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Antimicrobial Agents and Chemotherapy, May 2000, p. 1276-1283, Vol. 44, No. 5
Immunology1 and
Chemistry2 Laboratories, ICN Research
Department, ICN Pharmaceuticals, Inc., Costa Mesa, California 92626
Received 4 November 1999/Returned for modification 19 January
2000/Accepted 22 February 2000
The demonstrated utility of the nucleoside analog ribavirin in the
treatment of certain viral diseases can be ascribed to its multiple
distinct properties. These properties may vary in relative importance
in differing viral disease conditions and include the direct inhibition
of viral replication, the promotion of T-cell-mediated immune responses
via an enhanced type 1 cytokine response, and a reduction of
circulating alanine aminotransferase (ALT) levels associated with
hepatic injury. Ribavirin also has certain known toxicities, including
the induction of anemia upon chronic administration. To determine if
all these properties are linked, we compared the
D-nucleoside ribavirin to its L-enantiomer (ICN
17261) with regard to these properties. Strong similarities were seen
for these two compounds with respect to induction of type 1 cytokine
bias in vitro, enhancement of type 1 cytokine responses in vivo, and
the reduction of serum ALT levels in a murine hepatitis model. In
contrast, ICN 17261 had no in vitro antiviral activity against a panel
of RNA and DNA viruses, while ribavirin exhibited its characteristic
activity profile. Importantly, the preliminary in vivo toxicology
profile of ICN 17261 is significantly more favorable than that of
ribavirin. Administration of 180 mg of ICN 17261 per kg of body weight
to rats by oral gavage for 4 weeks generated substantial serum levels
of drug but no observable clinical pathology, whereas equivalent doses
of ribavirin induced a significant anemia and leukopenia. Thus,
structural modification of ribavirin can dissociate its
immunomodulatory properties from its antiviral and toxicologic
properties, resulting in a compound (ICN 17261) with interesting
therapeutic potential.
Ribavirin
(1- The therapeutic use of ribavirin is restricted by its toxicology
profile. Prolonged administration of ribavirin is frequently associated
with anemia, whose severity correlates with dose level and which is
reversible upon dose reduction or cessation of treatment. We sought to
identify compounds which would retain those properties deemed critical
for utility in the treatment of chronic HCV infection, but which would
not have the toxicity profile of ribavirin.
We have recently shown (34) that the
L-enantiomer of ribavirin, ICN 17261, has similar
type-1-cytokine-enhancing activity as ribavirin in vitro in activated
human T cells. The objective of this study is to expand on these
initial findings by performing a comparative analysis of ICN 17261 and
ribavirin relative to the aforementioned properties of ribavirin
(direct antiviral activity and cytotoxicity, immunomodulatory effects
in vitro and in vivo, effect on serum ALT, and preliminary toxicology
profile). The results from this study suggest that the bioactive
L-nucleoside ICN 17261 may offer a therapeutic advantage
over ribavirin for the treatment of some viral diseases.
Compounds.
ICN 17261 (1-
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The Ribavirin Analog ICN 17261 Demonstrates Reduced Toxicity and
Antiviral Effects with Retention of both Immunomodulatory Activity and
Reduction of Hepatitis-Induced Serum Alanine Aminotransferase
Levels
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-D-ribofuranosyl-1,2,4-triazole-3-carboxamide)
is a nucleoside analog that has demonstrated efficacy in treating viral disease as monotherapy (respiratory syncytial virus [RSV] [15]) and in combination with alpha interferon
(IFN-
) (hepatitis C virus [HCV] [27, 36]).
Ribavirin has multiple biologic properties that are favorable for
treating viral diseases. It can directly inhibit the replication of
many DNA and RNA viruses (38). More recently, studies have
shown that it can also act as an immunomodulator and thus promote
T-cell-mediated immunity against viral infection (18, 25, 30, 39,
40). The central focus of this effect of ribavirin is the
augmentation of antiviral type 1 cytokine expression (interleukin-2
[IL-2], gamma interferon [IFN-
], and tumor necrosis factor alpha
[TNF-]) and concomitant suppression of type 2 cytokine levels
[IL-4, IL-5, and IL-10] by activated T cells in both human and murine
systems. Finally, ribavirin, alone or in combination with IFN-, can
lower serum alanine aminotransferase (ALT) levels during the course of
treatment of HCV infection (11). Elevated serum ALT levels
are a marker for liver damage and progressive hepatitis, and hence the
ribavirin-mediated lowering of ALT levels is a distinct liver-specific
effect of this nucleoside analog.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-L-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a
new chemical entity and is the L-enantiomer of ribavirin. It is synthesized from
1,2,3,5-tetra-O-acetyl--L-ribofuranose and
methyl 1,2,4-triazole-3-carboxylate (34). ICN 17261 has a
molecular weight of 244.21 and is freely soluble in water. The structures of both ICN 17261 and ribavirin are shown in Fig.
1.
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FIG. 1.
Structures of ICN 17261 and ribavirin.
Animals. Six- to eight-week-old female BALB/c mice were purchased from Bantin and Kingman Universal (Fremont, Calif.).
In vitro studies (human).
Peripheral blood mononuclear cells
were isolated from healthy donors by density gradient centrifugation
followed by T-cell enrichment with Lymphokwik (One Lambda, Canoga Park,
Calif.). Contaminating monocytes were removed by adherence to plastic. Purified T cells comprised >99% CD2+, <1%
HLA-DR+, and <5% CD25+ and were maintained in
RPMI-AP5 (RPMI 1640 medium containing 5% autologous plasma, 1%
L-glutamine, 1% penicillin-streptomycin, and 0.05%
2-mercaptoethanol). For determination of cytokine protein levels, T
cells (106 cells in a volume of 1 ml) were activated by the
addition of either 400 ng of Staphylococcal enterotoxin B (SEB; Sigma,
St. Louis, Mo.) or 10 ng of phorbol myristate acetate (PMA) plus 0.5 µg of ionomycin (ION) (Calbiochem, La Jolla, Calif.) and were incubated in 24-well plates in the presence of 0 to 10 µM ICN 17261 or ribavirin for 48 h at 37°C and 5% CO2 in a
humidified incubator. Cell-free supernatants were taken and analyzed
for human cytokine levels, following appropriate dilution, by using enzyme-linked immunosorbent assay (ELISA) kits specific for IL-2, IFN-, TNF-, IL-4, and IL-5 (Biosource, Camarillo, Calif.). All ELISA results were expressed in picograms per milliliter.
In vitro studies (mouse).
In vitro experiments used lymph
node cells from contact allergen-primed BALB/c mice or spleen cells
from unsensitized BALB/c mice. Priming with contact allergen was
accomplished by application of 20 µl of 0.3% dinitrofluorobenzene
(DNFB) (Sigma) in acetone-olive oil, in the ratio 4:1, onto shaved
abdomens 5 days prior to sacrifice. Primed mice were sacrificed by
cervical dislocation and axillary/lateral axillary lymph nodes were
removed. Lymph node cell (LNC) suspensions were then prepared for
individual mice. Twenty-four-well plates were coated with 150 µl of a
25-µg/ml preparation of anti-mouse /-T cell receptor (TCR)
antibody (clone H57-597; Pharmingen, La Jolla, Calif.) for 90 min at
37°C and then plates were washed twice with cold phosphate-buffered
saline (PBS). LNC (2 × 106/well) were seeded in 1 ml
of complete Dulbecco modified Eagle medium (containing 4.5 g of
dextrose per liter [ICN Biomedicals, Costa Mesa, Calif.] and
supplemented with 10% fetal bovine serum [Hyclone, Logan, Utah], 1%
L-glutamine, 1% penicillin-streptomycin, 10 mM HEPES, 1×
nonessential amino acids, and 50 µM 2-mercaptoethanol) in the
presence of 0 or 2 µM ICN 17261 or ribavirin and cultured for 48 h at 37°C and 10% CO2 in a humidified incubator.
ELISAs from R & D Systems (Minneapolis, Minn.) or from Biosource. All ELISA results
were expressed in picograms per milliliter.
In spleen cell assays, T-cell proliferation was assessed by using a
colorimetric assay (MTT cell proliferation kit I; Boehringer Mannheim,
Indianapolis, Ind.) based on the conversion of the tetrazolium salt MTT
by mitochondrial dehydrogenases to a formazan dye which is readily
detectable by measuring the absorbance at 540 nm.
In vivo assay (contact hypersensitivity). Reactivity to DNFB was determined in BALB/c mice as previously described (41). Briefly, mice were sensitized by the application of 20 µl of 0.3% DNFB in 4:1 acetone-olive oil onto the shaved abdomens of naive mice. For optimal elicitation of contact hypersensitivity (CHS), the mice were challenged on both sides of each ear with 20 µl of 0.12% DNFB, 5 days after sensitization. Unsensitized mice were also challenged and used as controls in each experiment. After 24 h, ear thickness measurements were taken, and response to DNFB was assessed by subtracting postchallenge from prechallenge values. Where indicated, ICN 17261 or ribavirin, at a dose of 10 µg in 50 µl PBS (0.5 mg/kg), was administered by intraperitoneal (i.p.) injection at the time of challenge with DNFB. This dose of ribavirin gave maximal effect in preliminary optimization studies.
In vivo assay (SEB treatment in vivo). SEB was injected i.p. at a dose of 50 µg per mouse at day 0 into three groups of four mice. One group was injected with ICN 17261 and one group was injected with ribavirin, both at 10 µg in 50 µl PBS (0.5 mg/kg) i.p., 1 h prior to SEB injection. Three more groups of four mice were not treated with SEB but were injected with PBS, ICN 17261, and ribavirin, respectively. This dose of ribavirin gave maximal effect in preliminary optimization studies. All mice were anesthetized 24 h later with an appropriate dose of the inhalation anesthetic Penthrane (Abbott Labs, North Chicago, Ill.) and were exsanguinated by cardiac puncture to obtain whole blood. Serum was obtained from clotted blood and was used for determinations of nitric oxide production. Nitric oxide production was evaluated by measuring its stable end products, nitrite and nitrate. Total nitrite and nitrate levels were determined following the reduction of nitrate to nitrite through a nitrate reductase enzyme reaction followed by a colorimetric assay (Sigma) based on the reduction of total nitrite by Griess reagent to a purple azo compound.
In vivo assay (ConA-induced hepatitis).
BALB/c mice (six per
group) were injected intraperitoneally with a single dose of 20 µg (1 mg/kg) of ribavirin or ICN 17261 or with 200 µl of PBS 1 h prior
to intravenous tail vein injection with 0.3 mg of ConA (Calbiochem).
This dose of ribavirin gave maximal effect in preliminary optimization
studies. After 24 h, mice were anesthetized with Penthrane and
were exsanguinated by cardiac puncture to obtain whole blood. Serum was
obtained from clotted blood and was used for determinations of serum
ALT. Serum ALT levels were determined by using an enzyme activity assay
(Sigma). ALT catalyses the transamination of -ketoglutaric acid to
produce glutamic acid by using an amino group donated by the substrate alanine. This assay is based on the colorimetric measurement of the
products (pyruvic acid and glutamic acid).
Antiviral assays. In vitro testing for antiviral activity of ICN 17261 and ribavirin against influenza A and B viruses, parainfluenza viruses 1 and 3, and RSV (Institute of Antiviral Research, Utah State University, Logan, Utah) were performed as previously described (3, 4, 17). Anti-human immunodeficiency virus (HIV) activity was assessed by the National Cancer Institute (Bethesda, Md.) by using a procedure designed to detect agents acting at any stage of the virus reproductive cycle (43). Anti-hepatitis B virus (HBV) activity was monitored by measuring intracellular viral DNA levels following a 7-day culture of the 2.2.15 cell line (an HepG2 hepatoblastome cell line stably transfected by HBV DNA [19]) with and without various concentrations of ribavirin or ICN 17261. Viral DNA was evaluated by slot blot DNA hybridization similar to that described in Marion et al. (24) (Hepadnavirus Testing, Inc., Mountain View, Calif.). Anti-HIV activity and cytotoxicity for ribavirin were determined from previous data (2, 26).
Four-week oral gavage toxicity study.
A 4-week oral gavage
toxicity study was performed by Covance Laboratories Inc. (Madison,
Wis.). Male Crl:CD (SD) IGS BR rats were assigned to four groups (12 males/group in groups 1 to 3 and 15 males in group 4) and were given
the 29-day treatment regimen outlined in Table
1. The doses of ICN 17261, ribavirin
(used as positive control), and water control were given daily by oral gavage at a volume of 10 ml/kg. Food and water were provided ad libitum. Animals were observed twice daily for mortality and
moribundity. Body weight and food consumption data were collected
weekly. Blood samples were collected from all animals for drug level
determinations on days 30 and 60; plasma samples were analyzed by a
liquid chromatography-mass spectrometry methodology for ICN 17261 or
ribavirin (M. Larson, O. Oluyedun, and S. V. Ravavendran, Internal
Report 6937-101, Covance Laboratories, Inc., 1999). Further blood
samples were taken on days 11, 30, and 60 for hematology and clinical
chemistry. On day 30, 8 to 10 animals/group, and on day 60, four
animals/group, were fasted overnight, anesthetized, weighed,
exsanguinated, and necropsied. At necropsy, macroscopic observations
were recorded, and selected organs were weighed, collected, and frozen.
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Statistical analysis. Trend analysis was assessed by using analysis of variance where the main effects evaluated included donor (random effect), concentration, and nucleoside. Statistical significance, where relevant, was assessed by using the Student-Newman-Keuls multiple comparison method.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In vitro type 1 and type 2 cytokine synthesis in activated human T
cells.
Recently, we have shown that ribavirin can enhance
antiviral type 1 cytokines and suppress type 2 cytokine expression in
human T cells (40). Here we compared the influence of ICN
17261 and ribavirin on the cytokine pattern induced by the bacterial
superantigen SEB in human T cells from five normal donors. After
48 h of stimulation, secreted levels of type 1 cytokines IL-2,
TNF-, and IFN- were determined in the cell-free supernatants. Our
data, generated from ELISA analyses, show that ICN 17261, like
ribavirin, in the dose range 0.5 to 5 µM, augmented IL-2, IFN-,
and TNF- (Fig. 2). A significant
concentration-dependent effect was observed for both compounds on
IFN- and TNF- levels (P < 0.0001) but not on
IL-2 levels. Both compounds showed a significantly elevated cytokine
response at 2 and 5 µM for IFN- and TNF- (P < 0.05), with a peak effect at about 2 µM for all cytokines. No
significant differences were seen between nucleoside effects on all
cytokines. ICN 17261 induced a mean peak increase in IL-2, IFN-, and
TNF- of 42, 125, and 72% over activated control levels,
respectively. For ribavirin, the mean peak increase was 66, 84, and
51% over activated control levels, respectively. We were unable to
determine by ELISA whether levels of type 2 cytokines in SEB-stimulated T cells were suppressed by ICN 17261 or ribavirin, as levels of SEB-induced type 2 cytokines were below the level of immunoassay sensitivity.
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, and IFN- and the type 2 cytokines
IL-4 and IL-5 were determined in the cell-free supernatants. In Fig.
3, both ICN 17261 and ribavirin at 2.5 µM similarly augmented IL-2 (mean peak increase of 28 and 49%,
respectively) and TNF- (mean peak increase of 28 and 33%,
respectively) expression in PMA-ION-activated human T cells. A
significant positive concentration-dependent effect was observed for
both compounds on IL-2 and TNF- levels (P < 0.0002
and P < 0.003, respectively) but not IFN-. Both
compounds showed a significantly elevated cytokine response at 2 µM
for IL-2 and TNF- (P < 0.05). The effect on IL-2
and TNF- levels was not significantly different between both
nucleosides. ICN 17261 (mean peak increase of 73%) showed significant
enhancement of IFN- (P < 0.05), whereas ribavirin
did not (2%). This lack of enhancement of IFN- by ribavirin in
PMA-ION-activated human T cells has been observed previously
(40). A significant negative concentration-dependent effect
was observed for both compounds on IL-4 and IL-5 levels (P < 0.002 and P < 0.007, respectively). Both ICN
17261 and ribavirin at 2.5 µM significantly (P < 0.05) suppressed type 2 cytokines IL-4 (mean peak decreases of 38 and 26%, respectively) and IL-5 (mean peak decreases of 60 and 66%, respectively) (Fig. 3). These data show that following both physiologic (SEB) or pharmacologic (PMA-ION) T-cell activation, ribavirin and ICN
17261 have the property of inducing a type 1 cytokine bias in human T
cells.
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In vitro and in vivo effects of ICN 17261 on
type-1-cytokine-mediated immune responses in mice.
We investigated
the comparative effects of ICN 17261 and ribavirin on two
type-1-cytokine-mediated immune responses (1, 45) in vitro
and in vivo in mice. Firstly, we assessed the effect of ICN 17261 and
ribavirin on the in vitro cytokine and in vivo contact hypersensitivity
responses to the contact allergen DNFB. In vitro cytokine secretion in
individual LNC preparations from seven DNFB-primed mice was induced
with plate-bound anti-/-TCR antibody in vitro in the presence or
absence of 2 µM of ICN 17261 or ribavirin. The peak effect on
modulation of type 1 and 2 cytokine levels by ribavirin was previously
shown in mice to be at a concentration of 2 µM (39). It is
noteworthy that in this previous study (39) the enhancement
of contact hypersensitivity by ribavirin in BALB/c mice was dependent
on the suppression of IL-10. After 48 h, levels of type 1 cytokines IL-2 and IFN- and the type 2 cytokine IL-10 were
determined in the cell-free supernatants. In Fig.
4A, in LNC from DNFB-primed mice, both
ICN 17261 and ribavirin at 2 µM augmented IL-2 (mean peak increases
of 69 and 84%, respectively) and significantly (P < 0.05) augmented IFN- (mean peak increases of 27 and 24%,
respectively) while concomitantly and significantly (P < 0.05) suppressing the type 2 cytokine IL-10 (mean peak decreases of 37 and 55%, respectively).
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, IL-2, and splenocyte proliferation, as IFN- is one of the
critical mediators of this type of inflammatory response (1)
and T-cell proliferative responses are a functional effect of
nucleoside-mediated increase in IL-2 production (40). After
48 h, splenocyte proliferation and levels of type 1 cytokines IL-2
and IFN- in the cell-free supernatants were determined. Figure
5A shows the effect of ICN 17261 and
ribavirin in the dose range 0.125 to 10 µM on type 1 cytokine
production in BALB/c splenocytes. A significant positive
concentration-dependent effect was observed for both compounds on IL-2
and IFN- levels and on splenocyte proliferation (P < 0.0001, P < 0.0003, and P < 0.0001,
respectively). The mean peak increase (2 µM, P < 0.05 for both nucleosides) of proliferation and IL-2 and IFN-
levels over activated control for ICN 17261 was 89, 71, and 52%,
respectively, whereas ribavirin caused a mean peak increase of 61, 62, and 75%, respectively. Next, the functional effects of ICN 17261 and
ribavirin on inflammatory responses to SEB in vivo in BALB/c mice were
examined. Animals were treated with a single challenge i.p. with 50 µg of SEB 24 h prior to sacrifice. ICN 17261 or ribavirin (0.5 mg/kg) was given i.p. at the time of challenge. Modulation of
SEB-induced inflammatory responses in SEB-treated mice was determined
from serum levels of the inflammatory mediator, nitric oxide (total
nitrite) and was calculated following subtraction of responses in mice
treated with PBS. Total serum nitrite levels in SEB-treated BALB/c mice were significantly greater (P < 0.0001) than those
treated with PBS, ribavirin alone, or ICN 17261 alone in the absence of
SEB (Fig. 5B). Administration (i.p.) of both ICN 17261 and ribavirin enhanced serum nitrite levels in SEB-treated BALB/c mice (P < 0.0001) (Fig. 5B), giving mean percentage increases (±SD)
following ICN 17261 or ribavirin treatment of 63% ± 30% and 58% ± 20%, respectively, 24 h post-SEB treatment in vivo.
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Anti-inflammatory activities of ICN 17261 in ConA-induced
hepatitis.
Treatment of chronically HCV-infected patients
with ribavirin dramatically reduces serum ALT levels. Recently, a
new murine hepatitis model was developed in which liver-specific
inflammatory lesions are induced by injection of ConA (29).
Significantly, the hepatic injury appears to be a consequence of T-cell
activation (29). Here, using the ConA-induced hepatitis
model, we compared the influence of ICN 17261 and ribavirin on
hepatic-injury-induced serum ALT levels. BALB/c mice were injected i.p.
with either ICN 17261 (1 mg/kg), ribavirin (1 mg/kg), or PBS 1 h prior to intravenous injection of 0.3 mg of ConA. Mice were
exsanguinated 24 h later, and serum ALT levels were determined.
Figure 6 shows that both ICN 17261 and
ribavirin were able to substantially reduce ConA-induced serum ALT
levels from 1,896 U/ml to 969 ± 192 U/ml (49% inhibition) and
954 ± 179 U/ml (50% inhibition), respectively. Administration of
ribavirin or ICN 17261 (1 mg/kg) to normal mice did not affect serum
ALT levels when compared to untreated controls. Neither ribavirin nor
ICN 17261 interfere with the ALT assay, as serum samples from normal or
ConA-treated mice spiked with ribavirin or ICN 17261 showed
indistinguishable ALT concentrations from unspiked serum samples from
the same mice.
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Antiviral and cytotoxicity activities.
The antiviral
activities of ICN 17261 and ribavirin against a variety of viruses were
compared in vitro and are shown in Table 2. ICN 17261 showed no cytotoxicity and
little or no activity in any of the antiviral tests, whereas ribavirin
had the expected profile of antiviral activities and cytotoxicity.
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Preliminary toxicology data. A 4-week oral gavage toxicity study was performed to assess the toxicity of ICN 17261 in rats. The 29-day treatment regimen (Table 1) was followed by a 31-day recovery period in which no test compound was administered. Animals given 180 mg of ribavirin/kg/day had lower food consumption and lower body weights and body weight gains throughout treatment when compared with those of controls. During recovery, there was a trend of higher weight gains and similar food consumption compared to controls. In contrast, there were no apparent differences in body weight, body weight gain, or food consumption noted in animals given ICN 17261.
Administration of ICN 17261 had no effects on clinical pathology results. Administration of 180 mg of ribavirin/kg/day was associated with decreases in erythrocyte count, hemoglobin, hematocrit, leukocyte count (due to lower absolute neutrophil, lymphocyte, and eosinophil counts) (Table 3), and higher mean corpuscular hemoglobin concentration and platelet count. All of the effects of ribavirin were reversed following the 31-day recovery. The anemia and leukopenia caused by ribavirin were moderately severe. For example, the mean hematocrit at day 30 for the ribavirin-treated animals was 26% lower than that for the control animals (32 versus 43%). The mean leukocyte count for the ribavirin-treated animals was 55% lower than that of control animals (3,800 versus 8,400 cells per µl). The effect on leukocyte count was primarily due to lower absolute lymphocyte count (2,800 versus 7,100 cells per µl).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In the present study, we compared ribavirin and its
L-enantiomer, ICN 17261, with respect to key properties
associated with the clinical utility of ribavirin. We found that these
compounds have very similar activity in a variety of assays that
evaluate enhancement of the type 1 cytokine response by activated T
cells. These assays include both human and murine systems and both
pharmacologic and antigen-dependent activation in vitro. It is of note
that peak activity of both ICN 17261 and ribavirin in vitro was at 2 to
5 µM (0.6 to 1.3 µg/ml), a dose range which encompasses the reported steady-state ribavirin concentration range in patients administered ribavirin at 600 to 1,200 mg/day in combination with IFN- (13). Also, both ICN 17261 and ribavirin have
similar activity in two in vivo assays of type 1 cytokine activation, contact hypersensitivity responses to DNFB and in vivo responses to
SEB. Furthermore, both compounds reduced the levels of circulating liver enzymes in a murine inflammatory hepatitis model.
In contrast to these similarities, the two compounds also exhibited marked differences. ICN 17261 was less cytotoxic than ribavirin and was inactive against a range of viruses whose replication is normally inhibited by ribavirin in vitro. Importantly, ICN 17261 did not show apparent toxicity in rats following a 4-week multidose toxicology study. In contrast, ribavirin exhibited multiple toxicities, including the anemia commonly observed in clinical studies. Thus, the studies reported here demonstrate that a compound structurally related to ribavirin can also have multiple biologic properties, some remarkably similar (e.g., type 1 cytokine bias and lowering serum ALT) and others strikingly different (e.g., lack of direct antiviral activity and lack of apparent toxicity) to ribavirin.
The in vivo comparisons of these two enantiomers provide a basis for future work to establish their relative utilities. Drug levels were not assessed as part of the murine efficacy studies, which were short term and used i.p. injection to administer drug; the comparable efficacy of the two compounds in these immunological assays suggests adequate exposure to both compounds, but does not assure similar in vivo potency. Because poor bioavailability of ICN 17261 could explain the absence of toxicity observed in rats treated by gavage with this compound, we assessed the serum levels of ICN 17261 and ribavirin by a liquid chromatography-mass spectrometry methodology (M. Larson, O. Oluyedun, and S. V. Ravavendrun, Internal Report 6937-101, Covance Laboratories, Inc., 1999). The serum levels of ICN 17261 in these rats were proportional to dose and, at the highest dose, not significantly different from the serum levels of ribavirin that showed toxicity. Notwithstanding these encouraging results, the pharmacokinetics of ICN 17261 are of obvious interest and are the subject of ongoing investigations.
The common paradigm for the mechanism of action of antiviral nucleoside analogs involves transport into cells followed by enzymatic phosphorylation, generating nucleoside mono-, di-, and triphosphates. These phosphorylated products act to inhibit various functions critical to viral replication. Differences in enzymatic phosphorylation have been reported for enantiomeric guanosine nucleoside analogs (28). The mechanism(s) for immunomodulation by purine nucleoside analogs may not require phosphorylation (5, 14), and thus differences in phosphorylation may account for at least some of the distinguishing properties we report here for ribavirin and ICN 17261. However, additional work is needed to confirm this hypothesis.
The multiple properties ascribed to ribavirin provide potential utility
for the treatment of a range of viral diseases and make determination
of the optimal use of the compound more complex. Its demonstrable
inhibition of RSV replication in vitro, and the satisfactory clinical
performance of aerosol ribavirin in the treatment of RSV pneumonia in
pediatric patients, led to its adoption for this use. In contrast, the
absence of a clear inhibitory effect on the levels of circulating HCV
reduced interest in the use of ribavirin as monotherapy for this
disease, even though reproducible improvement in ALT levels was seen
(10, 23). The occurrence of anemia in some patients further
complicated its clinical use as monotherapy. However, clinical studies
using ribavirin in combination with IFN- 2b demonstrated major
improvement in the proportion of patients who clear chronic infection
with HCV compared to IFN- 2b alone (9, 27, 32), leading
to widespread adoption of this combination therapy. Thus, it is likely
that the relative importance of each of ribavirin's multiple
activities varies in the treatment of different viral diseases.
The absence of a substantial effect of ribavirin monotherapy on
circulating levels of HCV is consistent with a range of in vitro data.
Although direct assessment of inhibition of replication of HCV is
difficult, evaluation in primary hepatocytes showed ribavirin to be
inactive, whereas IFN- inhibited HCV replication in this system
(6). This result also is consistent with what is known
regarding the effects of ribavirin against HCV molecular targets. For
example, HCV utilizes an internal ribosome entry site element rather
than a 5'-cap structure to initiate translation, so any inhibition by
ribavirin of 5'-cap synthesis is unlikely to affect HCV replication.
Evaluation of the ability of ribavirin nucleotides to inhibit the HCV
helicase has not been reported; however, this enzyme is not selective
with regard to ribonucleoside triphosphates (33), and the
high intracellular concentrations of other ribonucleotides should
compete effectively to minimize inhibition by ribavirin triphosphate. A
more likely target is the HCV-dependent RNA polymerase, but the
available data are not clear on this point. One group has determined
that ribavirin triphosphate did not inhibit this enzyme in vitro (R. Bartenschlager, personal communication), while another has suggested
that high concentrations of ribavirin triphosphate in hepatocytes may
result in some inhibition of the HCV polymerase (16). It is
possible that modest inhibition of the HCV NS5b polymerase results in
an antiviral effect that is only clinically detectable when ribavirin
is combined with IFN-. Evaluation of HCV dynamics in combination
therapy showed no significant change in the viral clearance rate over a
4-week time frame compared to IFN- monotherapy (23); a
second study did observe differences later in therapy (31).
It is not clear that this late effect on viral titer results from
direct antiviral activity rather than immunologic mechanisms.
Also of interest is the fact that the clinical response to ribavirin monotherapy in patients chronically infected with hepatitis B virus (12) closely resembles the response to ribavirin monotherapy in patients chronically infected with HCV. In both diseases, ribavirin treatment suppresses liver damage (as measured by a reduction in levels of circulating ALT) with only minor reductions in the levels of circulating virus. As we report here, ribavirin has no in vitro activity against HBV, an observation that reinforces the conclusion that direct inhibition of viral replication by ribavirin does not play a significant role in its utility in treating viral hepatitis.
In contrast to all these data that indicate a minimal direct antiviral
effect of ribavirin in viral hepatitis, the role of immunomodulation by
ribavirin in chronic HCV infection may be significant. A robust,
multispecific T-cell response is seen in the majority of patients who
clear their HCV infections, either spontaneously or in response to
IFN- treatment (7, 20-22, 35, 37, 42, 44). Ribavirin and
IFN- combination treatment-induced control of viremia is also
associated with the development of HCV-specific T-cell responses with
enhanced IFN- and low IL-10 production (8). Such an
immune response is much less frequent in patients who remain
chronically infected following treatment. The favorable clinical
interaction of ribavirin and IFN- is thus consistent with ribavirin
enhancement of the type 1 cytokine response in stimulated T cells as
reported here and elsewhere (18). The recognition that
ribavirin can enhance the type 1 cytokine response provides a rationale
for combination use with IFN-, even in the absence of a direct
effect of ribavirin on HCV replication.
Thus, the known properties of ribavirin that appear to be of prime importance in the treatment of chronic viral hepatitis are the suppression of circulating ALT consequent to liver damage and the enhancement of a type 1 cytokine T-cell response. These properties are separable from the direct antiviral activity of ribavirin, and a compound possessing these properties (ICN 17261) has significantly reduced toxicity both in vitro and in vivo. ICN 17261 merits additional evaluation as a clinical candidate.
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ACKNOWLEDGMENTS |
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We thank Jace Collins for excellent administrative assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: ICN Research Dept., ICN Pharmaceuticals, Inc., 3300 Hyland Ave., Costa Mesa, CA 92626. Phone: (714) 545-0100, ext. 4109. Fax: (714) 668-3141. E-mail: rctam{at}icnpharm.com.
Present address: Averett Consulting, 26 Trinity, Irvine, CA 92612.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Antimicrobial Agents and Chemotherapy, May 2000, p. 1276-1283, Vol. 44, No. 5
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