Synthesis and Evaluation of Bile Acid-Ribavirin Conjugates as Prodrugs to Target the Liver

Article abstract of DOI:10.1002/jps.24375

Ribavirin is used to treat hepatitis C but causes serious hemolytic anemia. The objective of the study was to develop a ribavirin prodrug to achieve liver-specific drug delivery and to reduce its off-target effect in red blood cells (RBCs). The approach aimed to target the human sodium taurocholate cotransporting polypeptide (NTCP), which is a bile acid transporter predominately expressed in the liver. Six prodrugs with ribavirin conjugation at C-3 or C-24 of the bile acids were synthesized. In vitro uptake studies indicated that all six prodrugs were NTCP substrates. Metabolic studies in vitro indicated that ribavirin-l-Val-glycochenodeoxycholic acid (GCDCA) was able to release ribavirin in the mouse liver S9 fraction. Additionally, in vitro studies showed that ribavirin in RBC was reduced by 16.7-fold from prodrug compared with parent drug incubation. Moreover, almost no prodrug was present in RBC. In vivo study in mice also showed that ribavirin-l-Val-GCDCA could provide almost the same ribavirin exposure in the liver as ribavirin administration, but with about 1.8-fold less exposure of ribavirin in RBC, plasma, and kidney. Overall, the study suggested that ribavirin-l-Val-GCDCA has the potential to achieve ribavirin-specific liver delivery.

Full text of DOI:10.1002/jps.24375

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Synthesis and Evaluation of Bile Acid–Ribavirin Conjugates as
Prodrugs to Target the Liver
ZHONGQI DONG, QING LI, DONG GUO, YAN SHU, JAMES E. POLLI
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland 21201
Received 20 August 2014; revised 7 December 2014; accepted 8 January 2015
Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24375
ABSTRACT: Ribavirin is used to treat hepatitis C but causes serious hemolytic anemia. The objective of the study was to develop a ribavirin
prodrug to achieve liver-specific drug delivery and to reduce its off-target effect in red blood cells (RBCs). The approach aimed to target
the human sodium taurocholate cotransporting polypeptide (NTCP), which is a bile acid transporter predominately expressed in the liver.
Six prodrugs with ribavirin conjugation at C-3 or C-24 of the bile acids were synthesized. In vitro uptake studies indicated that all six
prodrugs were NTCP substrates. Metabolic studies in vitro indicated that ribavirin–L-Val–glycochenodeoxycholic acid (GCDCA) was able
to release ribavirin in the mouse liver S9 fraction. Additionally, in vitro studies showed that ribavirin in RBC was reduced by 16.7-fold from
prodrug compared with parent drug incubation. Moreover, almost no prodrug was present in RBC. In vivo study in mice also showed that
ribavirin–L-Val–GCDCA could provide almost the same ribavirin exposure in the liver as ribavirin administration, but with about 1.8-fold
less exposure of ribavirin in RBC, plasma, and kidney. Overall, the study suggested that ribavirin–L-Val–GCDCA has the potential to achieve
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ribavirin-specific liver delivery.
2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci
Keywords: bile acid transporters; hepatocytes; metabolism; NTCP; prodrugs; ribavirin; site-specific delivery
INTRODUCTION
involved in the enterohepatic circulation of bile acids. NTCP is
predominantly expressed at the basolateral membrane of hep-
atocytes and probably responsible for transporting more than
80% of conjugated bile acids from portal blood into the liver.8
Because of its liver specificity and high capacity for transport-
ing conjugated bile acids, bile acid conjugates have potential to
serve as prodrugs to achieve liver-specific drug delivery.9 Pre-
viously, bile acid conjugates were shown to deliver the drug of
interest to the liver.10,11 In the current study, we aim to develop
novel bile acid ribavirin prodrugs in order to reduce ribavirin
accumulation in RBCs and hence alleviate ribavirin’s most con-
cerned side effect of hemolytic anemia.
Briefly, six bile acid–ribavirin prodrugs were synthesized by
conjugating ribavirin to bile acids via an amino acid linker.
These prodrugs were then evaluated for their in vitro NTCP
uptake, metabolic stability, and potential of ribavirin accumu-
lation in RBCs. After in vitro evaluation, a prodrug of rib-
avirin conjugated to glycochenodeoxycholic acid (GCDCA) via
an L-valine linker was selected for in vivo assessment. The in
vivo distribution of ribavirin–L-Val–GCDCA was examined in
mice and compared with that of ribavirin itself. Results indi-
cated that this bile acid–ribavirin conjugate has the potential
to achieve liver-specific delivery of ribavirin and reduce its ac-
cumulation in nontarget organs.
Ribavirin is a guanosine analog for treating hepatitis C, which
is the disease that primarily affects the liver and may even-
tually cause liver cirrhosis or liver cancer.1,2 It is usually ad-
ministered along with interferon alpha or a protease inhibitor
(e.g., boceprevir) to enhance the sustained virological response,
where a higher dose of ribavirin is more effective.3 Recent stud-
ies suggest that ribavirin may also be used to treat rhinovirus
and acute myeloid leukemia.4,5 However, its usefulness is lim-
ited by the dose-dependent hemolytic anemia, which results in
dose reduction or treatment termination in approximate 20%
and 5% treated patients, respectively.6 This serious side effect
is caused by ribavirin accumulation in the anucleate red blood
cells (RBCs), where ribavirin undergoes irreversible phospho-
rylation. Ribavirin phosphorylation in RBCs causes depletion
of ATP in a competitive manner and exerts oxidative damage
on the cell membrane.7 Targeted delivery of ribavirin to the
liver may lower its accumulation in the RBCs and reduce its
dose-limiting side effects.
Human sodium taurocholate cotransporting polypeptide
(NTCP) is a sodium-dependent bile acid transporter that is
Abbreviations used: ASBT, sodium-dependent bile acid transporter; CA,
cholic acid; CDCA, chenodeoxycholic acid; DCM, dichloromethane; DEPC, di-
ethyl cyanophosphonate; DIPEA, N,N-diisopropylethylamine; DMF, dimethyl
formamide; DMP, 2,2-dimethoxypropane; EtOAc, ethyl acetate; GCDCA, gly-
cochenodeoxycholic acid; HBSS, Hank’s balanced salts solution; LTI, liver tar-
geting index; NTCP, sodium taurocholate cotransporting polypeptide; Pd, pal-
ladium; PdOH, palladium hydroxide; p-TsOH, p-toluenesulfonic acid; Pybop,
benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate; RBC, red
blood cell; SFB, sodium-free buffer; TEA, triethylamine; TFA, trifluoroacetic
acid; UDCA, ursodeoxycholic acid.
EXPERIMENTAL
Figure 1 illustrates the overall approach and scope of com-
pounds tested. Six bile acid–ribavirin prodrugs were collec-
tively evaluated for liver-specific delivery. It should be noted
that ribavirin itself is a prodrug, with the phosphorylated
form being the active moiety. Nevertheless, for simplicity, rib-
avirin is referred to as a drug and ribavirin conjugates as
prodrugs.
Correspondence to: James E. Polli (Telephone: +410-706-8292; Fax: +410-706-
5017; E-mail: jpolli@rx.umaryland.edu)
This article contains supplementary material available from the authors upon
request or via the Internet at http://onlinelibrary.wiley.com/.
Initially, five ribavirin conjugates were synthesized by con-
jugating the drug to CDCA, ursodeoxycholic acid (UDCA),
Journal of Pharmaceutical Sciences
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2015 Wiley Periodicals, Inc. and the American Pharmacists Association
Dong et al., JOURNAL OF PHARMACEUTICAL SCIENCES
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
was purchased from EMD Millipore (Billerica, Massachusetts).
All other chemicals were purchased from Sigma–Aldrich (St.
Louis, Missouri). Geneticin, fetal bovine serum (FBS), trypsin,
Dulbecco’s modified Eagle medium (DMEM), and pooled Balb-
c mice S9 fraction were obtained from Invitrogen (Rockville,
Maryland). Human whole blood was obtained from Innovative
Research (Novi, Michigan).
Synthetic Method Overview
Schemes 1 and 2 show the synthesis of ribavirin prodrug con-
jugated to the C-24 position of bile acids. Briefly, the hydroxyl
groups of ribavirin were initially protected with an acetonide
group. Following protection, the intermediate was conjugated
to an N-protected amino acid via an ester bond. After removal of
the acetonide group and the protecting group at the N-terminal,
the compound was conjugated to the amino acid’s carboxylic
acid at the C-24 position of CDCA, UDCA, or CA via amide
bond. Hydrogenation was carried out when necessary to yield
final prodrugs. Ribavirin was conjugated to the bile acids us-
ing either L-valine or L-glutamic acid, as shown in Schemes 1
and 2.
Scheme 3 shows the synthesis of ribavirin prodrugs conju-
gated at the C-3 position of bile acids. Briefly, the hydroxyl
groups of ribavirin were initially protected by benzylidene ac-
etals. The resulting intermediate was then conjugated to L-
valine via an ester bond. The compound was then coupled to
the hydroxyl group at the C-3 position of GCDCA benzyl ester
via a carbamate bond. Hydrogenation was carried out to re-
move the benzylidene acetals and the benzyl ester protection
group, yielding the final prodrug.
The identity and purity of all prodrugs were confirmed by
thin layer chromatography, mass spectrometry (MS), and nu-
clear magnetic resonance (Table S1).
Figure 1. Flow diagram of approach to synthesize and characterize
six bile acid–ribavirin prodrugs for liver-specific delivery.
Synthesis of Ribavirin Acetonide (Compound II)
or cholic acid (CA) at the C-24 position, via a linker. Pro-
drug was then screened in vitro for human NTCP-mediated
uptake across the cell membrane. All five were NTCP
substrates. CDCA–L-Glu-ribavirin and CDCA–L-Val–ribavirin
showed the higher normalized Jmax and were thus subjected
to ribavirin release assays in mouse S9 fraction. CDCA–L-
Val–ribavirin released more than 60% of ribavirin in mouse
S9 fraction, and was assessed for accumulation into human
RBCs as compared with ribavirin itself. Because of the unsatis-
fying CDCA–L-Val–ribavirin accumulation into human RBCs,
ribavirin–L-Val–GCDCA was synthesized to reduce passive pro-
drug permeability. This sixth prodrug was also assessed for
NTCP-mediated uptake, ribavirin release, and human RBCs
accumulation. The prodrug had the lowest RBC accumula-
tion and highest stability in whole blood, and was thus in-
travenously administrated to mice to evaluate the distribution
of both released ribavirin and intact prodrug. Its pharmacoki-
netics was compared with that of intravenously administrated
ribavirin.
To the suspension of ribavirin (compound I in Scheme 1; 2.0 g,
8.2 mmol) in acetone (40 mL), p-toluenesulfonic acid monohy-
drate (p-TsOH; 0.30 g, 1.58 mmol), and 2, 2-dimethoxypropane
(DMP; 2 mL, 16 mmol) were added. The reaction was heated
to 60°C and stirred for 6 h, then cooled to room tempera-
ture. The acetone was evaporated under vacuum and purified
by silica gel column chromatography using a mobile phase of
dichloromethane (DCM) and methanol (v/v, 14:1) to afford the
intermediate II as a white solid with 73% yield. MS showed an
appropriate peak of [M+Na] 307.2.
Synthesis of L-Valine–Ribavirin Conjugates of Bile Acids
(Compounds 1 and 2)
Intermediate II (1.2 g, 4.1 mmol) was coupled to N-Cbz–L-valine
(1.0 g, 4.1 mmol) using Pybop (2.1 g, 4.1 mmol) as the coupling
reagent in 10 mL dimethyl formamide (DMF) along with N,N-
diisopropylethylamine (DIPEA; 1.4 mL, 8.1 mmol). The reac-
tion was stirred at room temperature overnight. The reaction
was then quenched by adding 50 mL water followed by extrac-
tion with ethyl acetate (EtOAc) (3×). The combined organic
Materials
Ribavirin was purchased from Carbosynth (Berkshire, Eng- layer was washed with saturated sodium bicarbonate solution
land). CDCA was purchased from AK Scientific, Inc. (Union (1×), water (3×), brine (1×), and dried over anhydrous sodium
City, California). UDCA was purchased from Spectrum sulfate. The solvent was evaporated to yield a white solid inter-
Chemical (New Brunswick, New Jersey). Benzotriazol-1-yl- mediate (compound III; Scheme 1) with 99% yield. MS showed
oxytripyrrolidinophosphonium hexafluorophosphate (Pybop) an appropriate peak of [M+H] 518.9.
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
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Scheme 1. Synthesis of CDCA–L-Val–ribavirin (compound 1). (a) p-TsOH, DMP, acetone, 60°C, 6 h. (b) N-Cbz–L-valine, Pybop, DIPEA, DMF,
room temperature, overnight. (c) DCM–TEA (v/v, 1:9), room temperature, 48 h. (d) H2, Pd/C, room temperature, 4 h. (e) CDCA, TEA, DEPC,
DMF, room temperature, overnight.
Scheme 2. Synthesis of CDCA–L-Glu–ribavirin (compound 3). (f) Boc–Glu (OBzl)–OH, Pybop, DIPEA, DMF, room temperature, overnight. (g)
DCM–TFA (v/v, 1:9), room temperature, 48 h. (h) CDCA, TEA, DEPC, DMF, room temperature, overnight. (i) H2, Pd/C, room temperature, 4 h.
The intermediate III was dissolved in 5 mL DCM and trifluo- with silica gel column chromatography using a mobile phase of
roacetic acid (TFA; v/v, 1:9) and stirred at room temperature for DCM and methanol (v/v, 14:1) to yield a white solid intermedi-
48 h. After excess solvent evaporation under vacuum, saturated ate (compound IV; Scheme 1) with 20% yield.
sodium bicarbonate solution was added, followed by extraction
The intermediate IV was subjected to catalytic hydrogena-
with EtOAc (3×). The combined organic layer was washed with tion to remove the Cbz-protecting group using 10% palladium
water (3×), brine (1×), and dried over anhydrous sodium sul- (Pd)/charcoal in methanol for 4 h. The solution was filtered
fate. The solvent was evaporated and the residue was purified through Celite and the solvent was evaporated yielding a white
DOI 10.1002/jps.24375
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Scheme 3. Synthesis of ribavirin–L-Val–GCDCA (compound 6). (j) Benzaldehyde, ZnCl2, room temperature, 6 h. (k) Fmoc–L-valine, Pybop,
DIPEA, DMF, room temperature, overnight. (l) 5% piperidine, DMF, room temperature, 2 h. (m) CDCA–Gly–benzyl ester, triphosgene, pyridine,
DCM, room temperature, overnight. (n) H2, PdOH/C, room temperature, overnight.
solid intermediate (compound V; Scheme 1) with 97% yield. MS EtOAc (3×). The combined organic layer was washed with sat-
showed an appropriate peak of [M+H] 344.1.
urated sodium bicarbonate solution (1×), water (3×), brine
To the solution of the intermediate V (0.40 g, 1.2 mmol) in (1×), and dried over anhydrous sodium sulfate. The solvent
10 mL DMF, CDCA (0.46 g, 1.2 mmol) was added along with was evaporated to yield a white solid intermediate (compound
diethyl cyanophosphonate (DEPC; 0.2 mL, 1.3 mmol) and tri- VI; Scheme 2) with 47% yield. MS showed an appropriate peak
ethylamine (TEA; 1.7 mL, 12 mmol). The reaction was stirred of [M+Na] 626.1.
at room temperature overnight. The reaction was quenched
by adding 50 mL of water followed by extraction with EtOAc
(3×). The combined organic layer was washed with saturated
sodium bicarbonate solution (1×), water (3×), brine (1×), and
dried over anhydrous sodium sulfate. The solvent was evapo-
rated to yield a white solid. The crude product was further puri-
fied by silica gel column chromatography using a mobile phase
of DCM–methanol–acetic acid (v/v, 10:1:0.001) that yielded a
final white solid product (compound 1; Scheme 1). MS of com-
pound 1 showed appropriate peaks of [M+Na] 740.2 with 78%
yield. Compound 2 was synthesized in the same manner, but
with UDCA substituted for CDCA. MS of compound 2 showed
appropriate peaks of [M+Na] 740.2 with 81% yield.
The intermediate VI was dissolved in 5 mL DCM and TFA
(v/v, 1:9) and stirred at room temperature for 48 h to remove
both the acetonide- and Boc-protecting groups. Excess DCM
and TFA were evaporated under vacuum to yield an orange
oily intermediate (compound VII; Scheme 2), which was used
without further purification with 44% yield. MS showed an
appropriate peak of [M+H] 464.1.
To the solution of intermediate VII (0.13 g, 0.28 mmol) in
4 mL DMF, CDCA (0.11 g, 0.28 mmol) was added along with
DEPC (47 :L, 0.31 mmol) and TEA (390 :L, 2.8 mmol). The re-
action was stirred at room temperature overnight. The reaction
was quenched by adding 50 mL water followed by extraction
with EtOAc (3×). The combined organic layer was washed with
saturated sodium bicarbonate solution (1×), water (3×), brine
(1×), and dried over the anhydrous sodium sulfate. The solvent
was evaporated to yield a white solid intermediate (compound
VIII; Scheme 2) with 76% yield. MS showed an appropriate
peak of [M+H] 838.3.
Synthesis of L-Glutamate–Ribavirin Amide Conjugates of Bile
Acids (Compounds 3–5)
Intermediate II in Scheme 1 (1 g, 3.52 mmol) was coupled to
Boc–L-glutamic acid 5-benzyl ester (1.19 g, 3.52 mmol) using
Pybop (1.83 g, 3.52 mmol) as the coupling reagent in 10 mL
DMF along with DIPEA (1.22 mL, 7.04 mmol). The reaction
was stirred at room temperature overnight. The reaction was
quenched by adding 50 mL water followed by extraction with
Intermediate VIII was subjected to catalytic hydrogenation
to remove the 5-benzyl ester group using 10% Pd/charcoal in
methanol for 4 h. The solution was filtered through Celite
and the solvent was evaporated to give a white solid product.
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
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The crude product was further purified by preparative high-
The intermediate XII was subjected to catalytic hy-
performance liquid chromatography using a ZORBAX 300SB drogenation to remove the benzylidene acetal- and ben-
C18 PrepHT column (7 :m, 21.2 × 250 mm2; Agilent, Santa zyl ester-protecting groups using 20% palladium hydroxide
Clara, California). The mobile phase was composed of 68% (PdOH)/charcoal in methanol overnight. The solution was fil-
methanol and 32% water with a flow rate of 5 mL/min. UV tered through Celite and solvent was evaporated. The crude
absorbance was monitored at 210 nm. The fractions containing product was purified by C18 column chromatography using a
the product were collected and freeze dried to yield a final white mobile phase of water and methanol (v/v, 1.85:1), yielding a fi-
fluffy solid product (compound 3; Scheme 2). Compounds 4 and nal white solid product (compound 6; Scheme 3) with 58% yield.
5 were synthesized in the same manner, but with UDCA and MS showed an appropriate peak of [M−H] 817.4.
CA substituted for CDCA, respectively. MS showed appropriate
Cell Culture
peaks of [M−H] 746.3 with 88% yield, [M−H] 746.3 with 87%
yield, and [M−H] 762.3 with 75% yield.
Stably transfected human NTCP-HEK293 cells were grown in
atmosphere with 90% relative humidity and
a 37°C, 5% CO2
fed every 2 days as previously described.12 The medium was
composed of DMEM supplemented with 10% FBS, 100 units/mL
penicillin, 100 :g/mL streptomycin, and geneticin (1 mg/mL).
Cells were passaged when they reach 80% confluence.
Synthesis of Ribavirin Benzylidene Acetal (Compound IX)
To the suspension of ribavirin (compound I in Scheme 3; 2 g,
8.2 mmol) in benzaldehyde (20 mL), ZnCl2 (2 g, 14.8 mmol) was
added. The reaction was stirred at room temperature for 6 h and
poured into 250 mL diethyl ether, yielding a white precipitate.
After suction filtration, the precipitate was purified by silica
gel column chromatography using a mobile phase of DCM and
methanol (14:1) to give a white solid intermediate (compound
IX; Scheme 3) with 55% yield. MS showed an appropriate peak
of [M+Na] 355.1.
Uptake of Prodrugs by NTCP into Cells
Uptake studies were conducted on NTCP-HEK293 cells. Cells
were seeded at the density of 300,000 cells/well in 24-well Bio-
coat plates. On day 2 after seeding, cells were exposed to various
concentrations of prodrugs (0–500 :M), which were dissolved
in either Hank’s balanced salts solution (HBSS) with 137 mM
sodium chloride (pH 6.8) or sodium-free buffer (SFB) where
sodium chloride was replaced with tetraethylammonium chlo-
ride (pH 6.8). Cells were exposed to the incubation buffer solu-
tions for 5 min. The uptake was terminated by adding ice-cold
SFB buffer after incubation buffer was removed. Cells were
lysed using acetonitrile. After acetonitrile was evaporated, the
lysate were then dissolved in the acetonitrile–H2O (1:1) with
500 nM internal standard (i.e., taurocholate or carbamazepine).
The samples were stored at −80°C until further analysis.
In order to determine the prodrug kinetic parameters Kt
and Jmax, uptake data in HBSS buffer were fitted to Eq. (1),
where Kt is prodrug Michaelis–Menten constant; J is prodrug
flux; Jmax is the maximal flux of prodrug; S is the prodrug con-
centration; and Pp is the prodrug passive permeability that is
estimated from the uptake data in the SFB buffer as NTCP is
sodium-dependent transporter where no active transport takes
place in the absence of sodium. As a positive control, Jmax of
taurocholate, a prototypical native bile acid, was estimated
from taurocholate uptake studies at high-taurocholate concen-
trations where transporter was saturated (i.e., 200 :M) on each
occasion. Kt, Jmax, and Pp were calculated through nonlinear re-
gression using WinNonlin (Pharsight, Sunnyvale, California).
Jmax of each prodrug was normalized to the Jmax of taurocholate,
yielding normalized Jmax in order to accommodate the variation
in NTCP expression level across the studies.
Synthesis of L-Valine–Ribavirin Carbamate Conjugate of CDCA
(Compound 6)
Intermediate IX (1.49 g, 4.50 mmol) was then coupled to Fmoc–
L-valine (1.53 g, 4.50 mmol) using Pybop (2.34 g, 4.50 mmol)
in 15 mL DMF along with DIPEA (1.57 mL, 9.00 mmol). The
reaction was stirred at room temperature overnight. The reac-
tion was quenched by adding 50 mL water followed by extrac-
tion with EtOAc (3×). The combined organic layer was washed
with water (3×), brine (1×), and dried over anhydrous sodium
sulfate. The solvent was evaporated to yield a white solid inter-
mediate (compound X; Scheme 3) with 84% yield.
The intermediate X was dissolved in 10 mL of DMF with 5%
piperidine and reacted for 2 h to remove the Fmoc-protecting
group. The resulting product was acidified with 50 mL 2 M
HCl and extracted with 50 mL EtOAc. The aqueous layer was
immediately alkalized with saturated sodium bicarbonate so-
lution followed by extraction with EtOAc (3×). The combined
organic layer was washed with water (3×), brine (1×), and dried
over anhydrous sodium sulfate. The solvent was evaporated to
yield a white solid intermediate (compound XI; Scheme 3) with
55% yield.
Glycine benzyl ester was conjugated to CDCA with DEPC
as the coupling reagent, as described above with 72% yield. To
the solution of glycine benzyl ester, protected CDCA (1 g, 1.86
mmol) in 80 mL anhydrous DCM in the presence of the 0.5
mL pyridine (6.21 mmol), triphosgene (0.275 g, 0.93 mmol) in
anhydrous DCM was added dropwise. The reaction was stirred
at room temperature for 4 h and intermediate XI (0.80 g, 1.86
mmol) was subsequently added. The reaction was stirred for 48
h. After the solvent was evaporated, extraction was conducted
Jmax
S
J =
+ PpS
(1)
Kt + S
In Vitro Stability Study
with water and EtOAc (3×). The combined organic layer was Metabolic stability of prodrug was assessed in mouse liver
washed with brine (1×) and dried over anhydrous sodium sul- S9 fraction. Briefly, 5 :M of prodrug were incubated with
fate. The solvent was evaporated and the residue was subjected 1 mg/mL mouse liver S9 fraction supplemented with 1 mM
to further purification by silica gel column chromatography us- nicotinamide adenine dinucleotide phosphate in Dulbecco’s
ing a mobile phase of DCM and methanol (v/v, 33:1) to afford phosphate-buffered saline at 37°C. At designated time points
the intermediate XII (Scheme 3) as a white solid. MS showed (i.e., 0, 0.5, 1, 3, and 5 h), 100 :L samples were col-
an appropriate peak of [M+Na] 1019.3 with 32% yield.
lected and quenched immediately with 400 :L acetonitrile
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
containing 1 :M internal standard. Samples were votexed for ter centrifugation, 300 :L supernatant from each sample was
30 sand centrifuged at 20,000 g for 10 min. Supernatant was transferred to 24-well plates and evaporated overnight at room
dried overnight and reconstituted in 200 :L water and stores temperature. Dry extracts were reconstituted in 300 :L 5 mM
at −80°C until further analysis.
ammonium acetate buffer (pH 4.8). Samples were treated with
1 unit of phosphatase at 37°C overnight and stored at −80°C
until further analysis.
In Vitro Accumulation of Ribavirin and Prodrug in RBCs
In order to assess whether the drug achieved liver targeting,
liver targeting index (LTI) was calculated using Eq. (2).
Accumulation of the ribavirin, CDCA–L-Val–ribavirin and
ribavirin–L-Val–GCDCA in RBC was determined as previous
described with minor changes.13 Briefly, human RBCs were sep-
arated from plasma by centrifugation at 400 g at room temper-
ature for 10 min. Plasma was spiked with ribavirin or prodrug
and gently mixed with RBC and incubated at 37°C. The final
concentration of initial ribavirin or initial prodrug in the whole
blood was 100 :M. At designated time points (i.e., 0 and 5 h),
50 :L aliquots of whole blood were collected and centrifuged at
3500 g at 4°C for 2 min to separate RBCs from plasma. RBCs
were lysed by adding 100 :L 0.1% triton X-100. Protein was
precipitated with the addition of 300 :L and 400 :L of ace-
tonitrile containing 1 :M internal standard to the lysed RBC
and plasma, respectively. After votexing for 30 s, the samples
were centrifuged at 20,000 g for 10 minutes. After centrifu-
gation, 300-:L supernatant were transferred to 24-well plates
and evaporated overnight at room temperature. Dry extracts
were reconstituted in 200 :L 5 mM ammonium acetate buffer
(pH 4.8). RBC samples were treated with 1 unit of phosphatase
and incubated at 37°C overnight to convert phosphorylated rib-
avirin to ribavirin, which was measured. Both plasma and RBC
samples were stored at −80°C until further analysis.
AUC0−t in liver
AUC0−t in plasma
‘
LTI =
(2)
The LTI of ribavirin employed the AUC of ribavirin in the
liver and plasma over 8 h (t = 8 h).
Analytical Method
Both ribavirin and prodrugs were quantified by liquid
chromatography–mass spectrometry (LC–MS), which was per-
formed on Waters ACQUITY UPLC/TQD system (Waters, Mil-
ford, Massachusetts). All the samples were centrifuged at
20,000 g for 5 minutes and 5 :L supernatant were injected
into LC–MS. Taurocholate was used as the internal standard
for CA–L-Glu–ribavirin, whereas carbamazepine was used as
the internal standard for all other compounds.
To determine the prodrug concentration in the cell, a Luna
C8 (2) column (3 :m, 2.0 × 50 mm2; Torrance, California) was
used. A gradient mobile phase employed methanol and wa-
ter (v/v) with 0.1% formic acid, respectively. The flow rate was
0.7 mL/min and the compounds were detected under positive
electrospray ionization mode [M+H]+, except for CA–L-Glu–
ribavirin, which was detected under negative mode [M−H]−.
Multiple reaction monitoring was used for all methods. The
lower limit of quantification for all compounds was between 1
and 10 nM. Mass transitions and parameter of mass spectrom-
eter for all six prodrugs and internal standards are detailed in
Table 1.
In Vivo Distribution of Ribavirin and Prodrug in Mice
The study was conducted in accordance with the Guide for
the Care and Use of Laboratory Animals as adopted and pro-
mulgated by the U.S. National Institutes of Health (Bethesda,
Maryland) and was approved by the University of Maryland
Institutional Animal Care and Use Committee.
Normal nonfasted Balb/c mice (male, 12 weeks) were tail
injected (i.v. bolus) with a dose of 20 mg/kg (0.08 mmol/kg) rib-
avirin and 67 mg/kg (0.08 mmol/kg) ribavirin–L-Val–GCDCA,
respectively, to yield an equimolar amount of drug or prodrug.
The compounds were dissolved in 60% saline and 40% polyethy-
lene glycol (PEG 400). At designated time points (10 and 30 min,
and 1, 2, 4, and 8 h), mice (n = 3 per time point) were euthanized
by CO2 (carbon dioxide) asphyxiation, followed by cervical dislo-
cation. Blood was drawn immediately by cardiac puncture and
collected in heparinized vials. Samples were then centrifuged
at 3500 g and 4°C for 2 min to separate plasma and RBC. Liver,
small intestine, and kidney were isolated and weighed before
homogenization in 2 mL water using Digital SLP cell disruptor
(Branson Ultrasonics, Danbury, Connecticut).
Quantification of ribavirin and prodrug concentrations in
liver S9 fraction, RBC, and mice tissues was conducted using
the same mass transition as in Table 1. A Xterra RP18 column
Table 1. Parameters of Mass Spectrometer and Monitored Ion
Transitions for Ribavirin Prodrugs Quantification
Multiple Reaction
Collision
Compound
Monitoring Transition
Energy (V)
Ribavirin
245.13→112.87
237.13→193.99
16
26
Carbamazepine
(internal standard)
Taurocholate
514.26→79.96
100
Four-hundred microliter acetonitrile containing 1 :M inter-
(internal standard)
nal standard was added to 50 :L tissue homogenate or plasma CDCA–L-Val–ribavirina
718.67→97.00
718.67→97.00
748.54→97.06
748.54→97.06
762.33→128.01
819.48→96.93
35
35
72
72
68
74
UDCA–L-Val–ribavirina
to precipitate protein. Samples were votexed for 30 s and cen-
trifuged at 20,000 g for 10 minutes. After centrifugation, 300 :L
supernatant was transferred to 24-well plates and evaporated
overnight at room temperature. Dry extracts were reconsti-
CDCA–L-Glu–ribavirina
UDCA–L-Glu–ribavirina
CA–L-Glu–ribavirina
Ribavirin–L-Val–GCDCAa
tuted in 300 :L water and stored at −80°C until further anal-
ysis.
All compounds were detected under positive electrospray ionization mode
[M+H]+ except CA–L-Glu–ribavirin that was detected under negative mode
For RBC, 25-:L RBCs were first lysed by 100 :L 0.1% triton
X-100. Three-hundred microliter acetonitrile containing 1 :M
internal standard was then added to the lysate and votexed
[M−H]−
.
aAccuracy for CDCA–L-Val–ribavirin, UDCA–L-Val–ribavirin, CDCA–L-Glu–
ribavirin, UDCA–L-Glu–ribavirin, CA–L-Glu–ribavirin, and ribavirin–L-Val–
for 30 s. Samples were centrifuged at 20,000 g for 10 min. Af- GCDCA were 1.83%, 0.318%, −3.58%, 1.99%, 3.87%, and 0.508%, respectively.
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
7
(5 :m, 4.6 × 150 mm2; Waters) was used in order to separate en-
dogenous compound (most likely uridine) from the compounds
of interest.14 The mobile phase was composed of water and
methanol with 0.1% acetic acid. The flow rate was 1 mL/min.
The limit of quantification for all compounds was between 100
and 500 nM.
As ribavirin–L-Val–GCDCA was studied in vivo, the extrac-
tion efficiency of the ribavirin–L-Val–GCDCA from plasma,
RBC, liver, kidney, and small intestine was 98%, 99%,
100%, 98%, and 93% respectively. The accuracy of quantifi-
cation of ribavirin–L-Val–GCDCA in plasma, RBC, liver, kid-
ney, and small intestine was 1.96%, 7.77%, −6.92%, 8.37%,
and 0.121%, respectively. Ribavirin–L-Val–GCDCA stability in
plasma, RBC, liver, kidney, and small intestine for 30 min
on ice showed greater than 80% stability. Similarly, a freeze–
thaw cycle (−80°C and subsequent room temperature for 48 h)
of ribavirin–L-Val–GCDCA in plasma, RBC, liver, kidney, and
small intestine showed greater than 80% stability.
conjugates had much lower passive permeability than the neu-
tral L-valine conjugates, possibly because of the negative charge
on the linker.
Chenodeoxycholic acid–L-Val–ribavirin and CDCA–l-Glu–
ribavirin were the two compounds with the highest normalized
Jmax values and hence were subjected to further evaluation for
ribavirin release in mouse liver S9 fraction.
Release of Ribavirin from Prodrugs with C-24 Conjugation
In order to possess efficacy in the liver to treat hepatitis C,
ribavirin needs to be released from the prodrugs after hepatic
uptake by NTCP. Thus, mouse liver S9 fraction, which contains
a wide variety of liver enzymes, was used to assess ribavirin
release from the prodrugs. CDCA–L-Val–ribavirin and CDCA–
L-Glu–ribavirin were incubated with mouse liver S9 fraction
over 5 h. As shown in Figure 3, by 5 h, 63% of ribavirin were
released from the neutral CDCA–L-Val–ribavirin, whereas the
prodrug itself was largely degraded by 1 h. By contrast, very
little ribavirin (4%) was released from the anion CDCA–L-Glu–
ribavirin and 96% of the prodrug remained intact after 5 h. The
results suggest that CDCA–L-Val–ribavirin has the potential to
release the drug in the liver, whereas CDCA–L-Glu–ribavirin
showed little promise.
Statistical Analysis
Student’s t-test was used to evaluate difference of prodrug
uptake in the presence and absence of sodium. It was also
used to assess different ribavirin accumulation in RBCs
and plasma when ribavirin or prodrug (e.g., CDCA–L-Val–
ribavirin or ribavirin–L-Val–GCDCA) were incubated with hu-
man whole blood. A p value of less than 0.05 denoted statistical
significance.
Reduced Ribavirin Accumulation in RBCs with
CDCA–L-Val–Ribavirin
Given its susceptibility to release drug, CDCA–L-Val–ribavirin
was incubated with human whole blood to assess ribavirin and
prodrug distribution in plasma and RBC. The RBC is an off-
target of ribavirin therapy, with hemolytic anemia limiting rib-
avirin dosing. By 5 h, 42% of initial prodrug remained intact in
plasma (Fig. 4). Twenty-two percent of initial prodrug was dis-
tributed into RBC, and the prodrug remained intact in the RBC.
Relative to initial prodrug, 13% of prodrug was converted to
ribavirin and present in plasma and RBC, respectively. By con-
trast, the incubation of same molar amount of ribavirin itself
with human whole blood resulted in 25% of ribavirin in plasma
and 50% ribavirin accumulation in RBC. The comparison can
be translated that CDCA–L-Val–ribavirin was able to reduce
ribavirin accumulation in RBC by 3.8-fold (i.e., 13% vs. 50%).
However, 22% of the prodrug permeated into RBC, reflecting
the high-passive permeability of CDCA–L-Val–ribavirin. This
high-passive permeability may result in the passive diffusion
of the prodrug into organs or tissues other than the liver, as
observed here in RBC. This potential wide distribution may
promote prodrug hydrolysis before it enters the liver, thus re-
ducing the amount of ribavirin delivered into the liver.
RESULTS
NTCP Uptake of Prodrugs with C-24 Conjugation
Ribavirin was first conjugated to carboxylic acid at the
C-24 position of CDCA, UDCA, or CA using either L-
valine or L-glutamic acid as the linker to yield five neutral
or monoanionic compounds: CDCA–L-Val–ribavirin, UDCA–
L-Val–ribavirin, CDCA–Glu–ribavirin, UDCA–Glu–ribavirin,
and CA–L-Glu–ribavirin. Concentration-dependent uptake of
all five compounds using NTCP-HEK293-stably transfected
cells are shown in Figure 2. Studies were conducted in the
presence and absence of sodium, where uptake in the absence
of sodium served as the negative control as NTCP is a sodium-
dependent transporter.
Chenodeoxycholic acid–L-Val–ribavirin and UDCA–L-Val–
ribavirin are neutral compounds, whereas CDCA–L-Glu–
ribavirin, UDCA–L-Glu–ribavirin, and CA–L-Glu–ribavirin
each possess a single negative charge at the C-24 side chain. De-
spite these charge differences, all five compounds were NTCP
substrates, where flux differed (Student’s t-test, p < 0.05) be-
tween passive and total uptake at each concentration, except
for the lowest concentration (2.5 :M) of CA–L-Val–ribavirin.
In Vitro Characterization of Ribavirin–L-Val–GCDCA
In order to reduce a prodrug’s passive permeability as observed
Prodrug uptake kinetic parameters are listed in Table 2. for CDCA–L-Val–ribavirin and preserve its ability to release
The transport capacities (i.e., normalized Jmax) of all prodrugs ribavirin in the liver, ribavirin was conjugated to the C-3 po-
were significantly lower than that of the native substrate tau- sition of GCDCA using L-valine as the linker. GCDCA was se-
rocholate, with the normalized Jmax values ranging from 0.011 lected over CDCA as GCDCA has higher capacity to be taken
to 0.302. CDCA–L-Val–ribavirin provided the highest normal- up by NTCP than CDCA.15 This strategy retains the ester bond
ized Jmax (i.e., 0.302, or 30% that of taurocholate), followed by between L-valine and ribavirin and introduces one negative
CDCA–L-Glu–ribavirin (normalized Jmax = 0.075). The two pro- charge at the C-24 region to reduce the passive permeability,
drugs using L-valine as the linker yielded higher NTCP affinity as observed from the above for L-glutamic-linked prodrugs.
(i.e., lower Kt) than their corresponding L-glutamic acid-linked
Ribavirin–L-Val–GCDCA was assessed for NTCP uptake,
prodrugs. UDCA–L-Val–ribavirin had the highest affinity to- ribavirin release in mouse liver S9 fraction, and drug distribu-
ward NTCP, with a Kt value of 11.6 :M. The L-glutamic acid tion in human whole blood. As shown in Figure 5a, the passive
DOI 10.1002/jps.24375
Dong et al., JOURNAL OF PHARMACEUTICAL SCIENCES

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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Figure 2. Concentration-dependent uptake of (a) CDCA–L-Val–ribavirin, (b) UDCA–L-Val–ribavirin, (c) CDCA–Glu–ribavirin, (d) UDCA–Glu–
ribavirin, and (e) CA–L–Glu–ribavirin into NTCP-HEK293 cells for 5 min. Closed circles indicate total prodrug uptake by NTCP in the presence
of sodium. Open circles indicate passive uptake of the prodrug in the absence of sodium. The solid line indicates model fit of the total uptake.
Data are presented as mean ( SEM) of three measurements at each concentration.
permeability across HEK293 cells was reduced by 5.7-fold as After 5 h, 34% of ribavirin were released from the prodrug.
compared with CDCA–L-Val–ribavirin. Interestingly, the trans- The prodrug itself largely degraded within 1 h. Prodrug distri-
port capacity also decreased significantly (normalized Jmax
=
bution in human whole blood demonstrated that 85% of the
0.0538). However, the prodrug had much higher affinity to- prodrug remained intact in the plasma, whereas almost no
ward NTCP than CDCA–L-Val–ribavirin, with a Kt value of prodrug accumulated in the RBC (1%). Meanwhile, 3% of the
1.63 vs. 35.0 :M for CDCA–L-Val–ribavirin. Metabolic stability released ribavirin were present in both the plasma and RBC
in mouse liver S9 fraction (Fig. 5b) suggests that ribavirin–L- (Fig. 5c), which was 16.7-fold lower in RBC than when rib-
Val–GCDCA has the potential to release ribavirin in the liver. avirin alone was tested. On the basis of these in vitro studies,
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DOI 10.1002/jps.24375

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
9
Table 2. Sodium Taurocholate Cotransporting Polypeptide Uptake Parameters of CDCA–L-Glu–Ribavirin, UDCA–L-Glu–Ribavirin,
CA–L-Val–Ribavirin, CDCA–L-Val–Ribavirin, and UDCA–L-Val–Ribavirin
Jmax (pmol s−1 cm−2
)
Normalized Jmax
Pp × 106 (cm/s)
a
Prodrug
Kt (:M)
CDCA–L-Glu–ribavirin
UDCA–L-Glu–ribavirin
CA–L-Glu–ribavirin
CDCA–L-Val–ribavirin
UDCA–L-Val–ribavirin
182 26
244 41
659 261
35.0 7.4
11.6 1.4
0.145 0.009
0.0330 0.0026
0.0208 0.0053
0.592 0.044
0.0752 0.047
0.0171 0.0013
0.0108 0.0028
0.302 0.031
0.033 0.002
0.0567 0.0026
0.0154 0.0004
0.0274 0.0007
3.76 0.05
0.0654 0.0020
0.376 0.019
Data are summarized as mean ( SEM) of three measurements.
aJmax of each prodrug was normalized to Jmax of taurocholate that was measured in parallel to each study in order to accommodate the variation in NTCP
expression level across the studies.
Figure 4. Distribution of ribavirin and CDCA–L-Val–ribavirin in hu-
man whole blood at 37°C after 5 h. Ribavirin or CDCA–L-Val–ribavirin
(100 :M) was incubated with human whole blood for 0 and 5 h, followed
by quantification of ribavirin (if ribavirin incubated) or released rib-
avirin and intact prodrug (if prodrug incubated) in plasma and RBCs.
Closed bars indicate ribavirin incubation. The open bars indicate pro-
drug incubation (and hence prodrug or release ribavirin quantified).
Data are presented as mean ( SEM) of three measurements at each
time point. *Relative amount was reduced for CDCA–L-Val–ribavirin
incubation versus ribavirin incubation [Student’s t-test; p = 9.08 ×
Figure 3. Hydrolysis of (a) CDCA–L–Val–ribavirin and (b) CDCA–
L–Glu–ribavirin in mouse liver S9 fraction. For each prodrug, prodrug
10−3 (plasma) and p = 4.25 × 10−5 (RBC)].
(5 :M) was incubated with mouse liver S9 fraction (1 mg/mL) at 37°C
for 0, 0.5, 1, 3, and 5 h. Open circles indicate percentage of prodrug re-
maining intact. Closed circles indicate percentage of ribavirin appear-
ing in mouse S9 fraction, relative to initial prodrug. Data are presented
as mean ( SEM) of three measurements at each time point.
surement reflects the sum of ribavirin and phosphorylated rib-
avirin. Meanwhile, pilot studies here indicated that nonphos-
phorylated ribavirin was the predominant form in the liver
sample (Fig. S1). Hence, for liver samples, no treatment of phos-
phatase was needed, yet ribavirin concentration is interpreted
to reflect the sum of ribavirin and phosphorylated ribavirin.
As shown in Figure 6, in comparison to ribavirin, admin-
istration of ribavirin–L-Val–GCDCA resulted in less ribavirin
exposure in the plasma, RBC, and kidney, increased exposure
in the intestine, and similar exposure in the liver. The Tmax
ribavirin–L-Val–GCDCA possessed overall greater potential to
achieve in vivo liver targeting with reduced ribavirin accumu-
lation in RBC.
Pharmacokinetics of Ribavirin–L-Val–GCDCA and Ribavirin
in Mice
A pharmacokinetic study of ribavirin–L-Val–GCDCA and rib- of ribavirin in the liver was shifted from 30 to 120 min when
avirin was conducted in mice in order to assess the liver- prodrug was administered, likely because of the time required
targeting potential of the prodrug in vivo. Administration en- for ribavirin to be released from the prodrug. The AUC of both
tailed 0.08 mmol/kg intravenous tail vein injection in mice. ribavirin and ribavirin–L-Val–GCDCA was calculated for the
After prodrug or ribavirin administration, prodrug, and/or rib- plasma, RBC, liver, kidney, and intestine, as shown in Table 3.
avirin concentrations were measured in the plasma, RBC, liver, The LTI for ribavirin was 0.0125 from ribavirin–L-Val–GCDCA
kidney, and small intestine over 8 h.
administration and 0.00695 from ribavirin administration, in-
Previous studies suggested that ribavirin was present in dicating that the prodrug has achieved better ribavirin liver
RBCs in the form phosphorylated ribavirin.6 Thus, RBC sam- targeting by 1.80-fold. This enhancement was largely because
ples were treated with phosphatase, which converted phospho- of prodrug administration yielding about a 1.73-fold reduction
rylated ribavirin to ribavirin; total ribavirin concentration mea- in ribavirin exposure in plasma, compared with parent drug
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
administration. Prodrug and drug administration afforded
about the same ribavirin exposure in the liver.
DISCUSSION
In the current study, we have developed and characterized six
ribavirin prodrugs with the goal of achieving liver-specific de-
livery of ribavirin via targeting NTCP, thereby reducing toxic
ribavirin accumulation in the RBCs. Bile acid–ribavirin pro-
drugs were synthesized with either L-valine or L-glutamic acid
as the linker. Bile acids are native substrates of NTCP, and
both L-valine and L-glutamic acid are endogenous compounds
to impart either neutral or anionic character. Both in vitro and
in vivo tests were conducted to characterize the six prodrugs
among which ribavirin–L-Val–GCDCA was shown to achieve
ribavirin liver-specific delivery in mice.
NTCP Uptake
Human NTCP uptake studies were conducted to screen the
synthesized prodrugs for potential liver targeting. A previ-
ous study suggested that a negative charge at the C-24 side
chain of bile acids is essential for NTCP transport.16 Bile
acid conjugates with monoanionic side chains (i.e., CDCA–
L-Glu–ribavirin, UDCA–L-Glu–ribavirin, CA–L-Glu–ribavirin,
and ribavirin–L-Val–GCDCA) were substrates of NTCP. Inter-
estingly, conjugates with no charge on the C-24 side chain
(i.e., CDCA–L-Val–ribavirin and UDCA–L-Val–ribavirin) also
showed favorable uptake, with Kt values comparable to that of
taurocholate (i.e., 22.7 :M),12 suggesting that a monoanion at
the C-24 side chain may not be necessary for the transport by
human NTCP. Potentially, there may be unknown physicochem-
ical or structural variables other than charge that affect NTCP
transport, such as hydrophobicity that has been shown to im-
pact sodium-dependent bile acid transporter (ASBT) transport
activity.17
A previous study from our laboratory showed that bile acid
conjugates coupled at the C-3 position afford translocation by
NTCP.18 This C-3 coupling was also observed in the current
study where the C-3-conjugated prodrug (i.e., ribavirin–L-Val–
GCDCA) was a potent substrate with Kt of 1.63 :M. In terms
of transport capacity, all six prodrugs possess a significantly
reduced NTCP capacity compared with taurocholate. Of the six
prodrugs investigated, ribavirin–L-Val–GCDCA had the low-
est Kt value and a Jmax of and about 75% transport efficiency
(Jmax/Kt) of taurocholate, suggesting moderately efficient up-
take by NTCP.12 In addition, CDCA conjugates were shown to
have higher transport capacity (i.e., normalized Jmax) than their
corresponding UDCA or CA conjugates.
Figure 5. In vitro characterization of ribavirin–L-Val–GCDCA.
(a) From the NTCP uptake of ribavirin–L-Val–GCDCA, Kt = 1.63
Prodrug Metabolism in Mouse Liver S9 Fraction
(
0.70) :M, Jmax = 0.100( 0.004) (pmol s−1 cm−2), normalized Jmax
Metabolic stability studies were conducted to assess ribavirin
release when exposed to mouse liver S9 fraction in vitro. All
of the synthesized prodrugs possessed an ester bond between
the amino acid linker and ribavirin. However, only CDCA–L-
Val–ribavirin and ribavirin–L-Val–GCDCA showed favorable
drug release when exposed to mouse liver S9 fraction, whereas
CDCA–L-Glu–ribavirin was stable, suggesting that L-valine is
more susceptible to enzymatic hydrolysis than L-glutamic acid.
Although about 94% of CDCA–L-Val–ribavirin and ribavirin–
L-Val–GCDCA were degraded after 5 h, only 63% and 34% of
ribavirin was recovered, respectively. It has been reported that
= 0.0538( 0.0038) and passive permeability = 0.655( 0.025) × 10−6
cm/s. (b) Hydrolysis of ribavirin–L-Val–GCDCA in mouse liver S9 frac-
tion over 5 h. After 5 h, 34% of ribavirin was released from the
prodrug. (c) Distribution of released ribavirin and intact ribavirin–L-
Val–GCDCA in human whole blood at 37°C after 5 h. 16.7-fold lower
amounts of ribavirin accumulated in RBC when tested as prodrug com-
pared with ribavirin itself. Data are presented as mean ( SEM) of three
measurements at each time point. * denotes that relative amount was
reduced for ribavirin–L-Val–GCDCA incubation versus ribavirin incu-
bation [Student’s t-test, p = 1.24 × 10−3 (plasma), p = 1.22 × 10−5
(RBC)].
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
11
Figure 6. Pharmacokinetics of ribavirin and prodrug ribavirin–L-Val–GCDCA in mice after i.v. administration. Each data point represents
data from three mice i.v. injected with either 0.08 mmol/kg ribavirin or 0.08 mmol/kg ribavirin–L-Val–GCDCA. Plasma, RBC, liver, kidney, and
small intestine were collected at 0, 10, 30, 60, 120, 240, and 480 min. Panels a–e are concentrations of ribavirin and prodrug in the (a) plasma,
(b) RBC, (c) liver, (d) kidney, and (e) small intestine, respectively. Closed squares are the ribavirin concentration after ribavirin i.v. injection. Closed
circles are the concentration of released ribavirin from ribavirin–L-Val–GCDCA after i.v. prodrug injection. Open circles are the concentration of
intact ribavirin–L-Val–GCDCA after i.v. prodrug injection. Open circles and close circles represent data from the same mouse. Liver, kidney, and
small intestine concentrations were normalized by its tissue weight. Data are presented as mean ( SEM) of three measurements at each time
point.
DOI 10.1002/jps.24375
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RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Table 3. Exposure of Ribavirin and Prodrug Ribavirin–L-Val–GCDCA to Various Compartments and Tissues After i.v. Administration of Each
Ribavirin and Prodrug in Mice over 8 h
AUC0–8 h of Ribavirin After
AUC0–8 h of Ribavirin After Ribavirin
Administration (:M min or :mol
min/g Tissue)a
Ribavirin–L-Val–GCDCA
Administration (:M min or :mol
min/g Tissue)a
AUC0–8 h of Ribavirin–L-Val–GCDCA
After Its Administration (:M min or
:mol min/g Tissue)a
Compartment or Tissue
Plasma
RBC
Liver
Kidney
Intestine
3857
2940
53.6
10.4
8.47
2221
1788
55.6
5.43
16.6
600
547
3.28
0.476
7.03
Six time points (n = 3 mice per time point) were employed in measuring AUC.
aUnits are :M min for plasma and RBC and :mol min/g tissue for liver, intestine, and kidney.
there are two metabolic pathways for ribavirin.19 One is the
reversible phosphorylation pathway where ribavirin is phos-
phorylated to mono-, di-, and tri-phosphorylated ribavirin. The
other pathway is the degradation involving deribosylation and
amide hydrolysis to yield a triazole carboxylic acid metabo-
lite. Our results suggest that ribavirin might undergo an irre-
versible metabolism by 5 h, resulting in incomplete recovery of
ribavirin (Fig. S1).
40%, whereas an approximately equal amount of ribavirin was
seen in the liver as compared with the administration of rib-
avirin itself. It should be noted that the specific location of rib-
avirin exposure within the liver (e.g., hepatocyte, bile canali-
culi) was not evaluated. Ribavirin exposure was increased in
the intestine, perhaps because of the enterohepatic circulation
of bile acids where the apical ASBT may be responsible for re-
absorbing unmetabolized prodrug from the lumen of the small
intestine. Prodrug enterohepatic circulation could potentially
increase the amount of prodrug delivered to the liver, if the
prodrug is not quickly released in hepatocytes and instead is
circulated. However, prodrug hydrolysis in the intestine may
result in increased intestinal ribavirin exposure. Thus, a pro-
drug with faster hydrolysis in the liver may reduce ribavirin
exposure in the intestine.
Prodrug Distribution in Whole Blood
Both prodrug and ribavirin were incubated in human whole
blood to assess ribavirin accumulation in RBC and stability of
the prodrug in whole blood. It has been shown that ribavirin
is phosphorylated in RBC where triphosphorylated ribavirin is
the predominant metabolite.20 Phosphatase was used to con-
vert phosphorylated ribavirin to ribavirin so that ribavirin con-
centration measured in RBC by LC–MS represents all of the
ribavirin accumulated in cells.20 Ribavirin is actively trans-
ported into RBCs by equilibrative nucleoside transporter 1.21
Likely, the coupling of a bulky bile acid would hamper rib-
avirin’s uptake into RBC that represents an off-target in clin-
ical use of ribavirin. This reduced uptake was observed in our
study where both CDCA–L-Val–ribavirin and ribavirin–L-Val–
GCDCA caused significantly reduced ribavirin accumulation in
RBC. However, because of the high-passive permeability, 22%
of CDCA–L-Val–ribavirin was also present in RBC. The subse-
quent introduction of a single negative charge (i.e., ribavirin–L-
Val–GCDCA) decreased the prodrug’s passive permeability and
reduced the accumulation of prodrug to only 1%. Meanwhile,
the presence of prodrug in the plasma increased from 42% to
85% (CDCA–L-Val–ribavirin vs. ribavirin–L-Val–GCDCA).
CONCLUSIONS
Six bile acid–ribavirin prodrugs were synthesized. All six
demonstrated NTCP uptake, but had low-transport capacity
compared with that of taurocholate. Of these six prodrugs,
ribavirin–L-Val–GCDCA had the highest affinity toward NTCP,
whereas CDCA–L-Val–ribavirin had the highest transport ca-
pacity (i.e., normalized Jmax). Metabolic stability in mouse
liver S9 fraction indicated that CDCA–L-Val–ribavirin and
ribavirin–L-Val–GCDCA efficiently released ribavirin in the
liver. Of the two, ribavirin–L-Val–GCDCA had more favorable
drug distribution in human whole blood, with a high amount of
prodrug present in the plasma but a favorably low ribavirin ac-
cumulation in RBC. The in vivo pharmacokinetic study in mice
showed that i.v. administration of ribavirin–L-Val–GCDCA al-
lowed for greater liver targeting of ribavirin while reducing
ribavirin accumulation in RBC. Results indicate that a study
of oral administration of this prodrug for liver targeting is mer-
ited. Overall, the current study suggests that ribavirin–L-Val–
GCDCA has the potential to achieve liver-specific delivery of
ribavirin.
In Vivo Study in Mice
The potential advantage of targeting NTCP to achieve liver-
specific drug delivery motivated the in vivo evaluation of
ribavirin–L-Val–GCDCA. A prodrug of ribavirin that targets
NTCP may allow for preferential uptake into the liver. Hence,
with subsequent release of ribavirin from prodrug, ribavirin
may preferentially accumulate in the liver, which is the site
of drug action in treating hepatitis C. Correspondingly, liver
targeting has promise to minimize ribavirin accumulation in
ACKNOWLEDGMENTS
the RBC. Of course, all released ribavirin in the liver may This work was supported in part by National Institutes of
not be retained in the liver. A pharmacokinetic study was con- Health grant DK093406 and US FDA grant U01FD004320-
ducted to evaluate the liver-targeting potential of ribavirin–L- 01. The authors kindly acknowledge Dr. Keiser (University of
Val–GCDCA in vivo. The study here showed that ribavirin ex- Greifswald) for providing the human NTCP-HEK293 cell line
posure was reduced in the plasma, RBC, and kidney by about used in this study.
Dong et al., JOURNAL OF PHARMACEUTICAL SCIENCES
DOI 10.1002/jps.24375

RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
13
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Dong et al., JOURNAL OF PHARMACEUTICAL SCIENCES