Synthesis and in vitro evaluation of gabapentin prodrugs that target the human apical sodium-dependent bile acid transporter (hASBT)

Article abstract of DOI:10.1002/jps.22332

Gabapentin is a zwitterionic drug that exhibits low and variable oral absorption at therapeutic doses. The human apical sodium-dependent bile acid transporter (hASBT; SLC10A2) is a potential prodrug target to increase oral drug absorption. The objective w

Full text of DOI:10.1002/jps.22332

Synthesis and In Vitro Evaluation of Gabapentin Prodrugs That
Target the Human Apical Sodium-Dependent Bile Acid
Transporter (hASBT)
RANA RAIS, STEVEN FLETCHER, JAMES E. POLLI
Univerisity of Maryland School of Pharmacy, Baltimore, Maryland 21201
Received 4 May 2010; revised 16 June 2010; accepted 29 July 2010
Published online 16 September 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22332
ABSTRACT: Gabapentin is a zwitterionic drug that exhibits low and variable oral absorp-
tion at therapeutic doses. The human apical sodium-dependent bile acid transporter (hASBT;
SLC10A2) is a potential prodrug target to increase oral drug absorption. The objective was to
evaluate several bile acid conjugates of gabapentin as potential prodrugs that target hASBT.
Five analogues were synthesized and varied in ionic nature and the presence or absence of
glutamic acid linker between the bile acid and drug. Analogues were evaluated for their inhibi-
tion and uptake properties using stably transfected hASBT–MDCK cells. The two monoanionic
conjugates were potent hASBT substrates, with high affinity (Km of 16.3 and 5.99 :M) and high
capacity (Vmax of 0.656 and 0.842 pmol/cm2/s). The dianionic conjugate inhibited hASBT with
moderate potency but was not a substrate. The two monoanionic conjugates were catalytically
degraded in Caco-2 homogenate and rat liver microsomes. Each yielded gabapentin from pro-
drug. These two conjugates are novel prodrugs of gabapentin and illustrate prodrugs that can be
designed to target hASBT. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association
J Pharm Sci 100:1184–1195, 2011
Keywords: transporters; prodrugs; gabapentin; apical sodium-dependent bile acid trans-
porter; bile acid; active transport; bioavailability; permeability; stability
saturable amino acid transporters involved in its up-
take in the upper part of the small intestine.2,3 The
high doses required for neuropathic pain treatment
lead to saturation of transporter uptake.4–6 Rapid
gabapentin excretion and short half life (about 5–7 h)
warrants frequent dosing, causing noncompliance in
epileptic patients.7
INTRODUCTION
Gabapentin is a structural analogue of gamma-amino
butyric acid (GABA). It is indicated to treat neuro-
pathic pain and used as adjunctive therapy for par-
tial seizures in adults with epilepsy.1 Gabapentin ab-
sorption and pharmacokinetics are dose dependent
and highly variable between patients. In humans,
gabapentin bioavailability decreases from about 60%
at a 300 mg dose to about 35% or less at doses to treat
neuropathic pain (i.e., 1000 mg). Although the mech-
anism of uptake of gabapentin is still not clear, the
reasons for low and highly variable gabapentin ab-
sorption are speculated to be due to low capacity and
To overcome these pharmacokinetic limitations,
gabapentin enacarbil has been designed as
a
gabapentin prodrug. It is absorbed by high-capacity
nutrient transporters throughout the small and large
intestines, and is rapidly and extensively converted
by nonspecific esterases to gabapentin, delivering
dose-proportional exposure up to 2100 mg twice
daily.8 The prodrug also shows dose-proportional
gabapentin exposure at doses up to 6000 mg.9
Gabapentin enacarbil at 1200 mg significantly im-
proved restless legs syndrome symptoms compared
with placebo.10
Since the major limitation in gabapentin absorp-
tion results from its zwitterionic nature11 and its facil-
itated uptake by low-capacity transporters, an alter-
native approach to enhance gabapentin absorption is
through targeting high-capacity membrane transport
Abbreviations used: hASBT, human apical sodium-
dependent bile acid transporter; CDCA, chenodeoxycholic acid; glu-
CDCA, glutamic acid CDCA amide; OSU, N-hydroxysuccinimide;
OBT, benzotriazole; HBTU, benzotriazol-1-yloxytris-1,1,3,3 tetram-
ethyl uranium hexafluorophosphate; TEA, triethylamine; RT, room
temperature; MDCK, Madin–Darby canine kidney; CaCo-2, colon
adenocarcinoma cell line.
Correspondence to: James E. Polli (Telephone: +1-410-706-8292;
Fax: +1-410-706-5017; E-mail: jpolli@rx.umaryland.edu)
Journal of Pharmaceutical Sciences, Vol. 100, 1184–1195 (2011)
© 2010 Wiley-Liss, Inc. and the American Pharmacists Association
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BILE ACID PRODRUGS OF GABAPENTIN
1185
systems in the gut. The human apical sodium-
dependent bile acid transporter (hASBT; SLC10A2)
is one such potential target that can be exploited for
prodrug delivery. hASBT is a member of solute carrier
genetic superfamily and is involved in the absorption
of bile acids.12 It has high intestinal expression and
is a key player in enterohepatic circulation of bile
acids.13,14 hASBT transports 2 to 4 g of bile acids 6 to
10 times daily, giving an overall turnover rate of 20 to
30 g of bile salts per day, depicting the tremendous ef-
ficiency and capacity of this transporter. ASBT plays
a major role in cholesterol homeostasis and maintain-
ing the bile acid pool. A cholesterol-rich diet reduces
ASBT expression in mice and in Caco-2 cells. The
modulation of ASBT by cholesterol is an adaptive re-
sponse that decreases ASBT expression. hASBT has
been previously identified as a potential prodrug tar-
get to enhance oral drug absorption of low perme-
able drugs,15,16 although lack of a high-resolution
hASBT crystal structure has impeded understanding
of hASBT structural requirements.17
One prodrug strategy is to couple drug to a natu-
ral substrate to create a molecule that mimics the
three-dimensional structure of natural ligand of a
transporter.18 Bile acids are natural substrates for
hASBT. Their unique structural properties and the
large bile acid pool in the body affords them as suit-
able potential carriers for drug delivery. Various ap-
proaches have been employed to conjugate drugs to
bile acids to create novel molecules that retain recog-
nition by the bile acid transporters.15,16 One such ex-
ample is acyclovir, which has low oral bioavailability
because of low permeability. Acyclovir was coupled to
bile acid using valine as a linker to target hASBT.
Relative to uptake of acyclovir alone, the permeabil-
ity of the prodrug was 16-fold higher in cells in vitro.
In vivo, the oral bioavailability was enhanced twofold
as compared to the drug alone. This strategy clearly
indicated that ASBT can be used to enhance the oral
bioavailability of low-permeability drugs.19
Various structural prerequisites that are essen-
tial for recognition by hASBT have been determined
previously.17,20,21 It was demonstrated that monoan-
ionic, neutral, and cationic bile acid conjugates were
potent inhibitors of hASBT, indicating that a sin-
gle negative charge was not essential for binding to
hASBT, although was preferable for translocation. Di-
anionic compounds were neither inhibitors nor sub-
strates of hASBT. Based on the structural require-
ments, five gabapentin analogues were synthesized,
where drug was coupled to chenodeoxycholic acid
(CDCA). Among the five analogues, two were neu-
tral, two were monoanionic, and one was dianionic.
Since gabapentin shows incomplete and highly vari-
able uptake by amino acid transporter(s), high hASBT
efficiency and capacity was hypothesized to improve
gabapentin oral bioavailability.
EXPERIMENTAL SECTION
Materials
[3H]-Taurocholic acid (10:Ci/mM) was purchased
from American Radiolabeled Chemicals, Inc. (St.
Louis, Missouri). Taurocholate was obtained from
Sigma Aldrich (St. Louis, Missouri). CDCA was ob-
tained from TCI America (Portland, Oregon). Pro-
tected glutamic acid analogs were purchased from
Novabiochem (Gibbstown, New Jersey). Gabapentin
was obtained from spectrum chemicals (New
Brunswick, New Jersey). Geneticin, fetal bovine
serum (FBS), trypsin, and DMEM were purchased
from Invitrogen (Rockville, Maryland). All other
reagents and chemicals were of the highest purity
commercially available.
Synthesis of Gabapentin Analogues of
Chenodeoxycholic Acid
Synthetic schemes for the activation of carboxylic
acid have been described previously.17,21 Briefly, the
acid of bile acid was activated either by forming a
benzotriazole ester (OBT) or N-hydroxysuccinimide
ester (OSU) and separated by extraction. The acti-
vated bile acid was then coupled to "-benzyl ester
of glutamic acid through peptide-coupling chemistry
methods.17 Either the activated CDCA or its amide
with glutamic acid was then coupled to gabapentin
to obtain the monoanionic, neutral and dianionic con-
jugate. Gabapentin carboxylate was protected either
as its methyl ester or as its benzyl ester, as shown
in synthetic Schemes 1 and 2. The identity and pu-
rity of all final targets were confirmed by thin layer
chromatography (TLC), mass spectrometry (MS), and
one-dimensional (1D) C-13 NMR.
O
O
O
OH
O
O
O
HN
O
O
H
H
H2N
N
H
H2
N
CH3OH
O
CDCA-glu, TEA
HBTU, DMF
SOCl2
(1)
H
H
c
a
10%Pd/C, EtOH,
b
,
OH
HO
A
H
E
T
F
M
,
t
D
b
o
-
d
A
O
HO
O
C
O
O
HN
D
O
O
O
C
H
N
H
N
H
O
CH3
H
H
H
H
H
H
(3)
(2)
OH
HO
H
HO
OH
(a) 0ºC 1 h, RT 1 h, 70ºC overnight (b) 60ºC 24–48 h (c) H2 , 10% Pd/C, 40–50 psi 3–4 h (d) 60ºC 24–48 h.
Scheme 1. Synthesis of neutral and monoanionic conju-
gates of gabapentin methyl ester of glu-chenodeoxycholic
acid (CDCA) and CDCA. CDCA-"-benzyl-glu-gabapentin
methyl ester (1) was deprotected to yield the monoanionic
CDCA-glu-gabapentin methyl ester (2). CDCA-gabapentin-
methyl ester (3) is neutral and lacks a glutamic acid linker.
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1186
RAIS, FLETCHER, AND POLLI
OH
OH
O
O
N
O
O
O
N
OH
H2
benzyl alcohol
H2N
O
S
p-TsOH.H2O
S
O
OH
H2
O
O
O
O
O
O
HN
Toluene
b
O
O
H2N
H
H
O
S
OH
N
H
CDCA-glu, TEA
a
O
O
HBTU, DMF
a
O
OH
O
O
O
H
H
O
N
N
10%Pd/C, EtOH
b
OH
10%Pd/C, EtOH,
HO
H
CDCA-obt, TEA,
H
H
(4)
d
DMF
c
H
H
H
H
H
H
OH
HO
O
HO
OH
HO
OH
O
O
HN
H
N
H
O
H
(5)
H
H
(a) RT 12 h (b) 100º C 48 h (c) 60º C 48 h (d) H2 , 10% Pd/C, 40–50 psi 6–12 h.
OH
HO
H
Scheme 2. Synthesis of CDCA-gabapentin (4).
(a) 60º C 48 h (b) H2 , 10% Pd/C, 40–50 psi 6–12 h.
Scheme 3. Synthesis of dianionic gabapentin conjugate,
CDCA-glu-gabapentin (5).
Synthesis of Gabapentin Methyl Ester
To a magnetically stirred solution of gabapentin
(5.84 mM) in methanol on dry ice, 1.2 eq of thionyl
chloride (0.5 mL) was added dropwise. The reaction
was allowed to react for 1 h at 0◦C and then 1 h at
room temperature (RT). The reaction mixture was
then refluxed at 70◦C overnight. After the reaction
was complete, methanol was evaporated under re-
duced pressure and crude product was triturated in
ether. The resulting white crude material was ex-
tracted into 50 mL volumes of EtOAc (3×) and washed
with 15 mL of 1N NaHCO3 (3×), 15 mL of water
(3×), and 15 mL of brine (1×). The organic layer was
dried over Na2SO4 and filtered. Removal of EtOAc
under reduced pressure yielded a white fluffy solid.
MS showed appropriate peaks: [M + 1] 186.3, [M +
Na] 208.3.
Scheme 2 and 3 were employed to obtain compounds
4 and 5.
Compound 1. MS: [M + 1] 779.3. 13C NMR (DMSO-
d6): δ 11.65, 18.37, 20.26, 21.06, 21.06, 22.72, 23.16,
25.49, 26.95, 27.78, 30.56, 31.49, 31.59, 32.03, 32.29,
32.54, 32.54, 34.75, 34.85, 34.99, 35.32, 36.8, 39.10,
39.60, 39.78, 39.94, 40.11, 41.43, 41.92, 44.32, 50.02,
50.96, 51.77, 55.63, 65.77, 70.33, 127.70, 127.70,
127.99, 128.38,128.38, 136.03, 171.49, 171.80, 171.99,
172.95. Compound 2. MS: [M + 1] 689.6 13C NMR
(DMSO-d6): δ 11.65, 18.37, 20.24, 21.03, 21.03, 22.70,
23.15, 25.47, 27.26, 27.77, 30.55, 31.48, 31.82, 32.09,
32.26, 32.52, 32.52, 34.73, 34.82, 34.96, 35.30, 36.7,
39.60, 39.78, 39.94, 40.11, 41.41, 44.27, 41.90, 50.01,
50.93, 51.52, 55.64, 66.14, 70.31, 171.66, 171.80,
172.68, 173.57. Compound 3. MS: [M + 1] 560.3 13C
NMR (DMSO-d6): δ 11.65, 18.32, 20.29, 21.09, 21.09,
22.75, 23.20, 25.54, 27.15, 27.83, 30.60, 31.79, 32.32,
32.39, 32.54, 32.54, 34.78, 34.87, 34.97, 35.35, 36.88,
39.60, 39.78, 39.94, 41.46, 40.11 41.96, 50.07, 51.04,
55.72, 66.21, 70.38, 171.84, 172.88.
Synthesis of Bile Acid Conjugates of Gabapentin
The protected gabapentin methyl ester was coupled
to either OBT of CDCA or its amide with glutamic
acid, as presented in Scheme 1. After the reaction
was complete, the neutral compounds were extracted
with 60 mL volumes of EtOAc (3×), and washed with
15 mL of water (3×) and then 15 mL of brine (1×). The
organic layer was dried over Na2SO4 and filtered. Re-
moval of EtOAc under reduced pressure yielded crude
oily product. The conjugates were further purified by
flash column chromatography starting with ethylac-
etate and hexane (80:20), increasing to 100% ethylac-
etate. The products eluted out in ethylacetate. TLC
plates were stained using 10% w/v phosphomolybdic
acid in absolute ethanol, which showed single spots.
The neutral compounds (1 and 3) were used as such.
The monoanionic compound 2 was obtained via cat-
alytic hydrogenation to remove the "-benzyl ester
(10% Pd/charcoal in EtOH at 40–50 psi for 3–4 h).
MS showed appropriate peaks as given below. Sev-
eral attempts to hydrolyze methyl ester from 2 and 3
to obtain targets 4 and 5 were unsuccessful because
of simultaneous hydrolysis of amide bonds. Hence,
For all compounds, peaks 39.60, 39.78, and 39.94
were obscured by DMSO but were obtained by het-
eronuclear multiple bond correlation spectroscopy
(HMBC) of CDCA-glu-gabapentin and were also ev-
ident in C-13 NMR of compound 1.
Synthesis of Benzyl-Protected Conjugates of Gabapentin
In 20 mL of toluene, 1.71 g (10 mM) of gabapentin
and 1.1 eq (2 g) p-toluenesulfonic acid monohydrate
(PTSA) were magnetically stirred for 6 to 12 h.
Toluene was evaporated under reduced pressure,
which azeotropically removed all water from the re-
action vessel. To the pTsOH salt of gabapentin was
added 20 mL of benzyl alcohol, which was then heated
at 100◦C for 48 h. The solution was allowed to cool.
Fifty milliliters of ether was added, which led to a
white solid precipitate. The white solid was filtered
and washed several time with ether. MS showed
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BILE ACID PRODRUGS OF GABAPENTIN
1187
appropriate peak at [M + 1] 262.5. The protected ben-
zyl ester of gabapentin was coupled to either OBT of
CDCA or its amide with glutamic acid, as presented
in Schemes 2 and 3 which led to neutral conjugates.
These were then purified by flash column chromatog-
raphy as described above using a gradient of ethy-
lacetate and hexane. The monoanionic and dianionic
conjugates were then obtained by catalytic hydro-
genation to remove the "-benzyl ester (10% Pd/
charcoal in EtOH at 40 psi for 1 h). The mixture
was filtered over celite, and ethanol was evaporated
to yield white fluffy solids of conjugates 4 and 5.
Compound 4. MS: [M + 1] 546.3 13C NMR (DMSO-
d6): δ 11.60, 18.30, 20.22, 21.05, 21.05, 22.70, 23.13,
25.54, 27.19, 27.83, 30.54, 31.73, 32.26, 32.33, 32.60,
32.60, 34.72, 34.81, 34.91, 35.29, 36.53, 39.60, 39.78,
39.94, 40.48, 41.40, 41.90, 50.01, 55.63, 66.14, 70.33,
172.93, 173.16. Compound 5. MS: [M − 1] 673.5. 13C
NMR (DMSO-d6): δ 11.64, 18.38, 20.23, 21.03, 21.03,
22.68, 23.14, 25.33, 27.11, 27.79, 30.54, 31.48, 31.80,
32.09, 32.26, 32.56, 32.56, 34.73, 34.81, 34.97, 35.30,
36.2, 39.60, 39.78, 39.94, 40.11, 41.41, 44.27, 41.90,
50.01, 50.93, 55.64, 66.14, 70.31, 168.2, 172.2, 172.9,
173.9.
was added to lyse the cells (2 h), neutralized with
250 :L of 1N HCl, and counted for radioactivity us-
ing an LS6500 liquid scintillation counter (Beckmann
Instruments, Inc., Fullerton, California).
Inhibition data were regressed using a modified
Michaelis–Menten equation to determine the inhibi-
tion constant Ki, as previously described,20 including
parallel studies on each occasion to estimate Km, Vmax
and Pp for taurocholic acid.
,
Prodrug Uptake into hASBT–MDCK Cells
Uptake studies were performed in 12-well cell cul-
ture polystyrene plates (Corning Costar). The 12-well
plates were seeded with hASBT–MDCK at a density
of 1.5 × 106 cells/well and induced on day 4 with
10 mM sodium butyrate for 12 to 15 h prior to up-
take experiments. Uptake studies were performed in
a similar manner to above inhibition studies over
10 min.23 The concentration of conjugates was varied
from 1 to 250 :M for monoanions and 0 to 1000 :M for
the dianion. To terminate prodrug uptake, the cells
were washed three times with chilled sodium-free
buffer. Additional identical studies were performed
without sodium, to measure passive uptake of the
conjugate on each occasion. Also, on each occasion,
taurocholate Vmax and Pp were measured to normal-
ize for hASBT expression. Prodrug Vmax was divided
by taurocholate Vmax to yield normalized Vmax of pro-
drug (normVmax).
Binding Affinity Studies
Stably transfected hASBT–MDCK cells were cul-
tured as previously described.22 Briefly, the cells were
grown at 37◦C, 90% relative humidity, 5% CO2 atmo-
sphere, and fed every 2 days. Culture media consisted
on DMEM supplemented with 10% FBS, 50 units/mL
penicillin, and 50 :g/mL streptomycin. Geneticin was
added at 1 mg/mL to maintain selection pressure. The
cells were passaged after 4 days or after reaching 90%
confluency.
Quantification of Conjugates by LC/MS/MS
Cell lysis procedure and extraction assay have been
previously optimized in our laboratory using tau-
rocholate uptake into hASBT–MDCK monolayers.24
Briefly, the cells were lysed by adding 300 :L of ace-
tonitrile, which was then evaporated (2–3 h). The
conjugate was extracted using 1:1 acetonitrile and
HPLC grade water, spiked with internal standard.
The anilinyl conjugate of glu-CDCA was used as inter-
nal standard, as it bears structural similarity to the
prodrugs. The conjugate extract was collected from
the wells in silanized vials and stored in −80◦C until
analyzed.
The prodrug concentration was determined in the
cell lysate by liquid chromatography tandem MS
(LC/MS/MS). Compound uptake was quantified on a
Finnigan Surveyor HPLC system and a Finnigan TSQ
quantum Discovery Max mass spectrometer (Thermo
Fisher Scientific; Waltham, MA, USA). The column
was a Phenomenex Luna C8, 50 × 2 mm, 5:, heated
to 40◦C. The flow rate was 0.4 mL/min. The gradi-
ent of 5 to 95% acetonitrile was employed over 0.2
to 4 min, and the overall run time for all conjugates
was 8 min. The initial mobile phase concentration was
50:50 acetonitrile:water up to 0.2 min, followed by in-
crease in organic to 95%. The mobile phase included
The binding affinities of gabapentin bile acid con-
3
jugates were evaluated through cis-inhibition of H-
taurocholic acid uptake into hASBT–MDCK mono-
layers in 12-well plates (3.8 cm2, Corning Costar,
Corning, New York), as previously described.21 Be-
ing insoluble in buffer, even with 2.5% DMSO, neu-
tral compounds were not evaluated. Briefly, cells were
seeded at a density of 1.5 million/well. The cells were
induced on day 4 with 10 mM sodium butyrate for
about 12 to 17 h prior to experiment. The cells were
washed three times with Hank’s balanced salt solu-
tion [HBSS; Sigma Aldrich (St. Louis, MO, USA)],
followed by an addition of donor solution contain-
ing taurocholic acid (2.5 :M with 0.5 :Ci/mL 3H-
taurocholate) and conjugate (0–100 :M for monoan-
ions and 0–1000 :M for dianion). Inhibition stud-
ies were conducted at 37◦C for 10 min on an orbital
shaker (50 rpm). At the end of 10 min,23 donor solu-
tion was removed and the cells were washed three
times with chilled sodium-free buffer (where NaCl
was replaced by 137 mM tetraethylammonium chlo-
ride). Two hundred and fifty microliters of 1N NaOH
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RAIS, FLETCHER, AND POLLI
0.1% formic acid as a modifier for all prodrugs. Injec-
tion volumes were 10 :L. Detection was achieved un-
der positive ion electrospray tandem MS using [M +
H]+ peak, as a positive mode provided the greatest
sensitivity for the synthesized prodrugs. The multi-
ple reaction monitoring (MRM) transitions for inter-
nal standard were 597.3 > 83.7, 320.77; for compound
2, MRM transitions were 689.6 > 154.26, 191.64; for
compound 4, MRM transitions were 546.23 > 94.69,
153.78; and for compound 5, MRM transitions were
675.3 > 155.86, 266.31. The method was linear for
prodrugs over the concentration range of 1 to 5000 :M
for conjugate 2 (r2 = 0.9996), of 10 to 2500 :M for con-
jugate 4 (r2 = 0.998), of 25 to 2500 :M for conjugate
5 (r2 = 0.999).
LC/MS/MS Methods for Stability Studies
Amount of prodrug remaining was determined in a
similar manner as used earlier to quantify prodrug in
uptake studies (i.e., under same conditions). However,
the MRM transitions were 546.4 > 154.04, 510.44 for
4; 689.3 > 154.03, 165.21 for 2; and 597.3 > 83.92,
159.02 for internal standard.
To measure drug generation from prodrug,
gabapentin concentration was quantified via LC/MS/
MS. The column was aquasil C8, 5:, heated to 40◦C.
The mobile phase contained a mixture of 80:20 wa-
ter:acetonitrile with 0.1% formic acid, and the flow
rate was 0.4 mL/min. The internal standard was (S)-
(+)-aminocyclohexanepropionic acid at a concentra-
tion of 500 nM. The MRM transitions were 171.94 >
55.12, 126.13 for the internal standard; and 172.01 >
67.06, 154.12 for gabapentin. Total run times were
8 min. The method was linear over the concentration
range of 1 to 2500 :M (r2 of 0.998).
In Vitro Stability
Prodrugs were subjected to chemical stability as-
sessment, as well as metabolic stability assessment.
Chemical stability testing exposed prodrugs to 0.1 N
HCl acid for 1 h and pH 6.8 HBSS buffer for 1 h.
Compounds (5 :M) were incubated in acid or buffers
at 37◦C for 1 h in an orbital shaker. Sample was col-
lected after 1 h, diluted with 50% acetonitrile, and
analyzed by LC/ MS/MS, as described earlier.
RESULTS AND DISCUSSION
Synthesis of Gabapentin Prodrugs
Metabolic stability studies for the two monoan-
ions were performed using Caco-2 homogenate and
rat liver microsomes. Prodrug (1 :M) was incubated
with either Caco-2 homogenate or rat liver micro-
some (2 mg/mL) at 37◦C. Samples were quenched by
adding 200 :L of chilled acetonitrile spiked with in-
ternal standard. The samples were then centrifuged
at 10,000 rpm (i.e. 8944 g) at 4◦C for 5 min and stored
at −80◦C prior to analysis. The samples were ana-
lyzed for disappearance of prodrug and appearance of
drug by LC/MS/MS, as described below.
Regarding preparation of Caco-2 homogenates,
Caco-2 cells were grown in 225 cm2 flasks for 3 weeks
at 37◦C and 5% CO2. The cells were grown in Dul-
becco’s modified Eagle medium (DMEM), 10% FBS,
and 1% nonessential amino acids. The cells were
washed with 10 mL of Dulbecco’s phosphate buffered
saline (DPBS) three times. The cells were scraped off
the flasks on an ice tray and reconstituted in 10 mL of
DPBS. The cell suspension was transferred to a ster-
ile centrifuge tube and stored at −80◦C overnight.
The frozen cells were thawed in an ice bath and then
sonicated by using sonic dismembrator (model F60;
Fisher Scientific, Pittsburgh, Pennsylvania) at 5 to
6 W (RMS). Crude Caco-2 homogenates were cen-
trifuged at 10,000 rpm for 10 min at 4◦C. The 1.5-mL
aliquots of supernatant were collected in centrifuge
tubes and stored at −80◦ C until used. The total pro-
tein was quantified using the Bradford protein assay
(Bio-Rad Laboratories, Hercules, California) and was
about 0.4 mg/mL.
Five bile acid conjugates of gabapentin were syn-
thesized and included two neutral, two monoan-
ionic, and one dianionic compound. Compounds
1 to 5 are denoted CDCA-"-benzyl-glu-gabapentin
methyl ester, CDCA-glu-gabapentin methyl ester,
CDCA-gabapentin-methyl ester, CDCA-gabapentin,
and CDCA-glu-gabapentin, respectively.
Charge has previously been shown to impact con-
jugate inhibition and substrate activity for CDCA
conjugates.17 In Scheme 1, compound 2 was designed
by employing CDCA as the targeting moiety, and glu-
tamic acid as a linker to afford a single negative
charge near the bile acid’s C-24 position. Gabapentin
carboxylate was protected by methyl ester to have
an overall charge of −1 on compound 2. Compound 1
was the intermediate prior to deprotection of the ben-
zyl ester to yield compound 2, such that compound 1
is neutral. Compound 3 differs from compound 2: in
that compound 3 lacks the glutamic acid linker, and
hence is neutral. The two neutral conjugates 1 and 3
were insufficiently water soluble to allow for hASBT
testing.
Various attempts were made to obtain compounds
4 and 5 from compounds 2 and 3, respectively. How-
ever, selective hydrolysis of the methyl ester was not
achieved due to simultaneous cleavage of the amide
bond. Schemes 2 and 3 illustrate the alternative, suc-
cessful approach that yielded compounds 4 and 5.
Compounds 4 and 5 are monoanionic and dianionic,
respectively, and differ in the absence or presence of
a glutamic acid linker.
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BILE ACID PRODRUGS OF GABAPENTIN
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Table 1. Structures and Kinetic Parameters for Gabapentin Prodrugs
Compound
Number
Vmax
Normalized
PP × 106
R1
Charge
Ki (:M)
Km (:M)
(pmol/cm2/s)
Vmax
(cm/s)
4
−1
2.85 0.44
16.3 4.7
0.842 0.083
2.15 0.23
11.5 0.3
2
5
−1
6.46 0.92
5.99 1.45
0.656 0.030
1.68 0.09
1.11 0.01
−2
34.8 11.9
NS
NS
NS
0.109 0.008
Compounds are arranged in order of inhibition potency.
NS denotes compound was not a substrate.
monoanionic compounds are consistent with prior ob-
servations of monoanions to inhibit hASBT.17 The di-
anionic gabapentin conjugate inhibited hASBT with
moderate potency.
Inhibition of Taurocholate Uptake into
hASBT–MDCK Cells
The three negatively charged analogues, CDCA-
glu-gabapentin methyl ester (compound 2), CDCA-
gabapentin (compound 4), and CDCA-glu-gabapentin
(compound 5) were evaluated for the inhibition of
hASBT using taurocholate as a substrate. Each inhib-
ited taurocholate uptake into hASBT–MDCK mono-
layers in a concentration-dependent manner. Their
kinetic parameters are listed in Table 1 in order of
inhibitory potency. The two monoanions exhibited in-
hibitory potency similar to native bile acids (about
5 :M). The dianion showed moderate inhibitory po-
tency, with Ki = 34.8 :M. Figure 1 plots the inhi-
bition profiles. In panel A, neither gabapentin nor
gabapentin methyl ester inhibited taurocholate, while
CDCA-glu-gabapentin methyl ester decreased tau-
rocholate uptake. CDCA-gabapentin (panel B) and
CDCA-glu-gabapentin (panel C) each also inhibited
taurocholate uptake. Inhibition results of the two
Conjugate Uptake into hASBT–MDCK Cells
The gabapentin conjugates were further evaluated for
uptake into hASBT–MDCK cells. Figure 2 represents
the concentration-dependent uptake of potential pro-
drugs into hASBT–MDCK monolayers. In panels A
and B, CDCA-glu-gabapentin methyl ester uptake
and CDCA-gabapentin uptake were greater in the
presence of sodium than in the absence of sodium,
indicating active uptake by the transporter. In panel
C, the dianion CDCA-glu-gabapentin was equally low
in the presence and absence of sodium and was not a
substrate.
Kinetic parameters using the modified Michaelis—
Menten equation are listed in Table 1. For CDCA-
glu-gabapentin methyl ester, Km = 5.99 :M and
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1190
RAIS, FLETCHER, AND POLLI
0.12
0.1
a
CDCA-glu-gabapentin methyl
ester observed
0.08
0.06
0.04
0.02
0
CDCA-glu-gabapentin methyl
ester fitted
gabapentin
gabapentin methyl ester
0
20
40
60
80
100
Inhibitor concentration (µM)
b
0.10
0.09
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0.00
CDCA-gabapentin observed
CDCA-gabapentin fitted
0
20
40
60
80
100
Inhibitor concentration (µM)
0.12
0.10
0.08
0.06
0.04
0.02
0.00
c
CDCA-glu-gabapentin
observed
CDCA-glu-gabapentin
fitted
0
200
400
600
800
1000
Inhibitor concentration (µM)
Figure 1. Inhibition profiles of three potential gabapentin prodrugs. (a) CDCA-glu-gabapentin
methyl ester, (b) CDCA-gabapentin, and (c) CDCA-glu-gabapentin. Each inhibited taurocholate
uptake into hASBT-MDCK monolayers. In panel A, neither gabapentin nor gabapentin methyl
ester inhibited taurocholate.
Vmax = 0.656 pmol/cm2/s indicate high affinity and
capacity of hASBT for this substrate, suggesting the
compound to be a potential hASBT prodrug. In Table
1, the normalized Vmax value is Vmax for the conju-
gate normalized for functional ASBT expression, us-
ing taurocholate as the reference; conjugate Vmax is
divided by taurocholate Vmax from the same study
occasion to give conjugate normalized Vmax. The nor-
malized Vmax of CDCA-glu-gabapentin methyl ester
and CDCA-gabapentin were each about 2 (i.e., twofold
higher than taurocholate Vmax). As a reference, tau-
rocholate’s Km is about 5 :M, such that the Km of
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BILE ACID PRODRUGS OF GABAPENTIN
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1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
a
CDCA-glu-gabapentin methyl
ester total observed
CDCA-glu-gabapentin methyl
ester passive observed
0
50
100
150
200
250
Concentration (µM)
1.8
b
1.6
1.4
1.2
1
CDCA-gabapentin total
observed
CDCA-gabapentin passive
observed
0.8
0.6
0.4
0.2
0
0
20
40
60
80
100
Concentration (µM)
0.020
c
0.015
0.010
0.005
0.000
CDCA-glu-gabapentin total
observed
CDCA-glu-gabapentin passive
observed
0
20
40
60
80
100
Concentration (µM)
Figure 2. Concentration-dependent uptake of potential prodrugs into hASBT–MDCK mono-
layers. (a) CDCA-glu-gabapentin methyl ester uptake and (b) CDCA-gabapentin uptake were
greater in the presence of sodium (•) than in the absence of sodium (◦), indicating each to be
hASBT substrates. (c) CDCA-glu-gabapentin was not a substrate, as uptake was equally low in
the presence and absence of sodium.
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CDCA-glu-gabapentin methyl ester was the same as
taurocholate.
the apparent permeabilities of CDCA-gabapentin at
1, 2.5, 5, 10, 25, 50, and 100 :M were 188 × 10−6
48.2 × 10−6, 38.4 × 10−6, 31.6 × 10−6, 29.5 × 10−6
18.2 × 10−6, and 17.3 × 10−6 cm/s, respectively.
,
,
Like CDCA-glu-gabapentin methyl ester, CDCA-
gabapentin was a potent substrate, with Km
=
16.3 :M and Vmax = 0.842 pmol/cm2/s. The most no-
table difference between these two monoanions was
passive permeability, which was low for CDCA-glu-
gabapentin methyl ester (Pp = 1.11 × 10−6 cm/s) and
moderate for CDCA-gabapentin (Pp = 11.5×10−6 cm/
s). CDCA itself has a moderate-to-high passive per-
Overall, a single negative charge was preferred
for substrate translocation, and promoted aqueous
solubility. Although the dianionic compound bound
to hASBT with moderate potency, it was not a sub-
strate. These observations agree with previous find-
ings where other dianions were found not to be hASBT
substrates.17
meability of 20.7 × 10−6 20
,
but exhibits lower passive
permeability when conjugated to glutamic acid.17 The
lower passive permeability for CDCA-glu-gabapentin
methyl ester versus CDCA-gabapentin is not surpris-
ing due to conjugation in the former, while the lowest
passive permeability of the dianion (Pp = 0.109 ×
10−6 cm/s) was expected, given it has a negative
charge of two.
In Vitro Stability
For the two substrates, stability was conducted in
0.1 N HCl, HBSS buffer with pH = 6.8, Caco-2 ho-
mogenate, and rat liver microsomes. Each CDCA-glu-
gabapentin methyl ester and CDCA-gabapentin were
stable for more than 1 h in 0.1 N HCl and HBSS buffer
with pH = 6.8. Essentially no loss of either prodrug
was observed. For CDCA-glu-gabapentin methyl es-
In Table 1, the Ki and Km values for CDCA-
glu-gabapentin methyl ester were essentially iden-
tical, suggesting that substrate binding was the rate-
limiting step in its overall translocation by hASBT.
For CDCA-gabapentin, Ki was about sixfold more
potent than Km, implicating postbinding events as
the rate-limiting step(s) in CDCA-gabapentin up-
take. Postbinding events include steps that are re-
quired for substrate translocation but occur after
substrate binds to the transporter at the extracel-
lular surface; postbinding events include orienta-
tion of the transporter-substrate complex from ex-
tracellular orientation to intracellular orientation,
release of substrate from the transporter-substrate
complex into the cell, and re-orientation of the trans-
porter. With a viewpoint that Ki and Km reflect ki-
netics of substrate binding and overall translocation
(i.e., binding and postbinding events), respectively,
where smaller valued parameters denote faster ki-
netics, smaller Ki than Km, implicate that binding is
faster than postbinding events for CDCA-gabapentin
uptake.
The uptake results indicate high apparent perme-
abilities for the two-monoanionic compounds, includ-
ing higher prodrug permeability than gabapentin per-
meability. The apparent permeability of gabapentin
across MDCK monolayers was reported to be 0.73 ×
10−6 cm/s and 0.25 × 10−6 cm/s in apical-to-
basolateral and basolateral-to-apical directions, re-
spectively, which was described to be low and con-
sistent with poor passive absorption.6 The apparent
permeabilities of CDCA-glu-gabapentin methyl es-
ter and CDCA-gabapentin were several fold higher
than these gabapentin values. From uptake studies
(Fig. 2), the apparent permeabilities of CDCA-glu-
gabapentin methyl ester at 1, 2.5, 5, 10, 25, 50, 100,
and 250 :M were 87.5 × 10−6, 63.8 × 10−6, 43.3 ×
10−6, 33.9 × 10−6, 16.1 × 10−6, 12.7 × 10−6, 6.95 ×
10−6, and 3.62 × 10−6 cm/s, respectively. Similarly,
ter, about 104.7
9.1% and 114.6
3.92% was re-
maining in 0.1 N HCl and HBSS, respectively. Simi-
larly, for CDCA-gabapentin 102.9 8.9% and 89.3
4.9% was remaining in 0.1 N HCl and HBSS, respec-
tively.
In Figure 3, the stability profiles of CDCA-glu-
gabapentin methyl ester and CDCA-gabapentin in
Caco-2 homogenate are plotted. In panel A, only
68.2% of prodrug remained after 15 min. An equiva-
lent amount of gabapentin (32.6%) was produced after
15 min. Studies were also performed in rat liver mi-
crosomes (data not shown), where the loss of prodrug
was measured. After 2 h, 56.5% of prodrug remained.
The release of the drug in rat liver microsomes was
not feasible due to matrix effects (i.e., interference
of microsomal matrix with gabapentin quantification
by LC/MS). CDCA-glu-gabapentin methyl ester ex-
hibited slightly greater stability in rat liver micro-
somes. Compared to the observed chemical stability
in acid and buffer, prodrug was unstable in Caco-
2 homogenate and microsomes. Results from Caco-
2 homogenate and rat liver microsomes show that
CDCA-glu-gabapentin methyl ester was catalytically
degraded, yielding the parent drug.
The corresponding stability profiles of CDCA-
gabapentin are plotted in Figure 3 panel B. There
was general agreement between prodrug loss and
drug generation. After 15 min, 31.9% of prodrug de-
graded with a slightly less than equivalent amount
of gabapentin (17.1%) produced. CDCA-gabapentin
was more stable in rat liver microsomes compared
to Caco-2 homogenate. In rat liver microsomes, 56.9%
of prodrug remained after 2 h. Like for CDCA-glu-
gabapentin methyl ester, CDCA-gabapentin was cat-
alytically degraded and yielded drug from prodrug.
Hydrolysis rates were much higher in Caco-2 cell
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BILE ACID PRODRUGS OF GABAPENTIN
1193
120
100
80
60
40
20
0
a
CDCA-glu-gabapentin
methyl ester remaining (%)
gabapentin released (%)
0
5
10
Time (min)
15
120
100
80
60
40
20
0
b
CDCA-gabapentin
remaining (%)
gabapentin released (%)
0
5
10
Time (min)
15
Figure 3. Catalytic activation of prodrugs in Caco-2 homogenate. (a) CDCA-glu-gabapentin
methyl ester and (b) CDCA-gabapentin were evaluated. For each prodrug, prodrug concentration
declined (•), while gabapentin was generated (◦).
homogenates and liver microsomes as compared to
chemical stability in buffers, indicating enzymatic
bioconversion of the prodrugs.
Results showed that gabapentin was not a substrate
for these transporters, although it was found to be a
substrate for OCTN2. It has also been shown that the
amino acid transporter LAT1 is not significantly ex-
pressed in human intestine. Thus, the overall mecha-
nism for gabapentin’s highly variable and incomplete
absorption is still not clear.
A prodrug of gabapentin that increases gabapentin
absorption is in development.5,8 This prodrug was de-
signed to be recognized by a low-affinity high-capacity
nutrient transporter, such as monocarboxylate trans-
porter type I (MCT-1) expressed along the intes-
tine, and a high-affinity low-capacity, the sodium-
dependent multivitamin transporter (SMVT), which
is expressed largely in intestine and responsible
for the absorption of essential cofactors. Analysis of
the prodrug in MCT-1-specific component revealed a
Prodrug Design
Gabapentin is a model low-permeability drug because
of its zwitterionic nature. Gabapentin is structurally
similar to amino acids, which generally rely on amino
acid transporters for absorption. In vivo, gabapentin
is a substrate for saturable solute transporters in
the intestine and brain. Its dose-dependent pharma-
cokinetics limits its oral bioavailability, especially at
doses required to treat the neuropathic pain. In stud-
ies performed by Cundy et al.,6 gabapentin transport
was evaluated for various amino acid transporters,
such as B0,+, ATB0,+, and LAT2 in transfected oocytes.
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RAIS, FLETCHER, AND POLLI
saturable uptake with a Km of approximately 220 :M
and a Vmax of 6 pmol per oocyte. The prodrug was also
shown to be a substrate of SMVT with Km of 3 :M
and Vmax equal to only 40% of natural substrate bi-
otin. The prodrug showed low affinity for MCT-1 and
low capacity for SMVT.6
acid taurocholate. Additionally, each was chemically
stable but catalytically degraded, and yielded drug
from prodrug. Overall, these two conjugates are novel
prodrugs of gabapentin and illustrate that prodrugs
can be designed to target hASBT.
An alternative strategy is employed here where the
drug is coupled to a natural substrate for a trans-
porter, such that the drug-substrate conjugate mim-
ics the native substrate. Using CDCA as a targeting
moiety, the prodrugs were strategically designed to
target hASBT with the same high affinity and high
capacity as native bile acids. The following features
have previously been shown to be preferable for sub-
strate uptake by ASBT: (1) the presence of nega-
tive charge proximal to C-24, (2) the presence of
single negative charge, where two negative charges
hampered activity, and (3) hydrophobic moieties that
enhance interaction with ASBT. To achieve optimal
recognition by hASBT, we employed a “bile acid-linker
drug” strategy to design gabapentin prodrugs 2 and
5. A negative charge near C-24 was afforded by the
linker glutamic acid. The negative charge on the drug
was concealed by methyl ester protection on 2. Al-
ternatively, for 4, bile acid was directly coupled to
gabapentin and a negative charge was afforded by
drug.
Among five analogues, two were found to be po-
tential prodrugs that may increase gabapentin ab-
sorption via hASBT uptake: CDCA-glu-gabapentin
methyl ester and CDCA-gabapentin. hASBT translo-
cates several grams of bile acids daily with little
loss to feces. CDCA-glu-gabapentin methyl ester and
CDCA-gabapentin was each designed to be a monoan-
ion, with the negative charge within the vicinity of
C-24 of the bile acid. The native bile acid CDCA pos-
sesses a negative charge at C-24, while its glycine con-
jugated form, glycochenodeoxycolate possess a nega-
tive charge at C-25. For CDCA-glu-gabapentin methyl
ester, a negative charge was maintained at a position
favorable for uptake by hASBT (i.e., C-25). For CDCA-
gabapentin, gabapentin is a small molecule possess-
ing a carboxylate, such that directly coupling it to
CDCA maintained a negative charge, but at a slightly
distal location to C-24 (i.e., C-28).
ACKNOWLEDGMENTS
This work was support in part by National Institutes
of Health grants DK67530.
REFERENCES
1. Laird MA, Gidal BE. 2000. Use of gabapentin in the treat-
ment of neuropathic pain. Ann Pharmacother 34(6):802–
807.
2. Stewart BH, Kugler AR, Thompson PR, Bockbrader HN. 1993.
A saturable transport mechanism in the intestinal absorption
of gabapentin is the underlying cause of the lack of propor-
tionality between increasing dose and drug levels in plasma.
Pharm Res 10(2):276–281.
3. Uchino H, Kanai Y, Kim DK, Wempe MF, Chairoungdua A,
Morimoto E, Anders MW, Endou H. 2002. Transport of amino
acid-related compounds mediated by L-type amino acid trans-
porter 1 (LAT1): Insights into the mechanisms of substrate
recognition. Mol Pharmacol 61(4):729–737.
4. Gidal BE, Radulovic LL, Kruger S, Rutecki P, Pitterle M,
Bockbrader HN. 2000. Inter- and intra-subject variability in
gabapentin absorption and absolute bioavailability. Epilepsy
Res 40(2–3):123–127.
5. Cundy KC, Annamalai T, Bu L, De Vera J, Estrela J,
Luo W, Shirsat P, Torneros A, Yao F, Zou J, Barrett
RW, Gallop MA.
2004. XP13512 [(+/−)-1-([(alpha-
isobutanoyloxyethoxy)carbonyl] aminomethyl)-1-cyclohexane
acetic acid], a novel gabapentin prodrug: II. Improved oral
bioavailability, dose proportionality, and colonic absorption
compared with gabapentin in rats and monkeys. J Pharma-
col Exp Ther 311(1):324–333.
6. Cundy KC, Branch R, Chernov-Rogan T, Dias T, Estrada T,
Hold K, Koller K, Liu X, Mann A, Panuwat M, Raillard SP,
Upadhyay S, Wu QQ, Xiang JN, Yan H, Zerangue N, Zhou CX,
Barrett RW, Gallop MA. 2004. XP13512 [(+/−)-1-([(alpha-
isobutanoyloxyethoxy)carbonyl] aminomethyl)-1-cyclohexane
acetic acid], a novel gabapentin prodrug: I. Design, syn-
thesis, enzymatic conversion to gabapentin, and transport
by intestinal solute transporters. J Pharmacol Exp Ther
311(1):315–323.
7. Richter A, Anton SE, Koch P, Dennett SL. 2003. The im-
pact of reducing dose frequency on health outcomes. Clin Ther
25(8):2307–2335; discussion 2306.
8. Cundy KC, Sastry S, Luo W, Zou J, Moors TL, Canafax
DM.
2008. Clinical pharmacokinetics of XP13512, a
novel transported prodrug of gabapentin. J Clin Pharmacol
48(12):1378–1388.
Results from inhibition studies with taurocholate
demonstrated that the gabapentin conjugates are po-
tent inhibitors, with strong interaction with the trans-
porter. For definitive evidence of transport, uptake
studies were performed in a stably transfected cell
line and quantified using LC/MS/MS. The Km and
Vmax values for 2 and 4 indicate high transporter
affinity and capacity. The normalized Vmax showed
hASBT to possess higher transporter capacity for
these two conjugates than even for the native bile
9. Lal R, Sukbuntherng J, Luo W, Chen D, Vu A, Tovera J,
Cundy KC. 2009. Pharmacokinetics and tolerability of sin-
gle escalating doses of gabapentin enacarbil: A randomized-
sequence, double-blind, placebo-controlled crossover study in
healthy volunteers. Clin Ther 31(8):1776–1786.
10. Walters AS, Ondo WG, Kushida CA, Becker PM, Ellenbogen
AL, Canafax DM, Barrett RW. 2009. Gabapentin enacarbil
in restless legs syndrome: A phase 2b, 2-week, randomized,
double-blind, placebo-controlled trial. Clin Neuropharmacol
32(6):311–320.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011
DOI 10.1002/jps

BILE ACID PRODRUGS OF GABAPENTIN
1195
11. Madan J, Chawla G, Arora V, Malik R, Bansal AK. 2005.
Unbiased membrane permeability parameters for gabapentin
19. Tolle-Sander S, Lentz KA, Maeda DY, Coop A, Polli JE. 2004.
Increased acyclovir oral bioavailability via a bile acid conju-
gate. Mol Pharm 1(1):40–48.
20. Balakrishnan A, Wring SA, Polli JE. 2006. Interaction of
native bile acids with human apical sodium-dependent bile
acid transporter (hASBT): Influence of steroidal hydroxyla-
tion pattern and C-24 conjugation. Pharm Res 23(7):1451–
1459.
21. Gonzalez PM, Acharya C, Mackerell AD, Jr., Polli JE. 2009. In-
hibition requirements of the human apical sodium-dependent
bile acid transporter (hASBT) using aminopiperidine con-
jugates of glutamyl-bile acids. Pharm Res 26(7):1665–
1678.
22. Balakrishnan A, Sussman DJ, Polli JE. 2005. Development of
stably transfected monolayer overexpressing the human apical
sodium-dependent bile acid transporter (hASBT). Pharm Res
22(8):1269–U1213.
using boundary layer approach. AAPS
230.
J
7(1):E224–
12. Hagenbuch B, Dawson P. 2004. The sodium bile salt cotrans-
port family SLC10. Pflugers Arch 447(5):566–570.
13. St-Pierre MV, Kullak-Ublick GA, Hagenbuch B, Meier
PJ. 2001. Transport of bile acids in hepatic and non-hepatic
tissues. J Exp Biol 204(Pt 10):1673–1686.
14. Alrefai WA, Gill RK. 2007. Bile acid transporters: Struc-
ture, function, regulation and pathophysiological implications.
Pharm Res 24(10):1803–1823.
15. Balakrishnan A, Polli JE. 2006. Apical sodium dependent bile
acid transporter (ASBT, SLC10A2): A potential prodrug target.
Mol Pharm 3(3):223–230.
16. Sievanen E. 2007. Exploitation of bile acid transport systems
in prodrug design. Molecules 12(8):1859–1889.
17. Balakrishnan A, Wring SA, Coop A, Polli JE. 2006. Influence
of charge and steric bulk in the C-24 region on the interaction
of bile acids with human apical sodium-dependent bile acid
transporter. Mol Pharm 3(3):282–292.
23. Balakrishnan A, Sussman DJ, Polli JE. 2005. Development of
stably transfected monolayer overexpressing the human apical
sodium-dependent bile acid transporter (hASBT). Pharm Res
22(8):1269–1280.
18. Zhang EY, Phelps MA, Cheng C, Ekins S, Swaan PW. 2002.
Modeling of active transport systems. Adv Drug Deliv Rev
54(3):329–354.
24. Rais R, Gonzalez PM, Zheng X, Wring SA, Polli JE. 2008.
Method to screen substrates of apical sodium-dependent bile
acid transporter. AAPS J 10(4):596–605.
DOI 10.1002/jps
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