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
DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 3, MARCH 2011