l-Pal(3)-BBI in culture media after 24 hours of
incubation with Caco-2 cells grown in 6-well
cluster plates 43
18. An aliquot of the control uptake medium that
had not been incubated with cells was
incubated with FBS and was analyzed on a
Sephacryl S-200 column (40 ml) where the
sample was eluted with PBS and collected in
1 ml fractions 45
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List of Tables
Table Description Page
I. IV study of ^^l-B B I and ^^l-P al-BBI in mice
% intact polypeptide in blood 24
II. Relevant Pharmacokinetic Parameters Following
IV Administration of 1 2 5 I-BBI and 1 2 5 l-Pal-BBI 27
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1
INTRODUCTION:
One of the major factors limiting the design and development of
peptides as therapeutic agents is the oral bioavailability. Even after
entering the systemic circulation, there are several barriers to trans-
epithelial transport of proteins. One factor is the characteristic of the
capillary network. Those with the most continuous endothelial will inhibit
the transport of even the smallest peptides. Those of the kidneys act as a
barrier to the filtration of circulating molecules with a molecular weight
smaller than 60 kDa (Guyton, 1991). Small proteins will not re-enter the
circulation unless there is a specific reabsorption mechanism. By altering
the size and/or the physicochemical properties (Brenner et al., 1978) of a
protein, renal elimination can be delayed, as kidney filtration is a major
mechanism of clearance of very small peptides.
It has already been shown that the biodistribution of proteins can
be altered by chemically linking them to carrier ligands (Sezaki et al.,
1989). These carriers change the protein’s physicochemical properties by
altering their shape, charge and lipophilicity. There are many examples
where this technique has been used clinically, including the conjugation of
polyethylene glycol to asparaginase in cancer therapy or adenosine
deaminase in the treatment of severe combined immunodeficiency
disorder (Langer, 1990). When small unmodified protein drugs are
administered, multiple administrations or a continuous infusion is
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One protein under investigation is the Bowman-Birk protease inhibitor
(BBI). BBI, an 8 kDa polypeptide isolated from soybean (Bowman, 1944),
possesses both trypsin and chymotrypsin inhibitory activities (Steiner, 1972)
residing in two distinct domains (Odani and Ikenaka, 1978). It has been shown
that BBI can prevent radiation-induced transformation of C3H/10T1/2 cells ]n
vitro (Kennedy, 1993) and lung tumor development in mice in vivo (Kennedy,
1993; Witschi and Kennedy, 1989) and has the potential for use in humans as
an agent to prevent cancer. In order to improve absorption and tissue targeting
of BBI, modifications such as lipidization can be used to increase the stability of
the polypeptide from proteolysis and to increase its lipophilicity which could
increase the amount of transport via passive diffusion.
In order to be used as a potential drug for chemopreventive purposes, BBI
must be taken up by the epithelial cells of the target tissue or organ. The
physicochemical properties, as mentioned above, will determine the affinity of
BBI for the epithelial surface. Because the residues comprising the outer portion
of BBI are not predominantly positive, there is not a natural attraction between
the polypeptide and the negatively charged epithelial surface. With this limited
attraction, the most viable route of entry into the epithelial cells is passive
transport by way of fluid phase endocytosis. The concentration of BBI in the
extracellular space is the limiting factor to this inefficient process (Duncan, 1987).
Another route of transport which is also dependent on the extracellular
concentration is paracellular transport (Lee et al., 1990). The presence of tight
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junctions restricts passage to molecules less than 1 kDa (Lee, 1988; Artursson
and Magnusson, 1990), making BBI’s size of 8 kDa too large to include the
paracellular route as a possibility. Furthermore, paracellular transport would play
only a minor role in the transport of proteins because cellular junctions comprises
less than 0.5% of the mucosal surface area (Gonzalez-Mariscal, 1985). In a
transport process that require a high extracellular concentration, toxicity and
dosing limitations could make relying on this process unfeasible.
One method of transport which cannot generally be applied to proteins is
transcellular transport. In this case, the proteins would be taken up on one side
of the cell, be incorporated into vesicles and then be presented to the opposite
surface, with the vesicle membrane fusing with the cell membrane. At this point,
the protein would be released as the intact form from the vesicles ( Mostov and
Simister, 1985). Before this can occur, the protein molecules must reach the cell
surface. Mucosal barriers include negatively charged mucin, which forms gel like
structures in solution (Rhoades and Tanner, 1995). The thickness of this
secretion provides a physical barrier and restricts the amount of BBI which could
actually reach the cell surface,. Once at the surface, BBI would encounter the
lipid bilayer, another barrier to transport due to the hydrophilic nature of BBI.
Another defense that the mucosal surface has against penetration by proteins is
its enzymatic barrier (Lee, 1988). The mucosal endopeptidases and
exopeptidases can digest proteins from the end or middle, respectively, and the
smaller peptide fragments can then be cleared via renal elimination. Due to the
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numerous obstacles, proteins usually need specific carriers to accomplish
transcytosis, as in the case of insulin receptors for the transcytosis of insulin (van
Duers et al., 1989; King and Johnson, 1985).
In order to maintain a sufficient concentration in the vicinity of the target
tissue, the peptide drug would have to stay in circulation for a sufficiently long
period of time. One of the problems with using peptide drugs is that they are
frequently subjected to enzymatic or chemical degradation, resulting in very short
half-lives, and to rapid renal elimination of the resulting smaller peptide
fragments. One way to overcome the limitation of the short half-life is to give
frequent injections, but when clinical restrictions are considered, this would mean
administration by a trained medical professional, leading to higher medical costs
and reduced patient compliance. To overcome these problems, one approach is
the conjugation of long chain fatty acids to the peptide, which can increase the
stability of the peptide and increase the residence time in the blood. Fatty acids
have been used in the past as a method of modifying peptides, such as thyroid
releasing hormone (Muranishi et al., 1991), gastrin (Yodoya et al., 1994) and
insulin (Hashizume et al., 1992), but the emphasis of these studies has been to
measure the alteration of activity and/or mucosal absorption. While the
pharmacokinetics of a phospholipid-peptide conjugate has been determined
(Hostetler et al., 1994), the changes in pharmacokinetic parameters of fatty acid-
peptide conjugates has not previously been considered.
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We have recently developed a novel method for the preparation of fatty
acid conjugates of BBI. The molecule which is to be conjugated to BBI, which
could be a macromolecular earner, is not directly chemically linked to BBI.
Rather, a reducible disulfide spacer is covalently joins the BBI molecule and the
fatty acid(s). In spite of the presence of the fatty acid, the fatty acid conjugate of
BBI is water soluble, which enables the use of the conjugate in vivo.
One of the potential disadvantages of derivitizing peptides and proteins is
the loss of biological activity (Torchilin et al., 1980; Al-Obeido et al., 1992). The
BBI-palmitic acid conjugate, with an average of 3 palmitic acid moieties per BBI
molecule, Pal(3)-BBI, has been screened with an in vitro transformation assay
which has shown the conjugate to have retained its biological activity (Ekrami et
al., 1995). The preliminary in vitro data showed an increased uptake in the
human intestinal (Caco-2) cell model (Ekrami et al., 1995).
Before determining if there is a significant difference in the biodistribution
and pharmacokinetics of the fatty acid conjugate of BBI versus that of the native
polypeptide, it is important to screen the two polypeptides with an appropriate ]n
vitro system. In order to mimic the intestinal epithelium, the Caco-2 cell line was
chosen. Caco-2 cells are human colorectal carcinoma cells that have been used
previously as a model for human intestinal epithelial cells (Hildago et al., 1989,
Wilson et al., 1990). In culture, Caco-2 cells undergo spontaneous enterocytic
differentiation and exhibit epithelial characteristics (Pinto et al., 1983; Hildalgo et
al., 1989; Chantret et al., 1988). After the cells reach confluence as a monolayer
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on a microporous membrane, a brush border and microvilli (Hilgers et al., 1990)
is developed on the apical surface with enzymes normally found in the human
intestinal tract, such as alkaline phosphatase, aminopeptidase and sucrase
(Pinto et al., 1983). In addition to the presence of enzymes, the cells also
develop tight junctions Even though there are other cell lines which have a higher
TEER (trans-epithelial electrical resistance) when confluent, such as the MDCK
(Mandin-Darby Canine Kidney) cell line, the Caco-2 cells are the best model for
human intestinal epithelial cells because of the correlation between transport
across Caco-2 cells and Gl absorption (Artursson et al., 1991), which reduces
the amount of extrapolation to clinical usage.
In an attempt to alter the pharmacokinetics of a drug or to increase its
transport, an analog of a drug can be made by chemical modification of the
original drug, such as the covalent addition of an alkyl chain to increase the
lipophilicity. A potential detriment to chemical modification is a decrease in the
drug’s ability to bind to receptors. Prodrugs are an alternative approach, and our
Pal(3)-BBI is an example. The reversible disulfide linkage in protein conjugates
can be reduced at the cell surface after being transcytosed (Wan et al., 1990). If
the conjugate is internalized by the cell after association with the membrane, the
linkage can be reduced intracellularly (Taub et al., 1994). Once the disulfide
bonds are reduced, the active form of the drug is present to elicit the therapeutic
response, which is the desired outcome of the prodrug approach.
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7
MATERIALS AND METHODS:
Synthesis and radiolabeling of palmitic acid-conjugated BBI:
The synthetic procedure for the preparation of fatty acid-BBI conjugates
has been described previously (Ekrami et al., 1995). Briefly, dithiopyridine (I) was
reacted with cysteine (II) to produce PDC (III) (A). PDC was then acylated with
the active N-hydroxysuccinimide ester of palmitic acid (IV) to produce N-palmityl
CDP (Pal-CPD) (V) (B). N-succinimidyl propionate pyridine disulfide (SPDP) was
reacted with amine groups on BBI to introduce the exchangeable disulfide moiety
into the protein. After subsequent reduction with dithiothreitol (DTT), sulfhydryl-
containing BBI (BBI-SH) is reacted with Pal-PDC to form the desired conjugate,
Pal-BBI (VI) contained palmitic acid side groups linked to BBI via a reducible
disulfide bond (C). Scheme I represents the synthetic pathway. The reaction of
BBI with SPDP is pH sensitive, allowing the final number of palmitic acid
moieties to vary from 1 to 4.5. Each conjugate, containing varying numbers of
fatty acid additions, were synthesized separately, and hence required no further
separation from other conjugates. In this study, a conjugate with an average of 3
additions was synthesized.
After the conjugate was purified with an LH20 column, the Pal(3)-BBI and
native BBI were radioiodinated using the Chloramine-T method (McConahey and
Dixon, 1980). Namely, 2 mg of either native BBI or Pal(3)-BBI was dissolved in
0.8 ml of phosphate-buffered saline (PBS). To the protein solution, 0.4 p.Ci of
1 PS
Na I was added. The solution was mixed well. A Chloramine-T solution was
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< B )
^ V H
0 “ S' Sv ^
\= N v I
NH2
Q -s ’s v A N i
n H
(V)
(C) O . OH
i h sh . < w ?
N H
(VD (V)
O ^O H
0 = 8 * l PROTEIN j-S -S A
B B
(VII)
Scheme I. Dlthlopyridine (I) was reacted with cycteine (II) to produce PDC (III)
(A). PDC was then acylated with the active ester of palmitic acid (IV) to produce
Pal-PDC (V) (B). N-succinimidyl propionate pyridine disulfide (SPDP) was
reacted with BBI to produce a sulfhydryl-containing protein (BBI-SH) (VI), and
when reacted with Pal-PDC (V), produces the final product, Pal-BBI (VII), in
which each palmitic acid side chain is linked to BBI via a reducible disulfide
bond.
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prepared by dissolving 4 mg of Chloramine T in 0.5 ml of 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 for 10 minutes, with
gentle mixing every 2 minutes. A solution of sodium metabisulfite solution was
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 mixed well. The solution was
allowed to react for 10 minutes, with gentle mixing every 2 minutes. A potassium
iodide solution was prepared by dissolving 10 mg in 0.5 ml of water. An aliquot of
0.05 ml was added to the reaction mixture and the solution was mixed well. For
the native BBI as well as the fatty acid conjugate, the 1 25 l label resides on the
tyrosine residues within the BBI molecule and the fatty acid is not radiolabeled.
The iodinated protein conjugate was purified with a Sephadex G-25 (Sigma, St.
Louis, MO) column (10 ml) by eluting with PBS until one 9 ml were collected in
test tubes containing 1 ml each. Each fraction was analyzed in a gamma counter
to determine which fraction(s) contained the radiolabeled protein, which usually
eluted from fractions 3-4. Before free iodine eluted (from fractions 9-10), the
column was disposed of. The protein-containing fractions that were isolated were
subsequently dialyzed over night at 4°C using a dialysis tubing with a molecular
weight cut off of 3,000. After determining the final volume of protein solution
within the dialysis tubing, the specific activity was determined by dividing the total
radioactivity of the iodinated protein solution by the amount of protein in solution.
The specific activity was expressed in terms of pCi/mg and for sample analysis,
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10
was used to convert radioactivity in blood to amount of protein. This procedure
was earned out for both the native polypeptide and the palmitic acid conjugate.
Tissue culture technique:
The Caco-2 cell line was obtained from American Type Culture Collection
(Rockville, MD) and the method of maintaining the cells is a modification of one
used by Pinto, et al. (Pinto et al., 1983). The cell line is constantly maintained in
Dulbecco’s Modified Eagle’s Minimum Essential Medium (D-MEM) (Gibco, Grand
Island, NY) supplemented with 10% Fetal bovine serum (FBS) (Bio Whittaker,
Walkersville, MD), 1% nonessential amino acids (Gibco, Grand Island, NY), 1%
L-glutamine (Gibco, Grand Island, NY) and penicillin/streptomycin (Gemini
Bioproducts, Calabasas, CA) at a final concentration of about 1%., at 37°C in an
atmosphere of 5% CO2 and 90% humidity. The medium was changed every-
other day and the cells were grown until confluence was reached. The cells were
then transferred into a fresh T25 flask at a density of approximately one million
cells in 5 ml of fresh medium. For uptake studies, 6-well cluster plates with a well
2
diameter of 35 mm and a growth area of 9.5 cm were used (Costar, Cambridge,
5
MA) and cell cultures initiated by seeding 1.5 X 10 cells/2ml/well. The transport
study was performed on the day when the cells reached confluence. For
transport studies, Costar’s 24mm transwells with a porous cell culture insert
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containing a 0.4 pm nuclepore polycarbonate membrane (Costar, Cambridge,
5 2
MA) were used with cells seeded at a density of 1 X 10 cells/cm in the insert
with the same media 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 every other day. Confluence usually occurred
approximately six to seven days after passage. The TEER of the Caco-2
2
monolayer was approximately 500 Q/cm , which is sufficient to be used for the
determination of drug transport.
Uptake / affinity for cells:
The amount of radiolabeled polypeptide associating with two and one-half
week old Caco-2 cells was determined in 6-well cluster plates (Costar,
Cambridge, MA). Prior to the experiment, the FBS-containing medium was
replaced with serum-free cell culture medium. The cells were incubated in
serum-free medium for one-half hour as a pre-incubation prior to treatment
medium being used. Treatment medium consisted of 10 pg/ml of 1 2 5 l-labeled
protein, either as BBI or Pal(3)-BBI, in serum free medium and replaced the
serum-free medium after the one-half hour incubation. 2.0 ml of the treatment
medium was used to treat the Caco-2 cells. After one hour, the treatment
medium was removed by gently pipetting and the cells were washed three times
with ice-cold PBS. The cells were incubated for ten minutes at 37°C in 0.25 ml of
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12
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 200 X g for
ten minutes. The cell pellets were washed three times with fresh 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 both the
conjugate and the native BBI, this was done in duplicate. 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.
Transport across Caco-2 cells:
The amount of transcytosis of BBI or Pal(3)-BBI across the Caco-2 cell
monolayer was determined. The TEER was checked periodically during the time
course of the experiment, as leaky cellular junctions would allow larger
molecules to pass through and would give an inaccurate representation of the
transcytotic capability of the test systems.
Prior to the experiment, the FBS-containing medium was replaced with
serum-free medium. After one-half hour of pre-incubation, the serum-free
125
medium was replaced with treatment medium consisted of 10 pg/ml of I-
labeled protein, either as BBI or Pal(3)-BBI, in serum free medium. 1.5 ml of 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 1, 2, 3,4, 5 and 24 hours and was replaced with fresh medium. The
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13
TEER was measured at 3, 5 and 24 hours. At 24 hours, 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 rinses, the Transwell membranes were
removed from the inserts of Transwells and counted in a gamma counter to
determine the amount of radiolabeled protein associating with the cells. The
basal medium from each time point was analyzed on a 20 ml Sephadex G-50
column and eluted with PBS until one and one-half column volumes was
collected in 1 ml fractions. During the 24 hour time course of the experiment, the
cells were maintained at 37°C in an atmosphere of 5% CO2 and 90% humidity
except for the time to replace the basal medium.
Uptake / cellular processing in Caco-2 cells:
The procedure for this experiment is similar to the uptake / cellular affinity
experiment except that the cells were incubated with treatment medium for
twenty-four hours instead of one hour. At the end of the twenty-four hour
incubation period, a 0.1 ml aliquot of the medium was incubated with 0.9 ml of
FBS for 15 minutes and was run on a 40 ml Sephacryl S-200 column
(Pharmacia, Uppsula, Sweden) for analysis. An equal volume of treatment
medium that had not been incubated with cells, but had been kept in the same
conditions, was used as a control.
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Pharmacokinetic study:
125 125
The pharmacokinetic studies of l-BBI and l-Pal(3)-BBI were
conducted in CF-1 mice and was compliant with the “Principles of Laboratory
Animal Care” (NIH publication #85-23, revised 1985). The mice, 6 weeks old
and weighing between 20 and 25 g each, were separated into two groups of
twenty-four. Prior to dosing, the mice were placed under a heat lamp for twenty
to thirty minutes, which caused sufficient vasodilation to make dosing easier. The
mice were injected intravenously via the tail vein with unlabeled BBI spiked with
a 1 2 5 l-BBl label or unlabeled Pal(3)-BBI spiked with a 1 2 5 |-Pal(3)-BBI label,
respectively, at a dose of 3 mg/kg and with a specific radioactivity of
g
approximately 1X10 cpm/dose for each compound. The volume of the injected
dose was 0.1 ml. Three animals from each group were sacrificed at 5,10, 20,
60,120, 240, 360, and 480 minutes post-injection, respectively. The animals
were anesthetized with diethyl ether and blood (0.5 -1.0 ml) was collected by
cardiac puncture. Liver, kidneys, lungs, spleen, stomach, intestines, and colon
were removed, and the organs were rinsed with fresh, isotonic phosphate-
buffered saline (PBS, pH 7). After rinsing, each whole organ was placed in a test
tube, the organ-associated radioactivity was determined in a gamma counter
(Packard, Meriden, CT) and the results presented as mean percent injected
dose per tissue ± S.D. vs. time (minutes). A 0.2 ml aliquot of blood was counted
for radioactivity to determine the concentration. A value of 2.1 ml was used as
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
15
the total blood volume per mouse to determine the percent of the injected dose
(Davies and Moms, 1993).
Analysis of whole-blood radioactivity composition:
To each 0.2 ml aliquot blood sample, 0.8 ml of distilled water was added.
After vortexing, the blood was incubated in a 37°C water bath for 10 minutes to
lyse the red blood cells. Blood was pooled by combining a 0.33 ml aliquot from
the three animals of the same group and same time point. The pooled blood was
centrifuged at 200 X g for 10 minutes to remove cell ghosts. To determine the
percent of the total radioactivity corresponding to intact protein, a 0.8 ml aliquot
of the blood supernatant was applied to a size exclusion Sephadex G-50 column
(20 ml) and eluted with PBS, pH 7. One and one-half column volumes (30 ml)
were collected in 1 ml fractions, and the radioactivity in each fraction was
determined, assuming that intact protein eluted at void volume (10 ml), whereas
degradation products eluted at column volume (20 ml). The concentration of
intact polypeptide was calculated by multiplying the total concentration by the
percentage of intact polypeptide.
Determination of pharmacokinetic parameters:
The pharmacokinetic analysis of the blood concentration vs. time data
was performed using a two-compartment model with the RSTRIP program
(Micromath, Salt Lake City, Utah). This program determines the mean residence
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
16
time (MRT) and calculates the area under the concentration vs. time curve
125 125
(AUC) using the trapezoidal rule and estimates the half-life of l-BBI and I-
Pal(3)-BBI in blood using a non-linear, weighted, least squares regression. The
parameters were determined based on intact protein at each time point for both
groups, as described above. The apparent blood clearance (Cl) and steady state
volume of distribution (Vd) were calculated with the parameters from RSTRIP
using the following equations:
Cl = Dose / AUC
Vd = Cl * MRT
The in vitro and in vivo binding of BBI and Pal(3)-BBI to albumin, serum
and plasma:
To determine the in vitro binding, a sample of 2 mg of albumin was
incubated with 5 jig 1 2 5 I-BBI or 1 2 5 |-Pal(3)-BBI. Once it was determined that there
was a difference in the binding of BBI when compared to that of Pal(3)-BBI, the
same procedure was performed with mouse serum, another preparation was
125 125
made that contained a combination of 10 ng l-BBI or l-Pal(3)-BBI and 0.3
ml FBS. A third preparation contained a combination of 10 pg 1 2 5 I-BBI or 1 2 5 |-
Pal(3)-BBI and 0.175 ml of mouse serum. The samples were incubated at 37°C
for 1 hour and diluted to a final volume of 1 ml with PBS. The samples were
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
analyzed on a 40 ml Sephacryl S-200 column, and eluted with PBS. The sample
was collected in 1 ml fractions in test tubes until one and one-half column
volumes were collected. Each fraction was analyzed by determining the
absorbance at 280 nm, after the samples were transferred to a quartz cuvette, to
quantify the presence of protein, and also in a gamma counter in the collection
test tubes.
To determine the in vivo binding of the polypeptide to plasma proteins,
125
one l-Pal(3)-BBI-treated mouse was sacrificed 2 hours post-injection and the
blood was removed, transferred to test tubes containing 0.1 ml of heparin, and
125
centrifuged to isolate the plasma. An aliquot of plasma from l-Pal(3)-BBI-
treated mouse was analyzed in the same manner as the above in vitro samples.
RESULTS:
Cellular uptake:
Caco-2 cells were incubated with 1 2 5 I-BBI or 1 2 5 |-Pal(1 )-BBI or 1 2 5 |-Pal(4)-
BBI for one hour, and the cell-associated radioactivity, and hence polypeptide,
was measured. As shown in Figure 1, there was minimal association of native
BBI with the cells, while as the number of fatty acid residues per BBI increased
from one to four, there was a corresponding increase in affinity of the conjugate
for the cells.
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18
Transport:
To determine if the increase in cellular uptake would result in the transport
125 125
of the conjugate across Caco-2 cells, the transcytosis of l-BBI or l-Pal(2)-
BBI in Transwells was determined. Figure 2 shows the analysis of the 24 hour
basal medium. The peak at fraction 9 corresponds to 8.6 ng of intact 1 2 5 l-Pal(3)-
125
BBI, while there was no intact l-BBI transported. It was not determined
whether the chemical nature of this fraction was that of intact conjugate, or free
BBI.
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19
Pal(1)-BBI Pal(4)-BBI
Fig. 1. Amount of cell-associated BBI in Caco-2 cells in culture. Cells were
incubated with 1 2 5 I-BBI, 1 2 5 l-Pal(1)-BBI or 1 2 5 l-Pal(4)-BBI for one hour, rinsed,
and counted in a gamma counter. Values are represented as ng of BBI per well
and with standard deviations indicated as error bars (n=3).
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20
10000
8000 -
6000 '
1 4000
O
2000
-2000
0 10 20 30 40
Eluate Volume (mL)
Fig. 2. Transport of 1 2 5 I-BBI and 1 2 5 l-Pal(2)-BBI across filter-grown Caco-2 cells.
Cells were incubated with radiolabeled BBI, either as the native polypeptide (□)
or as the Pal(2)-BBI conjugate (♦). The 24 hour transport media for each
compound were analyzed on separate Sephadex G-50 columns (20 ml). The
samples were eluted with PBS and collected in 1 ml fractions, which were
counted in a gamma counter.
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21
Pharmacokinetics:
At the completion of the animal experiment, the isolated organs were
counted in a gamma counter. Organs with significant levels ofradioactivity are
125 125
presented as mean percent injected dose per tissue ± S.D. for l-BBI or I-
Pal(3)-BBI treatments. The spleen, and lungs had less than 2% of the injected
dose at any time point, and are therefore not shown.
125
In order to properly determine the pharmacokinetic parameters of l-BBI
and its palmitic acid conjugate, the amount of intact polypeptide had to be
determined. In order to do this, the blood was analyzed on a 20 ml size exclusion
Sephadex G-50 column (Fig. 3) to distinguish the intact polypeptide from smaller,
degradation products. Table I shows the amount of intact polypeptide (the ratio
of the CPM in the intact protein peak to the total CPM applied to the column, with
125 125
the column recovery greater than 95%) as a function of time for l-BBI and I-
125
Pal(3)-BBI. The amount of radioactivity in the last two time points from the I-
BBI dose was insufficient to be quantified on the Sephadex G-50 column. At 4
hours, compared with the total amount of polypeptide in the blood, 6.8% was
intact in the blood, so a conservative estimate of 6.6% intact polypeptide was
used for the 6 and 8 hour time points. As this was a conservative estimate, the
125 125
differences in the AUC and t-j/2p for l-BBI and l-Pal(3)-BBI are potentially
even greater. The amount of intact polypeptide (ng) per ml of blood vs. time
(minutes) was used to determine the pharmacokinetic parameters.
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22
5000.0
4500.0
4000.0
3500.0
3000.0
2 2500.0
O 2000.0
1500.0
1000.0
500.0
0.0 ■
-500.0 (
Eluate Volume (mL)
Fig. 3. Blood analysis on a Sephadex G-50 (20 ml) column to determine the
amount of intact protein in each time point. With the amount of intact protein
known, the pharmacokinetic parameters could be determined. This
representative blood analysis is from the 2 hour time point of iv-administered
l-Pal(3)-BBI, where the intact compound eluted at fraction 8 and the
degradation products eluted at fraction 20.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
25 30
125 125
Table I. IV study of l-BBI and l-Pal-BBI in mice
a
% intact polypeptide in blood
Time (min) BBI Pal-BBI
5 80.1 96.0
10 61.3 92.5
20 27.9 83.9
60 14.3 69.5
120 8.6 69.4
240 6.8 58.4
360 b 61.6
480 b 45.8
“Intact” refers to the % of radioactivity found at the Sephadex G-50 column
void volume when compared to the total amount of radioactivity in the blood
sample analyzed on the column.
k The amount of radioactivity of the sample was not sufficient to be analyzed
the Sephadex G-50 column.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Fig. 4 shows the curve fit graph generated by RSTRIP. Table II shows the final
125 125
pharmacokinetic parameters for both the l-BBI and l-Pal(3)-BBI treatment.
125
After the single dose, the distribution half-life (t-|/2a) for l-BBI is approximately
125
5.4 minutes, while for l-Pal(3)-BBI it is approximately 11.1 minutes. Once
distribution has occurred, the terminal half live (t-|/2p) are 105.4 minutes and
125
176.0 minutes, respectively. The mean residence time (MRT) of l-Pal(3)-BBI
125
is 218.6 minutes, which is 2.8-fold higher than that of l-BBI, 77.3 minutes. The
area under the curve (AUC) is the PK parameter with the most dramatic
difference. By conjugating three palmitic acids to BBI, the AUC increases by a
125 125
factor of 10.8. For both l-BBI and l-Pal(3)-BBI administration, there was
less than 2% of the injected dose in the lungs and spleen at all time points.
125
In the blood samples from l-BBI-treated mice (Fig. 5), the level of radioactivity
in blood was 24.3% of the injected dose at 5 minutes post-dose and declined
125
biexponentially with a terminal value of 1.1 % at 480 minutes. In l-Pal(3)-BBI-
treated mice, blood levels were 58.3% of the injected dose at 5 minutes post
dose and 5.8% of the injected dose remained at 480 minutes. When the levels in
the kidneys (Fig. 6) were compared with those in the liver (Fig. 7), there was a
125 125
dramatic, yet understandable difference between I- BBI and l-Pal(3)-BBI.
125
The levels in the kidneys for l-BBI started off at 35% of the injected dose, and
decreased to 1.4% over the 480 minute time course. There was a steady and
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
25
irv
ra
O J
u
1 0'
, t i
1 0 ’
n
i ime iminj
Fig. 4. Curve-fit of concentration (mg) versus time (min) for l-BBI ( • ) and I-
Pal(3)-BBI (■). From this curve, the pharmacokinetic parameters were
determined by RSTRIP. Each time point represents an average value from three
animals.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
26
Table II. Relevant Pharmacokinetic Parameters Following IV Administration of
1 2 5 I-BBI and 1 2 5 l-Pal-BBI
BBI Pal-BBI
MRT (min) 77.3 218.6
AUC
(min.pg/ml)
163.8 1773.5
CIT (ml/min.kg) 18.3 1.69
a-t-i/2 (min)
5.42 11.06
p-t-i/2 (min)
105.4 176.0
Vd (ml/kg) 1415.0 369.8
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
27
70
o
C O
o
Q
■ o
a
o
a t
c
o
c
a >
o
0 )
a.
0 100 200 300 400 500
Time (min)
Fig. 5. Total radioactivity in blood as percent of the total injected dose following
iv administration of 1 2 5 I-BBI (O) and l-Pal-BBI (•) in mice. Each mouse was
administered 3 mg/kg, either as the free polypeptide or as the palmitic acid
conjugate. Each point represents an average of three animals. The standard
deviations are represented with error bars, or are smaller than the symbols.
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28
80
70
60
50
40
30
20
10
0
0 100 200 300 400 500
Time (min)
Fig. 6. Total radioactivity in kidneys as percent of the total injected dose
following iv administration of 1 2 5 I-BBI (O) and 1 2 5 l-Pal-BBI ( • ) in mice. Each
mouse was administered 3 mg/kg, either as the free polypeptide or as the
palmitic acid conjugate. Each point represents an average of three animals. The
standard deviations are represented with error bars, or are smaller than the
symbols.
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29
o
0
o
O
■ o
£
u
0
'e
40
o
c
0
U
a *
0
Q .
O-
500 0 100 200 300 400
Time (min)
Fig. 7. Total radioactivity in the liver as percent of the total injected dose
following iv administration of 1 2 5 I-BBI (O) and 1 2 5 l-Pal-BBI (• ) in mice. Each
mouse was administered 3 mg/kg, either as the free polypeptide or as the
palmitic acid conjugate. Each point represents an average of three animals. The
standard deviations are represented with error bars, or are smaller than the
symbols.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
pronounced clearance during the first 60 minutes post-injection. In comparison,
the levels in the liver started off at 4% as the maximum and decreased gradually
125
over time. On the other hand, for l-Pal(3)-BBI the comparison profile was
practically reversed. The maximum level in the kidney (2.7%) were at the first
time point and decreased over time. In the liver, the levels were at almost 42% of
the injected dose initially. For the first 10 minutes, the levels were relatively
constant, but from 10 to 60 minutes, there was a rapid clearance followed by a
more gradual clearance from the liver over the remainder of the study. For the Gl
tract (stomach, intestines and colon considered together), there was an increase
until 120 minutes, at which time the maximum levels were reached, then the
levels decreased (Fig. 8). This trend was seen in both treatments.
Evaluation of binding:
To determine if albumin was responsible for the altered pharmacokinetics,
125 125
albumin was incubated with l-BBI or l-Pal(3)-BBI and analyzed on a 40 ml
Sephacryl column. The absorbance readings showed that albumin was eluted at
fraction 22, while native BBI emerges later at fraction 32 (Fig. 9). On the other
hand, when the palmitic acid conjugate was incubated with albumin, the
radioactivity and absorbance peaks eluted identically in fractions 12-22 (Fig. 10).
When 1 2 5 I-BBI was incubated with FBS, BBI eluted at fraction 30, while the
albumin peak eluted predictably at fraction 21 (Fig. 11). In a similar incubation
with 1 2 5 l-Pal(3)-BBI, the albumin peak was unchanged, but the polypeptide
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31
o
Q
• o
a »
**
o
a »
c
a >
o
w
0
a.
80
70
60
50
40
30
20
10
0
0 100 200 300 400 500
Time (min)
Fig. 8. Total radioactivity in the Gl (stomach, small and large intestines and
colon were combined) tract as percent of the total injected dose following iv
administration of 1 2 5 I-BBI (O) and liK > l-Pal-BBI ( • ) in mice. Each mouse was
administered 3 mg/kg, either as the free polypeptide or as the palmitic acid
conjugate. Each point represents an average of three animals. The standard
deviations are represented with error bars, or are smaller than the symbols.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
32
s
Q .
O
4900
3900
2900
1900
900
-100
0.40
0.15
- 0.10
0 10 20 30 40 50 60
E
c
s
C M ,
0 )
J D
<
Eluate Volume (mL)
Fig. 9. In vitro binding of 1 2 5 I-BBI to albumin. Analysis on a Sephacryl
column (40 ml) where sample was eluted with PBS and collected in 1
fractions. Each fraction was counted in a gamma counter (□ ) and the
absorbance was read 280 nm (♦).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
C O E
33
5000 r0.50
4000
3000 0.25
2000
1000 0.00
0
-1000 -0.25
0 10 20 30 40 50 60
Eluate Volume (mL)
Fig. 10. In vitro binding of 1 2 5 l-Pal(3)-BBI to albumin. Analysis on a Sephacryl S-
200 column (40 ml) where sample was eluted with PBS and collected in 1 ml
fractions. Each fraction was counted in a gamma counter (□ ) and the
absorbance was read 280 nm (♦).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
40000
r 2.o
30000
O 20000
10000
Eluate Volume (mL)
Fig. 11. In vitro binding of 1 2 5 I-BBI to fetal bovine serum. Analysis on a
Sephacryl S-200 column (40 ml) where the sample was eluted with PBS and
collected in 1 ml fractions. Each fraction was counted in a gamma counter (o)
and the absorbance was read 280 nm (•).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
35
conjugate peak emerged from the column at fraction 15 (Fig. 12). The binding of
125 125
l-BBI or l-Pal(3)-BBI was also tested in previously-isolated mouse serum
for a more precise correlation with the in vivo conditions. The elution profile of
125
l-BBI indicated that native BBI did not bind to serum proteins (Fig. 13), while
125
l-Pal(3)-BBI bound to a serum protein with a molecular weight higher than that
of albumin (Fig. 14). Plasma was isolated and analyzed in the same manner as
the above in vitro samples. The elution profile of a plasma sample, which was
125
isolated from a single mouse that was injected with l-Pal(3)-BBI and sacrificed
125
2 hours post-injection, was similar to that of the samples with l-Pal(3)-BBI and
either FBS or mouse serum (Fig. 15). The recovery of radioactivity from the
column was greater than 95% for every sample analyzed.
Having chosen a conjugate with three fatty acids per BBI, and having a
method of distinguishing between BBI and Pal(3)-BBI, there was an interest in
repeating the uptake experiment and try to evaluate the cellular processing of the
conjugate and determine if the disulfide bond is cleaved at the cell surface,
releasing free BBI into the medium. The uptake experiment was done in the
same manner as before, except for the fact that the cells were exposed to the
treatment medium for 24 hours instead on one hour. At the end of 24 hours, after
the cells were rinsed and removed from the plates with trypsin/EDTA, the cell
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
36
£
a
40000
r2.0
■1.5
30000
■ 1.0
20000
0.5
10000
0.0
0 -knmnmiP— r-
0 10 20
-0.5
30 40
CM
«
.o
Eluate Volume (mL)
Fig. 12. In vitro binding of 1 2 5 l-Pal(3)-BBI to fetal bovine serum. Analysis on a
Sephacryl S-200 column (40 ml) where sample was eluted with PBS and
collected in 1 ml fractions. Each fraction was counted in a gamma counter (o )
and the absorbance was read 280 nm (•).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
37
15000
s
a.
o
5000
-5000
- 1.0
- 0.8
- 0.4
* 0.0
0 20 40
E
e
s
N ,
m
<
Eluate Volum e (ml)
Fig. 13. In vitro binding of 1 2 5 I-BBI to serum isolated from a mouse. Analysis on
a Sephacryl S-200 column (40 ml) where sample was eluted with PBS and
collected in 1 ml fractions. Each fraction was counted in a gamma counter (□ )
and the absorbance was read 280 nm (♦).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
38
r 1 -2
■ 1.0
15000
• 0.8
s
0 L
o
•0.4
5000
• 0.0
-5000
40 0 20 60 80
E
c
o
CO
C M
«
■D
<
Eluate Volume (ml)
Fig. 14. In vitro binding of 1 2 5 l-Pal(3)-BBI to serum isolated from a mouse.
Analysis on a Sephacryl S-200 column (40 ml) where sample was eluted with
PBS and collected in 1 ml fractions. Each fraction was counted in a gamma
counter (□) and the absorbance was read 280 nm (♦).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
39
40000
r 2 .o
30000
' 1.0
20000
0.5
10000
0.0
20 30 40 50 60 10
E
c
§
2.
c ri
A
<
Eluate Volume (ml)
Fig. 15. In vivo binding of 1 2 5 l-Pal(3)-BBI to serum proteins in a mouse that was
injected with the conjugate by the iv route via the tail vein. Analysis on a
Sephacryl S-200 column (40 ml) where sample was eluted with PBS and
collected in 1 ml fractions. Each fraction was counted in a gamma counter (o)
and the absorbance was read 280 nm (•).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
40
2 0
■ BBI
■ Pal(3)-BBI
0.0435%
Trypsin-removable Cell-associated
Fig. 16. Amount of 1 2 5 I-BBI and 1 2 5 l-Pal(3)-BBI that associates with the
extracellular matrix (trypsin removable) and the cellular matrix (cell-associated)
of Caco-2 cells after 24 hours of exposure. Amounts are expressed as per cent
of total polypeptide that the cells were exposed to.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
pellet was counted as a separate fraction from the trypsin solution after
centrifugation. Fig. 16 depicts the amount of BBI or Pa!(3)-BBI that was removed
by the trypsin, as well as the amount still associated with the cells. The
polypeptide that was removable by trypsin treatment was considered cell
surface-bound BBI, while the amount remaining with the cells after the rinses
and trypsin treatment was BBI either associated with the lipid bilayer, or had
been internalized into the cells. An aliquot of the 24 hour treatment medium was
incubated with FBS and analyzed on a Sephacryl S-200 column. As a control, an
aliquot of the treatment medium that had not been used in the experiment, but
had been kept in the same conditions of 37°C in an atmosphere of 5% CO2 and
90% humidity for the same 24 hour period. The 24 hour uptake medium analysis
(Fig. 17) showed that about 30% of the sample consisted of degradation
products eluting at fraction 47. The albumin peak was at fraction 21, while 1 2 5 l-
Pal(3)-BBI was visible at fraction 15. In contrast to the uptake medium, the
amount of degradation products in the control medium (Fig. 18) was only 9%.
Also, the peak at fraction 30, which corresponds with intact BBI, was larger
(14.32 %) in the uptake medium than in the control medium (7.98 %).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
42
s
a .
o
40000
■3.5
30000
-2.5
20000
10000
0.5
0
-10000 -0.5
0 10 20 30 40 50 60
o
c o
c o
<
Eluate Vol. (mL)
Fig. 17. 1 2 5 l-Pal(3)-BBI in culture media after 24 hours of incubation with Caco-2
cells grown in 6-well cluster plates. An aliquot of the medium was incubated with
FBS and was analyzed on a Sephacryl S-200 column (40 ml) where the sample
was eluted with PBS and collected in 1 ml fractions. Each fraction was counted
in a gamma counter (□) and the absorbance was read 280 nm (♦ ).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
43
80000
-3.5
60000
-2.5
40000
S
o.
o
-1.5
20000
-0.5
•20000
•0.5
0 10 20 30 40 50 60
Eluate Volume (mL)
Fig. 18. An aliquot of the control uptake medium that had not been incubated
with cells was incubated with FBS and was analyzed on a Sephacryl S-200
column (40 ml) where the sample was eluted with PBS and collected in 1 ml
fractions. Each fraction was counted in a gamma counter (□ ) and the
absorbance was read 280 nm (♦).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
DISCUSSION:
I. Can lipidization increase cellular uptake and transcellular transport of
peptides?
!n the beginning of this project, fatty acid-BBI conjugates containing
different numbers of palmitic acids per BBI molecule were considered. It was
intended to show was that the number of fatty acids would make a difference in
the conjugate’s ability to associate with the cells surface as a first step to being
internalized and subsequently transported. Indeed, it was demonstrated that as
the number of palmitic acids per BBI molecule increased, there was a
corresponding increase in the amount of association with Caco-2 cells. As the
number of fatty acids was increased, the lipophilic nature of the conjugate
increased and lead to a greater attraction to the lipid bilayer of the cells.
When a fatty acid conjugate containing two palmitic acids per BBI
molecule was compared with the native polypeptide in terms of transport across
125
Caco-2 cells, the l-BBI elution profile showed no radioactive peak at fraction
10, which indicates that intact protein was not being transported across the
125
Caco-2 cell layers. On the other hand, when l-Pal(2)-BBI was incubated with
Caco-2 cells in the same manner, there was radioactivity eluting at the fraction
corresponding to intact polypeptide. The amount that was intact was determined
to be 8.6 ng, which was 0.057% of the amount incubated with the cells Although
the amount was small, it was encouraging enough for us to proceed with an
animal model. We chose to work with mice, but the small blood volume of mice
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
45
limited the number of time points that each animal could contribute to one. To
compensate for this, each time point represents an average of three animals. In
retrospect, rats might have been a more reasonable choice because of the ability
to take serial blood samples and get a complete pharmacokinetic profile for each
animal.
Can lipidization improve the pharmacokinetics of a peptide?
125
We demonstrate that by chemically linking palmitic acid to l-BBI, more
than a 10-fold increase in the AUC can be achieved, an important
125
pharmacokinetic parameter. In a subsequent experiment, l-Pal(3)-BBI was
shown to bind to a serum protein, most likely to lipoproteins, with a higher
molecular weight than that of albumin. The binding to the plasma proteins
125
explains the increase in AUC of the conjugate over the native l-BBI. The half-
125 125
lives of l-BBI and l-Pal(3)-BBI are not remarkably different, but that is
125
because only the free form of l-Pal(3)-BBI is able to leave the circulation and
be eliminated.
The binding of Pal(3)-BBI conjugate to serum protein(s) may explain the
increase of conjugate uptake by hepatocytes and the decrease of elimination via
the kidneys; consequently, an increase in AUC is observed. Once released from
125
the serum proteins, the unbound form of l-Pal(3)-BBI would be chemically less
125
stable and would be eliminated by the kidneys in a similar manner to l-BBI
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
125
due to the similarity in size (9 kDa versus 8 kDa). It is also likely that free l-BBI
released from the fatty acid conjugate upon reduction of the disulfide linkage in
the blood or organs would subsequently be eliminated by the kidneys.
Generally, when the lipophilicity of a drug is increased, the volume of
distribution (Vd) increases because the drug will tend to leave the central
125
compartment and enter the periphery. However, in the case of l-Pal(3)-BBI,
125
when compared with native l-BBI, the Vd is lower. This can be explained by
the binding of the conjugate to serum proteins, as described above. Only the
unbound form is able to leave the central compartment.
An amphiphilic block copolymer of ethylene oxide (hydrophilic) and
propylene oxide (hydrophobic), and the surface-active polymer proxanol, was
used to modify BBI previously (Gladysheva et al., 1995). Due to a slight increase
in hydrophilicity over BBI, their monoaldehyde form of proxanol on BBI (BBI-PE)
was found to have reduced interactions with membranes, while our Pal(3)-BBI
conjugate demonstrated an increase in uptake of our conjugate in Caco-2 cells
(Ekrami et al., 1995) as well as a 10.8-fold higher increase in AUC in mice.
III. Can lipidization change the binding of peptides to plasma proteins?
From the animal experiment, it is clear that the palmitic acid-BBI
conjugate is associating or complexing with a constituent of the blood which may
be responsible for the increase of the plasma t-j/2 - Because albumin is the
predominant plasma protein, and because it is well-known that albumin is the
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natural carrier for fatty acids (Reynolds et al., 1968), it was first assumed that
125 125
Pal(3)-BBI was also bound to albumin. When l-BBI and l-Pal(3)-BBI are
125
incubated with albumin, l-BBI elutes from Sephacryl S-200 column sufficiently
125
later than albumin, giving evidence to the fact that l-BBI does not form a
125
complex with albumin. On the other hand, when l-Pal(3)-BBI is incubated with
pure bovine albumin, there is a shift in the elution time of the conjugate,
indicating that a complex formation occurs. A complex forms between albumin
and Pal(3)-BBI that does not significantly change the elution profile of the
albumin (Shen et al., 1996) and these results suggest that the elution profile is
characteristic of the serum protein. The fact that the BBI conjugate does not add
significantly to the weight of complex when compared with the serum protein
alone, implies that the analysis of the serum protein to determine its identity can
be based solely on molecular weight. One beneficial piece of information
obtained from this albumin-binding screening was the elution profile of albumin,
as detected in the absorbance of the fractions at 280 nm.
125
Since there are more constituents to blood than albumin alone, l-BBI
125
and l-Pal(3)-BBI were incubated with the fetal bovine serum that is normally
125
used as a supplement to our tissue culture medium. The l-BBI showed had
125
similar elution profile to the sample with albumin alone. In contrast, the I-
125
Pal(3)-BBI showed an interesting shift in elution time to an earlier fraction. I-
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
48
Pal(3)-BBI eluted at fraction 15, which indicates that the size of the eluting
molecules are larger than the exclusion size of the gel. Based on Sephacryl S-
200 size-exclusion chromatography, which can separate molecular weights
ranging from 5000 - 200,000, it is concluded that the molecular weight of the
Pal(3)-BBI-carrying protein(s) must be larger than 200,000. Plasma proteins fall
into this category, and include LDLs, HDLs, etc. It also indicates that the
molecular weight is larger than that of albumin. The shift in elution time is
125
suggesting that l-Pal(3)-BBI’s affinity for this unknown element of serum is
greater than that its affinity for albumin. The only clue to its identity is that the
molecular weight is higher than 67,500, which is that of albumin.
In order to develop a link between the in vitro analysis described thus far,
125
a single mouse was given an iv administration of l-Pal(3)-BBI and the plasma
that was isolated two hours post-injection was analyzed. The binding observed in
125
plasma which was isolated from l-Pal(3)-BBI iv-injected mice was consistent
with the binding in FBS and previously isolated mouse serum. This similarity
125
indicates that the element that l-Pal(3)-BBI binds to in serum can be
reproduced in vitro, making further analysis more convenient and less costly than
the in vivo counterpart.
The analysis of the blood from the pharmacokinetics study had been with
a Sephadex G-50 column in which BBI and Pal(3)-BBI have the same elution
profile, but it was now clear that with a Sephacryl S-200 column, if the
polypeptide or conjugate are in serum, they can be distinguished based on
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
49
elution time due to complex formation of Pal(3)-BBI with serum proteins. The
chemical nature of the polypeptide could now be determined.
IV. What is the intracellular fate of a lipidized peptide?
With this new method of analysis using a Sephacryl S-200 column and
incubation of samples in FBS, it was interesting to take a closer look at the
cellular processing of the palmitic acid conjugate in tissue culture uptake studies.
After 24 hours of exposure of Caco-2 cells to 1 2 5 l-Pal(3)-BBI, the 24 hour
treatment medium was incubated with FBS and analyzed on a Sephacryl S-200
column. The same analysis was performed on the control medium. There were a
few differences in the medium that had been incubated with the Caco-2 cells.
There was an increase in the amount of degradation produces, as indicated by a
large peak at fraction 47. Also, there was a larger peak at fraction 30, which most
likely corresponds with BBI being released into the medium following reduction of
the disulfide bond of the conjugate. Analysis of the control medium assured us
that the presence of free BBI in the medium, as well as degradation products,
was not due to heat-generated degradation or disulfide bond reduction. Rather,
the presence of these compounds was due to the cellular processing of 1 2 5 l-
Pal(3)-BBI over the time course of exposure during incubation.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
50
Conclusion:
In conclusion, we have shown that it is possible to decrease the
hydrophilic nature of BBI and to increase its affinity for cells through conjugation
with palmitic acid. The number of fatty acid moieties will influence the
physicochemical properties, size and lipophilicity, which controls the ability for
the conjugate to be taken up by cells, and to be transported across cells layers. It
might also change the efflux mechanism of internalized conjugate, although the
mechanism of efflux of the internalized fatty acid conjugate was not determined.
An alternative thought to the lipophilicity being the main determinant of transport
is that the rate limiting step is dehydration of the solute in order for it to enter the
hydrophobic, non-hydrogen bonding interior of the membrane (Conradi et al.,
1991). The researchers determined that solute-solvent interactions via hydrogen
bonding was a strong determinant of membrane permeability (Conradi et al.,
1991). They also argued that intermolecular hydrogen bonding leads to self
association of the peptide, forming large nondiffusible aggregates in solution.
Through conjugation, it is possible to achieve selective tissue targeting
and a change in tissue distribution. In the case of Pal(3)-BBI, there was an
increase in hepatic levels, so for liver disease or tumors in this region, this would
be a way of increasing the amount of drug reaching the desired area and
decreasing systemic toxicity. It is thought that fatty acids are taken up across
enterocytes by a specific and saturable process at the plasma membrane (Potter
et al., 1989) by a 40 kDa plasma membrane fatty acid-binding protein (Stremmel,
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
1988). Hepatocytes are rich in lipoprotein receptors, and if the Pal(3)-BBI
conjugate is binding to lipoproteins, this would also explain the increased levels
in the liver, but no specific experiments were done to determine whether it was
the presence of fatty acids or binding of the conjugate to lipoproteins that
contributed to the change in distribution. While the pharmacokinetic results
determined for BBI and Pal(3)-BBI do not apply to oral dosing, altering the
cellular affinity and targeting ability of peptides is critical if the oral route is to ever
be considered for peptide drugs. From a clinical perspective, the oral route is the
most desirable from the standpoint of self-administration.
The pharmacokinetic parameters of peptide drugs can be favorably
altered to increase systemic circulation and to reduce elimination. This is
especially important in the clinical setting, where the frequency of administration
of a therapeutic agent are minimized if possible. The 10-fold increase in AUC of
our conjugate over that of the unmodified polypeptide is promising.
The lipid modification of the polypeptide BBI has increased its lipophilicity,
as demonstrated in our previous studies in Caco-2 cells (Ekrami et al., 1995),
and has increased its AUC, making Pal(3)-BBI a better drug candidate than its
native counterpart, BBI. By derivitizing BBI with palmitic acid, we have changed
its pharmacokinetic parameters, and prolonged its persistence in the blood. For
BBI and other peptide or protein drugs, this could mean a longer duration of
action and higher tissue exposure, and is a viable approach in drug design.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
It has already been determined that reduction of the disulfide bond occurs
intracellularly (Shen et al., 1985), but it would also be useful to determine which
serum protein the palmitic acid conjugate has such a strong affinity. In order to
determine the plasma protein that the BBI conjugate is binding with, there are
various established procedures in which the serum lipoprotein classes and
subclasses can be separated based on a density gradient (Pietzsch et al., 1995,
Murdoch et al., 1994). The use of a vertical rotor and speeds up to 625,000 X g
shortened the run times from 48-72 hours to2-3 hours. With this knowledge, our
conjugate can be modified to further optimize its tissue targeting ability as well as
to further increase its retention in systemic circulation.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
53
References:
Al-Obeido, F., Hruby, V.J., Yaghoubi, N., Marwan, M.M., and Hadley, M.E.
(1992). Synthesis and biological activities of fatty acid conjugates of a cyclic
lactam alpha-melanotropin. J. Med. Chem. 3 5 :118-123.
Artursson, P. and Magnusson, C. (1990). Epithelial transport of drugs in cell
culture. II: Effects if extracellular calcium concentration on the paracellular
transport of drugs of different lipophilicities across monolayers of intestinal
epithelial (Caco2) cells. J. Pharm. Sci. 79: 595-600.
Bowman, D.E. (1944). Fractions derived from soy beans and navy beans which
retard tryptic digestion of casein. Proc. Soc. Exp. Biol. Med. 57:139-140.
Brenner, B., Hostetter, T., and Humes, D. (1978). Molecular basis of proteinuria
of glomerular origin. New Eng. J. Med. 298: 826-833.
Chantret, I., Barbat, A., and et al. (1988). Epithelium polarity, villin expression,
and enterocytic differentiation of cultured human colon carcinoma cells: a survey
of twenty cell lines. Cancer Res. 4 8 :1936-1942.
Conradi, R.A., Hilgers, A.R., Ho, N.F.H., and Burton, P.S. (1991). The influence
of peptide structure on transport across Caco-2 cells. Pharm. Res. 8 : 1453-
1460.
Davies, B. and Moms, T. (1993). Physiological parameters in laboratory animals
and humans. Pharm. Res. 1 0 ,1093-1095.
Davis, F.F., Kazo, G.M., Nucci, M.L., and Abuchowski, A. (1991). Reduction of
immunogenicity and extension of circulation half-life of peptides and proteins. In
Peptide and Protein Drug Delivery. V.H.L. Lee, ed. (New York: Marcel Dekker,
Inc.), pp. 831-880.
Ducan, R. (1987). Selective endocytosis of macromolecular drug carriers. In:
Controlled Drug Delivery, 2n d edition. (New York: Marcel Dekker), pp. 581-621.
Ekrami, H.M., Kennedy, A.R., and Shen, W. (1995). Water-soluble fatty acid
derivatives as acylating agents for reversible lipidization of polypeptides. FEBS
Letters 371: 283-286.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
54
Gladysheva, I.P., Polekhina, O.V., Shen, W.C., Shevchenko, A.A., Kazanskaya,
N.F., and Larionova, N.I. (1995). Structure and biological properties of Bowman-
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M. (1995). Very fast ultracentrifugation of serum lipoproteins: influence on
lipoprotein separation and composition. Biochim. Biophys. Acta 1 2 5 4 :77-88.
Pinto, M., Robine-Leon, S., Appay, M., Kedinger, M., Triadou, N., Kussaulx, E.,
Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J., and Zweibaum, A. (1983).
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Potter, B.J., Sorrentino, D., and Berk, P.D. (1989). Mechanisms of cellular
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Pharmacokinetics and biodistribution of Pal-BBI, a fatty acid-polypeptide
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
56
References:
Al-Obeido, F., Hruby, V.J., Yaghoubi, N., Marwan, M.M., and Hadley, M.E.
(1992). Synthesis and biological activities of fatty acid conjugates of a
cyclic lactam alpha-melanotropin. J. Med. Chem. 3 5 :118-123.
Artursson, P. and Magnusson, C. (1990). Epithelial transport of drugs in
cell culture. II: Effects of extracellular calcium concentration on the
paracellular transport of drugs of different lipophilicities across monolayers
of intestinal epithelial (Caco2) cells. J. Pharm. Sci. 79: 595-600.
Bowman, D.E. (1944). Fractions derived from soy beans and navy beans
which retard tryptic digestion of casein. Proc. Soc. Exp. Biol. Med. 57:
139-140.
Brenner, B., Hostetter, T., and Humes, D. (1978). Molecular basis of
proteinuria of glomerular origin. New Eng. J. Med. 298: 826-833.
Chantret, I., Barbat, A., and et al. (1988). Epithelium polarity, villin
expression, and enterocytic differentiation of cultured human colon
carcinoma cells: a survey of twenty cell lines. Cancer Res. 4 8 :1936-1942.
Conradi, R.A., Hilgers, A.R., Ho, N.F.H., and Burton, P.S. (1991). The
influence of peptide structure on transport across Caco-2 cells. Pharm.
Res. 8 : 1453-1460.
Davies, B. and Morris, T. (1993). Physiological parameters in laboratory
animals and humans. Pharm. Res. 1 0 ,1093-1095.
Davis, F.F., Kazo, G.M., Nucci, M.L., and Abuchowski, A. (1991).
Reduction of immunogenicity and extension of circulation half-life of
peptides and proteins. In Peptide and Protein Drug Delivery. V.H.L. Lee,
ed. (New York: Marcel Dekker, Inc.), pp. 831-880.
Ducan, R. (1987). Selective endocytosis of macromolecular drug earners.
In: Controlled Drug Delivery, 2n d edition. (New York: Marcel Dekker), pp.
581-621.
Ekrami, H.M., Kennedy, A.R., and Shen, W. (1995). Water-soluble fatty
acid derivatives as acylating agents for reversible lipidization of
polypeptides. FEBS Letters 371: 283-286.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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Honeycutt, Laura Roxann
(author)
Core Title
Alteration of the in vitro and in vivo processing of a polypeptide, BBI, through conjugation with palmitic acid
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Graduate School
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Master of Science
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Pharmaceutical Sciences
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Chemistry, pharmaceutical,Health Sciences, Pharmacy,OAI-PMH Harvest
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Shen, Wei-Chiang (
committee chair
), Koda, Robert T. (
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