Dual-action mechanism of viramidine functioning as a prodrug and as a catabolic inhibitor for ribavirin

Article abstract of DOI:10.1128/AAC.48.10.4006-4008.2004

An investigational nucleoside analogue drug, viramidine, has recently emerged as a potentially safer alternative to ribavirin for the treatment of hepatitis C viral infection. We have reported that viramidine mainly functions as a prodrug of ribavirin that is enriched in the liver. This in vitro study further explores viramidine's activity against nucleoside pbosphorylase, a host enzyme that is responsible for phosphorolysis of ribavirin in vivo. Our experiments show that viramidine inhibits ribavirin phosphorolysis with a K _i of 2.5 μM. This result suggests that viramidine may act through a dual-action mechanism by serving as a prodrug of ribavirin and concomitantly as an inhibitor for nucleoside phosphorylase catabolism of ribavirin.

Full text of DOI:10.1128/AAC.48.10.4006-4008.2004

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2004, p. 4006–4008
0066-4804/04/$08.00ϩ0 DOI: 10.1128/AAC.48.10.4006–4008.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 48, No. 10
Dual-Action Mechanism of Viramidine Functioning as a
Prodrug and as a Catabolic Inhibitor for Ribavirin
Jim Zhen Wu,* Gary Larson, and Zhi Hong
Drug Discovery, R&D, Valeant Pharmaceuticals International, Costa Mesa, California
Received 24 March 2004/Returned for modification 25 May 2004/Accepted 8 June 2004
An investigational nucleoside analogue drug, viramidine, has recently emerged as a potentially safer alter-
native to ribavirin for the treatment of hepatitis C viral infection. We have reported that viramidine mainly
functions as a prodrug of ribavirin that is enriched in the liver. This in vitro study further explores viramidine’s
activity against nucleoside phosphorylase, a host enzyme that is responsible for phosphorolysis of ribavirin in
vivo. Our experiments show that viramidine inhibits ribavirin phosphorolysis with a Ki of 2.5 ␮M. This result
suggests that viramidine may act through a dual-action mechanism by serving as a prodrug of ribavirin and
concomitantly as an inhibitor for nucleoside phosphorylase catabolism of ribavirin.
Hepatitis C virus (HCV) is the major causative agent of
non-A, non-B virus-induced hepatitis (2). An insidious and
deadly disease, hepatitis C is responsible for an emerging pan-
demic of chronic liver diseases. There are 170 million infected
individuals worldwide and approximately 4 million virus carri-
ers in the United States alone. Unresolved acute HCV infec-
tion may progress to a chronic disease that could persist for
decades. As many as 20% of infected individuals eventually
develop liver cirrhosis, with 1 to 5% subsequently progressing
to hepatocellular carcinoma (12). This accounts for nearly
10,000 annual deaths in the United States. The current stan-
dard for treatment is a combination therapy of subcutaneous
pegylated alpha interferon with the oral nucleoside drug riba-
virin (6). The sustained viral response, defined as an undetect-
able viral load at 6 months after cessation of therapy, is around
54 to 56% for the combination therapy. Moreover, this treat-
ment has many adverse effects, including serious influenza-like
symptoms from alpha interferon and hemolytic anemia due to
the accumulation of ribavirin 5Ј-phosphates in red blood cells
(RBCs). These undesirable side effects can lead to dose reduc-
tion and discontinuation of the combination therapy (9). In an
effort to specifically deliver more ribavirin to the liver and
reduce the trapping of ribavirin metabolites in RBCs, thereby
improving the therapeutic index, a number of ribavirin deriv-
atives have been explored. One promising compound that has
emerged is the 3-carboxamidine derivative of ribavirin, known
as viramidine. Viramidine exhibits in vitro and in vivo antiviral
and immunomodulatory activities comparable to those of riba-
virin (1). Recent studies revealed that viramidine mainly acts
as a prodrug and is converted to ribavirin by adenosine deami-
nase (Fig. 1) (14). Animal studies indicate that viramidine is
not efficiently taken up by RBCs like ribavirin (5). In contrast,
viramidine has a better liver-targeting property and is enriched
in the liver twice as much as ribavirin (13). Owing to this
favorable property of enrichment in the liver, as well as a
reduced exposure to the risk of hemolysis development, vira-
midine appears to be a safer alternative to ribavirin, which
could potentially provide improved clinical benefits to HCV
patients. Viramidine is currently in phase 3 clinical trials with
pegylated alpha interferon for the treatment of active chronic
HCV infection.
Purine nucleoside phosphorylase has been reported to me-
tabolize ribavirin to triazole nucleobase in vivo as illustrated in
Fig. 1 (7). Conversely, viramidine is not a substrate but an
inhibitor for nucleoside phosphorylase (11). Therefore, we rea-
son that viramidine could potentially prevent ribavirin from
catabolism by inhibiting nucleoside phosphorylase. To investi-
gate this novel concept, a purine nucleoside phosphorylase
from human blood was obtained from Sigma. A radiochemical-
based thin-layer chromatography (TLC) assay was developed
to monitor the conversion of [5-14C]ribavirin (54 mCi/mmol;
Moravek Biochemicals, Brea, Calif.) to [5-14C]triazole nucleo-
base. In the assay, nucleoside phosphorylase (2.5 U/ml) was
added to 10 ␮l of 1ϫ Dulbecco’s phosphate-buffered saline,
pH 7.4, containing various concentration of ribavirin. The as-
say mixture was incubated for 10 min at 30°C and then was
stopped by heating at 90°C for 1 min. The assay mixture was
briefly clarified by centrifugation. Four microliters of the re-
action mixture was applied to a silica gel 60 TLC plate (Selecto
Scientific, Suwanee, Ga.), which was then developed in a sol-
vent system of chloroform-methanol-acetic acid (85:15:5). The
TLC plate was dried and autoradiographed overnight. Prod-
ucts on the TLC plate were analyzed and quantified with a
PhosphorImager. With this assay, we found that nucleoside
phosphorylase indeed catalyzes phosphorolysis of ribavirin as
previously reported (7). However, under similar conditions,
[5-14C]viramidine (56 mCi/mmol; Moravek Biochemicals) was
not hydrolyzed, indicating that viramidine is not a substrate for
purine nucleoside phosphorylase.
Further steady-state kinetic analysis showed that the reac-
tion of ribavirin phosphorolysis was linear for the first 15 min
and it quickly reached equilibrium within half an hour (data
not shown). At equilibrium, approximately 40% of the ribavi-
rin was converted, confirming that the phosphorolysis process
is reversible and nucleoside phosphorylase catalyzes the reac-
tion in both directions. The initial velocity at various concen-
trations of ribavirin (0.2 to 2 mM plus 0.054 ␮Ci of [5-14C]riba-
* Corresponding author. Mailing address: Drug Discovery, R&D,
Valeant Pharmaceuticals International, 3300 Hyland Ave., Costa
Mesa, CA 92626. Phone: (714) 545-0100, ext. 3024. Fax: (714) 668-
3142. E-mail: jwu@valeant.com.
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VOL. 48, 2004
NOTES
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FIG. 1. Schematic diagram depicting viramidine as a prodrug and as a catabolic inhibitor for ribavirin. Ribavirin is subject to either 5Ј
phosphorylation by nucleoside and nucleotide kinases or degradation to nucleobase by purine nucleoside phosphorylase. In addition to functioning
as a prodrug of ribavirin, viramidine could directly inhibit nucleoside phosphorylase and prevent or slow down the catabolism of the newly
converted ribavirin, thereby providing more ribavirin for phosphorylation.
virin) was determined and applied to the Michaelis-Menten
equation with a nonlinear least-squares fit to calculate the kcat
and Km values for the reaction. The Km for ribavirin was
determined to be 0.76 Ϯ 0.08 mM from an average of three
results (Fig. 2A). To calculate kcat, the human blood purine
nucleoside phosphorylase from Sigma was analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and its pu-
rity was estimated at around 60%. With the assumption that
this commercial nucleoside phosphorylase is fully active, the
kcat was then calculated as 33 Ϯ 3 minϪ1. Thus, ribavirin is a
decent substrate for purine nucleoside phosphorylase, with a
catalytic efficiency of 43 minϪ1.mMϪ1 (kcat/Km).
Inhibition of ribavirin phosphorolysis by viramidine was
studied by varying the inhibitor’s concentration from 1 to 25
␮M with the ribavirin concentration fixed at 100 ␮M. Applying
the initial velocities at different inhibitor concentrations to a
Dixon plot yielded a Ki of 2.5 Ϯ 0.1 ␮M for viramidine (Fig.
2B). This is similar to the reported Ki for viramidine when
viramidine was tested against human lymphoblast purine nu-
cleoside phosphorylase with inosine as a substrate (11). In
addition, we investigated viramidine 5Ј-monophosphate
(VMP), a major metabolite of viramidine, as an inhibitor for
nucleoside phosphorylase. Inhibition of VMP against human
blood purine nucleoside phosphorylase was performed by ti-
trating VMP from 10 to 1,250 ␮M against a fixed concentration
of ribavirin (100 ␮M). From a Dixon plot, the Ki for VMP was
calculated to be around 250 ␮M. This result indicates that
VMP inhibits purine nucleoside phosphorylase about 100-fold
weaker than does viramidine. The weak inhibitory activity of
VMP may not be physiologically relevant. From these studies,
we conclude that viramidine is a potent inhibitor for purine
nucleoside phosphorylase and it is capable of preventing riba-
virin phosphorolysis in vitro.
Previous drug action mechanism studies suggest that vira-
midine confers the majority of its antiviral activity through the
prodrug mechanism. Its immunomodulatory activity observed
in peripheral blood mononuclear cells or in vivo animal models
is likely derived from ribavirin that is generated from deami-
nation of viramidine (10). This study further reveals a potential
self-potentiating catabolic inhibition mechanism of viramidine.
Our in vitro data convincingly demonstrated that viramidine
inhibits ribavirin phosphorolysis with good potency. This in
vitro study has significant in vivo implications, considering the
oral delivery route of viramidine through the stomach and
intestines and drug transportation from the plasma to the liver,
in some of which nucleoside phosphorylase is highly expressed
FIG. 2. Kinetic analysis of ribavirin phosphorolysis catalyzed by nucleoside phosphorylase and inhibition of the reaction by viramidine. The
initial velocity of a reaction was determined by a PhosphorImager to integrate the pixel number of the product band on TLC. Kinetic parameters
reported are the average of three sets of results. One representative data set is shown here. (A) Michaelis-Menten curve of ribavirin phosphorolysis
catalyzed by human blood purine nucleoside phosphorylase (2.5 U/ml). The data were used to calculate the Km and kcat values for the reaction.
(B) Dixon plot of inhibition of ribavirin phosphorolysis by viramidine (1 to 25 ␮M). In the experiment, the ribavirin concentration was constant
(100 ␮M). The plot was used to calculate the Ki value for viramidine.

4008
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
(8). The observed in vitro potency of viramidine (Ki ϭ 2.5 ␮M)
is achievable by this delivery route on the basis of pharmaco-
kinetic analysis of animals (3). It is reasonable to assume that
viramidine can accumulate to a level that is sufficient to sup-
press nucleoside phosphorylase activity in vivo. Consistent with
this postulation, a previous study indicated that viramidine is
capable of suppressing nucleoside phosphorylase activity in cell
cultures (11).
may warrant further clinical considerations with a combination
therapy of ribavirin and a nucleoside phosphorylase inhibitor,
such as viramidine, to achieve higher potency and efficacy in
the treatment of chronic HCV infection. This study also pro-
vides a new concept in the design of bifunctional prodrugs.
We acknowledge J. Shim, C.-C. Lin, W. Zhong, D. Smith, R. Tam,
and H. Walker for helpful discussion and suggestions.
Ribavirin undergoes three metabolic pathways in vivo (7).
Two major routes include conversion to active 5Ј-phosphate
derivatives and catabolism to triazole nucleobase (Fig. 1).
Pharmacokinetic analysis of ribavirin administered to animals
indicated that most of the ribavirin is degraded and excreted
from urea. Of the remaining drug that is distributed around
various parts of the animal, a significant amount exists in the
form of triazole nucleobase (3, 4). Inhibiting ribavirin phos-
phorolysis represents a logical strategy to enhance the drug’s
stability, thereby delivering more active metabolites for effi-
cacy. This study demonstrates that viramidine can directly in-
hibit nucleoside phosphorylase, the enzyme that is believed to
be responsible for ribavirin catabolism. Taken together, the
mode of action of viramidine in anti-HCV therapy is likely
bipartite: it serves as a prodrug of ribavirin and concomitantly
as a direct inhibitor for nucleoside phosphorylase to prevent or
slow down the degradation of the newly formed ribavirin.
Like ribavirin, viramidine undergoes 5Ј phosphorylation in
vivo (4). Our in vitro studies show that VMP is only a weak
inhibitor for nucleoside phosphorylase. Thus, the viramidine
effect on the stability of ribavirin is likely transient. The timing
may be right since the stabilization of ribavirin is mostly
needed before its conversion to more stable 5Ј-phosphate de-
rivatives. However, viramidine is different from a conventional
drug metabolism inhibitor that could be included in a drug
formulation to suppress undesirable drug metabolism. Virami-
dine is eventually metabolized to ribavirin or 5Ј-phosphates
without causing long-lasting damage to nucleoside phosphor-
ylase. Since prodrug conversion and drug metabolism are a
dynamic process, further studies are needed to quantify the
extent of the contribution made by viramidine as a catabolic
inhibitor to the stability and potency of ribavirin in vivo. Nev-
ertheless, the proposed dual-action mechanism of viramidine
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