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The eco-geno-toxicological impacts of the most widely used antiviral drugs against SARS-CoV2 - ribavirin, ritonavir, nirmatrelvir and tenofovir - were investigated in freshwater organisms. Ribavirin and tenofovir exhibited the highest acute toxicity in the rotifer Brachionus calyciflorus at concentrations of a few mg/L while ritonavir and nirmatrelvir showed similar effects at tens of mg/L; acute toxicity of ribavirin was also observed in the crustacean Ceriodaphnia dubia at similar concentrations. In contrast, the crustacean Thamnocephalus platyurus showed the lowest sensitivity to the antiviral drugs tested with no sublethal effects. Chronic toxicity tests revelead that these antivirals induced effects in consumers at concentrations of environmental concern (ng-µg/L). Ribavirin showed the highest toxicity to the alga Raphidocelis subcapitata, while ritonavir showed the highest toxicity to B. calyciflorus and C. dubia. DNA damage and oxidative stress were observed in C. dubia at 0.001 µg/L and 0.1 µg/L when exposed to ritonavir and nirmatrelvir respectively, and at 1 µg/L when exposed to ribavirin and tenofovir. Toxic and genotoxic environmental risks were assessed with risk quotients for ritonavir, tenofovir and ribavirin exceeding the threshold of 1, indicating significant environmental concern., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Residual antiviral drugs in wastewater may increase the risk of generating transformation products (TPs) during wastewater treatment. Therefore, chlorination behavior and toxicity evolution are essential to understand the secondary ecological risk associated with their TPs. Herein, chlorination kinetics, transformation pathways, and secondary risks of ribavirin (RBV), one of the most commonly used broad-spectrum antivirals, were investigated. The pH-dependent second-order rate constants k increased from 0.18 M -1 ·s -1 (pH 5.8) to 1.53 M -1 ·s -1 (pH 8.0) due to neutral RBV and ClO - as dominant species. 12 TPs were identified using high-resolution mass spectrometry in a nontargeted approach, of which 6 TPs were reported for the first time, and their chlorination pathways were elucidated. The luminescence inhibition rate of Vibrio fischeri exposed to chlorinated RBV solution was positively correlated with initial free active chlorine, probably due to the accumulation of toxic TPs. Quantitative structure-activity relationship prediction identified 7 TPs with elevated toxicity, concentrating on developmental toxicity and bioconcentration factors, which explained the increased toxicity of chlorinated RBV. Overall, this study highlights the urgent need to minimize the discharge of toxic chlorinated TPs into aquatic environments and contributes to environmental risk control in future pandemics and regions with high consumption of antivirals., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The rapid spread of coronavirus disease 2019 has increased the consumption of some antiviral drugs, wherein these are discharged into wastewater, posing risks to the ecosystem and human health. Therefore, efforts are being made for the development of advanced oxidation processes (AOPs) to remediate water containing these pharmaceuticals. Here, the toxicity evolution of the antiviral drug ribavirin (RBV) was systematically investigated during its degradation via the UV/TiO 2 /H 2 O 2 advanced oxidation process. Under optimal conditions, RBV was almost completely eliminated within 20 min, although the mineralization rate was inadequate. Zebrafish embryo testing revealed that the ecotoxicity of the treated RBV solutions increased at some stages and decreased as the reaction time increased, which may be attributed to the formation and decomposition of various transformation products (TPs). Liquid chromatography-mass spectrometry analysis along with density functional theory calculations helped identify possible toxicity increase-causing TPs, and quantitative structure activity relationship prediction revealed that most TPs exhibit higher toxicity than the parent compound. The findings of this study suggest that, in addition to the removal rate of organics, the potential ecotoxicity of treated effluents should also be considered when AOPs are applied in wastewater treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ribavirin is an antiviral drug showing high and delayed toxicity to the destructive agricultural pest Spodoptera litura . Larvae fed with artificial diets containing ribavirin could not molt successfully and showed abnormal phenotypes, including cuticle melanization and heavy wrinkle of the newly formed procuticle. RNA-Seq analysis suggested that ribavirin has great negative influence on cuticle. Quantitative real-time-polymerase chain reaction results indicated that ribavirin treatment decreased the expression of key genes in juvenile hormone (JH) biosynthesis ( CYP15C1 and JH acid methyltransferase) and most cuticle protein genes, whereas the genes in melanin biosynthesis and bursicon genes were upregulated by ribavirin treatment. These results coincided with the decreased titer of JH I, JH II, and JH III determined by enzyme-linked immunosorbent assay, the much thinner procuticle layer exhibited by histopathological examination, and the cuticle melanization after ribavirin treatment. These results provided a valuable theoretical basis for the creation of green insecticides targeting JH and the development of new insecticide derivatives from 1,2,4-triazole.

Direct-acting antivirals have revolutionized the treatment of chronic hepatitis C. Sofosbuvir and simeprevir are prescribed worldwide. However, there is a scarcity of information regarding their genotoxicity. Therefore, the present study assessed the cytotoxic and genotoxic effects of sofosbuvir and simeprevir, alone and combined with ribavirin. HepG2 cells were analyzed using the in vitro cytokinesis-block micronucleus cytome assay. Cells were treated for 24 h with sofosbuvir (0.011-1.511 mM), simeprevir (0.156-5.0 µM), and their combinations with ribavirin (0.250-4.0 mM). No significant differences were observed in the nuclear division cytotoxicity index, reflecting the absence of cytotoxic effects associated to sofosbuvir. However, the highest concentration of simeprevir showed a significant difference for the nuclear division cytotoxicity index. Moreover, significant results were observed for nuclear division cytotoxicity index in two combinations of sofosbuvir plus ribavirin and only in the highest combination of simeprevir plus ribavirin. Additionally, our results showed that sofosbuvir did not increase the frequency of chromosomal damage, but simeprevir significantly increased the frequency of micronuclei at the highest concentrations. The combination index demonstrated that both sofosbuvir and simeprevir produced antagonism to the genotoxic effects of ribavirin. In conclusion, our results showed that simeprevir, but not sofosbuvir, has genotoxic effects in HepG2 cells.

Ribavirin has been proven to be an antiviral treatment, whereas there are still risks of hemolysis and congenital malformation. Abnormal cardiac development contributes to the occurrence and development of many heart diseases. However, there is so far no evidence that ribavirin induces human cardiac developmental toxicity. Herein, we employed the cardiac differentiation model of human induced pluripotent stem cells (hiPSCs) to determine the impact of ribavirin on heart development. Our data showed that ribavirin at clinically high concentrations (5 and 10 μM) significantly inhibited the proliferation and differentiation of hiPSCs from mesoderm to cardiac progenitor cells and cardiac progenitor cells to cardiomyocytes, but not from pluripotent status to mesoderm. Meanwhile, DCFH-DA staining revealed that ribavirin could increase ROS content in the mid-phase of differentiation. In addition, ribavirin treatment (1, 5 and 10 μM) remarkably caused DNA damage which was shown by the increase of γH2AX-positive cells and upregulation of the p53 during the differentiation of hiPSCs from mesoderm to cardiac progenitor cells. Moreover, exposuring to ribavirin (5 and 10 μM) markedly upregulated the expression of lncRNAs Gas5 in both mid-phase and late phase of differentiation and HBL1 in the mid-phase. In conclusion, our results suggest that ribavirin is detrimental in cardiac differentiation of hiPSCs, which may be associated with DNA damage, upregulated p53 and increased Gas5. It may provide the evidence for the rational clinical application of ribavirin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

SRO-91 is a non-natural ribofuranosyl-1,2,3-triazole C-nucleoside obtained by a synthetic sequence involving a C-alkynyl glycosylation mediated by metallic indium and a Huisgen cycloaddition for the construction of the triazole. Its structure is close to the one of ribavirin, a drug presenting a broad-spectrum against viral infections. SRO-91 antitumor activities were investigated on 9 strains of tumor cells and IC 50 of the order of 1 μM were obtained on A431 epidermoid carcinoma cells and B16F10 skin melanoma cells. In addition, studies of ovarian tumor cell inhibitions show an interesting activity in regard to the need for new drugs for this pathology. Finally, cytotoxicity and mouse toxicity studies reveal a favorable therapeutic index for SRO-91., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

The prevalence of hepatitis C virus/tuberculosis (HCV/TB) coinfection has not been estimated globally but few studies highlight the risk of hepatotoxicity following TB treatment or HCV treatment. Previously reported data highlights the risk of drug-induced hepatotoxicity associated with three of the first-line anti-TB agents: rifampin, isoniazid, and pyrazinamide specifically in patients coinfected with HIV and HCV. Thus far, direct-acting antiviral (DAA) drug-induced hepatotoxicity has not been reported in the literature but herein, we observed an unusual case of HCV virological breakthrough and hepatoxicity during treatment with DAA drugs in a patient who has previously been successfully treated for TB., (© 2019 Wiley Periodicals, Inc.)

Ribavirin is an important component of the treatment for hepatitis C virus (HCV) infection and, in combination with the new direct-acting antiviral (DAA) agents, comprises the major current therapeutic regimens. This study evaluated the cytotoxicity and chromosomal instability induced by ribavirin using the in vitro cytokinesis-block micronucleus cytome (CBMN-Cyt) assay in two cell lines with different expression levels of drug-metabolizing enzymes: human hepatocellular carcinoma cells (HepG2) and Chinese hamster ovary (CHO-K1) cells. HepG2 cells were treated with nine concentrations (from 15.3 μg/ml to 3.9 mg/ml) and CHO-K1 cells were exposed to eight concentrations (from 15.3 μg/ml to 1.9 mg/ml) of ribavirin for 24 h. Ribavirin inhibited cell proliferation in both cell lines, but at different concentrations: 3.9 mg/ml in HepG2 and 244.2 μg/ml in CHO-K1 cells. No significant differences were observed regarding aspects of cell death in HepG2 and CHO-K1 cells, reflecting the absence of cytotoxic effects associated to ribavirin. Ribavirin did not increase the frequency of nucleoplasmic bridges (NPBs) and nuclear bud (NBUD). However, when compared to the negative control, a significant increase in micronuclei (MNi) frequency was observed in both cell lines. However, chromosomal instability was induced by higher concentrations of ribavirin in HepG2 cells (from 61.1 to 976.8 μg/ml), compared with CHO-K1 cells (15.3 and 30.5 μg/ml). These results demonstrate the potential of ribavirin to promote chromosomal instability, and suggest that cells with different expressions of drug-metabolizing enzymes show different susceptibility to ribavirin effects.

Ribavirin has been found to enhance the anti HCV potential of pegylated interferon and sovaldi. However its use was associated with impact on the hemopoietic system and iron status. The hemopoietic toxicity, sometimes forced patients to reduce the dosage or to discontinue treatment in rare occasions. The main purpose of the present study was to assess the potential of black seed oil, a known potent antioxidant, to ameliorate the negative impact of ribavirin on hematological indices, iron status and natural immunity in rats. Twenty four female albino rats were equally divided into four groups as follows: Group1, served as negative control and received an oral dose of (0.5 ml) saline. Group 2, served as positive control and were given 30 mg/kg/day doses of Ribavirin for / 5 days/ week for 4 weeks. Group 3, which were orally fed a 1ml/kg/ of black seed oil for 5 day/week for 4 weeks. Group 4, which were orally given the above mentioned doses of black seed oil plus ribavirin / 5 days/ week for 4 weeks. The results demonstrated that treatment with ribavirin induced significant decrease in absolute neutrophils count, RBCs count, haemoglobin concentrations (Hb), Packed Cell Volume percentage (PCV%) and liver total iron binding capacity (TIBC). On the other hand, it induced significant increases in serum and liver iron, liver ferritin, transferring saturation % compared to control group. Black seed oil treatment increase absolute neutrophil count and restored RBCs count, Hb concentration and PCV percentage to normal control level. It also significantly reduced serum iron and liver total iron in animals treated ribavirin. Black seed oil supplementation also enhanced serum IgM and IgG concentrations in ribavirin treated rats. In conclusion, black seed oil had the potential to ameliorate the negative effect of ribavirin on the hemopoietic system offering better chances for completion of HCV treatment course with full dose. It also has proven to have the ability to reduce serum and liver iron load and enhance natural IgM and IgG levels.

Introduction: Significant teratogenic effects have been demonstrated in all animal species exposed to ribavirin. Ribavirin is prescribed for chronic hepatitis C and is contraindicated in women who are pregnant and in the male sexual partners of women who are pregnant. Both sexes are advised to avoid pregnancy for 6 months after exposure. The Ribavirin Pregnancy Registry was established in 2003 to monitor pregnancy exposures to ribavirin for signals of possible human teratogenicity., Methods: This voluntary registry enrolls pregnant women with prenatal exposure to ribavirin. Exposure is classified as direct-women taking ribavirin during pregnancy or the 6 months prior to conception-or indirect-women exposed through sexual contact, 6 months prior to or during pregnancy, with a man who is taking or has taken ribavirin in the past 6 months. Women are followed until delivery and infants for 1 year. When enrollment is complete, birth defect rates will be compared with the Metropolitan Atlanta Congenital Defects Program's published rate of 2.67. Using data collected since inception in 2003 through February 2016, preliminary rates were calculated., Results: The registry has enrolled 272 pregnant women, with 180 live births: there were seven birth defect cases among 85 directly exposed women [7/85 (8.2%) (95% confidence interval (CI) 3.4-16.2)] and four birth defect cases among 95 indirectly exposed women [4/95 (4.2%) (95% CI 1.2-10.4)]. Of the 11 infants, nine had structural defects and two had chromosomal anomalies. Patterns suggesting a common etiology or relationship with ribavirin exposure are not seen., Conclusion: Based on the patterns of birth defects reported, preliminary findings do not suggest a clear signal of human teratogenicity for ribavirin. However, the current sample size is insufficient for definitive conclusions, and ribavirin exposure should be avoided during pregnancy and during the 6 months prior to pregnancy, in accordance with prescribing information., Clinical Trial Registration: ClinicalTrials.gov identifier: NCT00114712.

Effects of ribavirin on the structure of peritubular sheath (PS) of seminiferous tubules and on testicular functions were studied. We found that ribavirin at a dose of 4 mg/kg/day for 4 weeks produced a significant reduction in testosterone level (6.3 ± 0.2; P < 0.001) and in spermatogenic score count (3.8 ± 0.2; P < 0.001) compared to control values. The thickness of PS (17.8 ± 1.13) and tubular lumen perimeter (1024.7 ± 67) was significantly increased compared to controls (10.7 ± 0.70; P < 0.001 and 808 ± 25; P = 0.004, respectively). The length of germinal epithelium (411.8 ± 39) and tubular external diameters (1661.8 ± 115) was significantly reduced compared to control values (708.4 ± 40; P < 0.001 and 2358.8 ± 169; P < 0.001, respectively). The basement membranes (BMs) were thickened with great deposition of collagen. Myoid cells showed altered structure and extracellular matrix revealed disorganization by excessive collagen I and IV accumulation. Testicular damage was established histologically. Evidence of apoptosis was detected in germ cells. There was a significant increase in integrated density of Casp-3 expression (38,121,743 ± 1,763,420; P < 0.001) in seminiferous tubules compared to control (24,788,409 ± 1,900,140). It is concluded that ribavirin can cause alterations of the testicular function and structure with increased apoptosis in the tissues after 4 weeks of administration. The damaging effect could be persuaded by destruction of the peritubular sheath.

Moroxydine hydrochloride (Mor) is known to have multi-antiviral activities against DNA and RNA viruses but very little information exists on its pharmacology. The paper was undertaken to explore the antiviral response and antiapoptotic mechanism of Mor against grass carp reovirus (GCRV) in Ctenopharyngodon idella kidney (CIK) cells. The results showed that exposing GCRV-infected cell to 6.3 μg mL(-1) of Mor for 96 h avoid ca. 50% apoptosis. Meanwhile, Mor had lower cytotoxicity than ribavirin (Rib) as the value of safe concentration was threefold higher than effective concentration and the compound could ensure sufficient into and out of cells within 4 h when tested at the maximal safe concentration. Mor blocked the GCRV-induced cytopathic effects and eliminated nucleocapsids in CIK cells to keep the normal morphological structure. Moreover, the expressions of viral protein genes were significantly inhibited especially the guanylyl transferase and RNA-dependent RNA polymerase related expression. Furthermore, GCRV caused Bcl-2 down-regulation and Bax mitochondrial translocation was prevented by treatment of CIK cells with Mor. The downstream effector, caspase activity was also significantly inhibited in Mor treated cells. The potential mechanism might be that mitochondrial apoptotic signals were not activated by the intervention of Mor for targeting viral gene expression. Taken together, Mor showed high anti-GCRV activity and had been proved as a secure and promising agent in viral controlling in aquaculture industry., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Macromolecular prodrugs (MPs) are a powerful tool to alleviate side-effects and improve the efficacy of the broad-spectrum antiviral agent ribavirin. In this work, we sought an understanding of what makes an optimal formulation within the macromolecular parameter space--nature of the polymer carrier, average molar mass, drug loading, or a good combination thereof. A panel of MPs based on biocompatible synthetic vinylic and (meth)acrylic polymers was tested in an anti-inflammatory assay with relevance to alleviating inflammation in the liver during hepatitis C infection. Pristine polymer carriers proved to have a pronounced anti-inflammatory activity, a notion which may prove significant in developing MPs for antiviral and anticancer treatments. With conjugated ribavirin, MPs revealed enhanced activity but also higher toxicity. Therapeutic windows and therapeutic indices were determined and discussed to reveal the most potent formulation and those with optimized safety. Polymers were also tested as inhibitors of replication of the hepatitis C viral RNA using a subgenomic viral replicon system. For the first time, negatively charged polymers are revealed to have an intracellular activity against hepatitis C virus replication. Concerted activity of the polymer and ribavirin afforded MPs which significantly increased the therapeutic index of ribavirin-based treatment. Taken together, the systematic investigation of the macromolecular space identified lead candidates with high efficacy and concurrent direct activity against the hepatitis C virus and inflammation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Mice lacking the type I interferon receptor (IFNAR-/- mice) reproduce relevant aspects of Crimean-Congo hemorrhagic fever (CCHF) in humans, including liver damage. We aimed at characterizing the liver pathology in CCHF virus-infected IFNAR-/- mice by immunohistochemistry and employed the model to evaluate the antiviral efficacy of ribavirin, arbidol, and T-705 against CCHF virus., Methodology/principal Findings: CCHF virus-infected IFNAR-/- mice died 2-6 days post infection with elevated aminotransferase levels and high virus titers in blood and organs. Main pathological alteration was acute hepatitis with extensive bridging necrosis, reactive hepatocyte proliferation, and mild to moderate inflammatory response with monocyte/macrophage activation. Virus-infected and apoptotic hepatocytes clustered in the necrotic areas. Ribavirin, arbidol, and T-705 suppressed virus replication in vitro by ≥3 log units (IC50 0.6-2.8 µg/ml; IC90 1.2-4.7 µg/ml). Ribavirin [100 mg/(kg×d)] did not increase the survival rate of IFNAR-/- mice, but prolonged the time to death (p<0.001) and reduced the aminotransferase levels and the virus titers. Arbidol [150 mg/(kg×d)] had no efficacy in vivo. Animals treated with T-705 at 1 h [15, 30, and 300 mg/(kg×d)] or up to 2 days [300 mg/(kg×d)] post infection survived, showed no signs of disease, and had no virus in blood and organs. Co-administration of ribavirin and T-705 yielded beneficial rather than adverse effects., Conclusions/significance: Activated hepatic macrophages and monocyte-derived cells may play a role in the proinflammatory cytokine response in CCHF. Clustering of infected hepatocytes in necrotic areas without marked inflammation suggests viral cytopathic effects. T-705 is highly potent against CCHF virus in vitro and in vivo. Its in vivo efficacy exceeds that of the current standard drug for treatment of CCHF, ribavirin.

Ribavirin (1-β-d-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a widely used broad-spectrum antiviral drug. Recently, several reports revealed genotoxic effects of ribavirin in vivo and in vitro, which were correlated with the production of reactive oxygen species (ROS). This study aimed to evaluate the genotoxicity of ribavirin and to investigate the role of the natural antioxidant silymarin to modulate this genotoxicity. Male albino mice (age, 8-10 weeks) were injected intraperitoneally (i.p.) with ribavirin at three dose levels (20, 75 and 130 mg/kg bw) either in a single injection (acute treatment) or in multiple injections on five consecutive days (sub-acute treatment). Other comparable groups were treated with silymarin (70 mg/kg bw) 1h before the injection with ribavirin. Mice were sacrificed at different sampling times (24, 48 and 72 h) after the last ribavirin treatment. Micronucleus (MN) and single-strand conformation polymorphism (SSCP) assays were used to assess genotoxic and cytotoxic effects of ribavirin and to evaluate the protective effect of the pre-treatment with silymarin. Our results reveal genotoxic and cytotoxic effects of ribavirin in the MN assay. Pre-treatment with silymarin reduced the toxicity of ribavirin. In the SSCP assay, ribavirin treatment did not induce any mutations in the two selected sites in the D-loop of mitochondrial DNA (mtDNA)., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Newcastle Disease Virus (NDV) causes a serious infectious disease in birds that results in severe losses in the worldwide poultry industry. Despite vaccination, NDV outbreaks have increased the necessity of alternative prevention and control measures. Several recent studies focused on antiviral compounds obtained from natural resources. Many extracts from marine organisms have been isolated and tested for pharmacological purposes, and their antiviral activity has been demonstrated in vitro and in vivo. Fucoidan is a sulfated polysaccharide present in the cell wall matrix of brown algae that has been demonstrated to inhibit certain enveloped viruses with low toxicity. This study evaluated the potential antiviral activity and the mechanism of action of fucoidan from Cladosiphon okamuranus against NDV in the Vero cell line., Methods: The cytotoxicity of fucoidan was determined by the MTT assay. To study its antiviral activity, fusion and plaque-forming unit (PFU) inhibition assays were conducted. The mechanism of action was determined by time of addition, fusion inhibition, and penetration assays. The NDV vaccine strain (La Sota) was used in the fusion inhibition assays. PFU and Western blot experiments were performed using a wild-type lentogenic NDV strain., Results: Fucoidan exhibited antiviral activity against NDV La Sota, with an obtained IS50 >2000. In time of addition studies, we observed viral inhibition in the early stages of infection (0-60 min post-infection). The inhibition of viral penetration experiments with a wild-type NDV strain supported this result, as these experiments demonstrated a 48% decrease in viral infection as well as reduced HN protein expression. Ribavirin, which was used as an antiviral control, exhibited lower antiviral activity than fucoidan and high toxicity at active doses. In the fusion assays, the number of syncytia was significantly reduced (70% inhibition) when fucoidan was added before cleavage of the fusion protein, perhaps indicating a specific interaction between fucoidan and the F0 protein., Conclusion: The results of this study suggest that fucoidan from C. okamuranus represents a potential low-toxicity antiviral compound for the poultry industry, and our findings provide a better understanding of the mode of action of sulfated polysaccharides.

The ability to extinguish a viral population of fixed reproductive capacity by causing small changes in the mutation rate is referred to as lethal mutagenesis and is a corollary of population genetics theory. Here we show that coxsackievirus B3 (CVB3) exhibits reduced mutational robustness relative to poliovirus, manifesting in enhanced sensitivity of CVB3 to lethal mutagens that is dependent on the size of the viral population. We suggest that mutational robustness may be a useful measure of the sensitivity of a virus to lethal mutagenesis.

Background & Aims: Genetic variation of inosine triphosphatase (ITPA) causing an accumulation of inosine triphosphate (ITP) has been shown to protect patients against ribavirin (RBV)-induced anemia during treatment for chronic hepatitis C infection by genome-wide association study (GWAS). However, the biologic mechanism by which this occurs is unknown., Methods: We examined whether ITP can be used by adenosine triphosphatase (ATPase) in human erythrocytes or recombinant human adenylosuccinate synthase (ADSS). RBV-induced adenosine triphosphate (ATP) reduction in erythrocytes was compared with the genetically determined low or normal activity of ITPA, leading respectively to high or normal ITP levels., Results: Although ITP is not used directly by human erythrocyte ATPase, it can be used for ATP biosynthesis via ADSS in place of guanosine triphosphate (GTP). With RBV challenge, erythrocyte ATP reduction was more severe in the wild-type ITPA genotype than in the hemolysis protective ITPA genotype. This difference also remains after inhibiting adenosine uptake using nitrobenzylmercaptopurine riboside (NBMPR). Interestingly, the alleviation of ATP reduction by the hemolysis protective ITPA genotype was canceled by the ADSS inhibitor 6-mercaptoethanol (6-MP)., Conclusions: ITP confers protection against RBV-induced ATP reduction by substituting for erythrocyte GTP, which is depleted by RBV, in the biosynthesis of ATP. Because patients with excess ITP appear largely protected against anemia, these results confirm that RBV-induced anemia is due primarily to the effect of the drug on GTP and consequently ATP levels in erythrocytes., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objectives: To study the association between trough ribavirin concentration (C(trough)) with sustained virological response (SVR) and haemoglobin (Hb) decrease in HIV/hepatitis C virus (HCV)-co-infected (HIV+/HCV+) patients treated with anti-HCV therapy., Methods: HIV+/HCV+ patients treated with ribavirin and pegylated interferon were prospectively evaluated. Qualitative and quantitative HCV-RNA, Hb levels and ribavirin C(trough) were measured at baseline and weeks 2, 4, 12, 24, 36 and 48 during therapy. HCV-RNA was also measured at 24 weeks after the end of therapy. Efficacy analysis was performed on patients with a definitive virological outcome (SVR, relapser and non-responder), whereas for toxicity analysis, dropouts were considered until the last available observation., Results: Fifty-two patients (54.7% with genotype 1 or 4) were included. Overall, no correlation between ribavirin C(trough) and early virological response (EVR) nor SVR was found. However, in patients with genotype 1 or 4, ribavirin C(trough) was independently associated with EVR (P = 0.036) and SVR (P = 0.046). A ribavirin C(trough) cut-off of 1600 ng/mL was found to be associated with both EVR (chi(2) = 5.69, P = 0.028) and SVR (chi(2)=4.2, P = 0.04). Higher ribavirin C(trough) correlated with Hb decrease (R = -0.361, P = 0.009) and was independently associated with an Hb decrease of >4 g/dL (P = 0.009). Receiver operating characteristic (ROC) analysis indicated that a ribavirin C(trough) of >2300 ng/mL was associated with an Hb decrease of >4 g/dL (chi(2) = 8.08, P = 0.01)., Conclusions: Our study confirmed a relationship between ribavirin exposure and both efficacy and toxicity. Moreover, we found ribavirin C(trough) cut-offs for both SVR in genotypes 1 and 4 and overall haematological toxicity. These findings deserve further clinical evaluation.

Chronic hepatitis C (CHC) and end-stage liver disease are becoming an increasingly common cause of mortality in patients with congenital bleeding disorders, especially in the HIV-coinfected group. Combination of pegylated interferon (Peg-IFN) and ribavirin has recently become the treatment of choice for CHC. In this study, we evaluated the safety and efficacy of combination therapy with Peg-IFN plus ribavirin for the treatment of CHC in human immunodeficiency virus (HIV)- and HIV+ patients with congenital bleeding disorders. Between 2000 and 2004, 50 (18-68 years old) patients with CHC (19 HIV+) from two hemophilia centers were included in the study. They were treated with weekly subcutaneous administration of Peg-INF-alpha combined with 800-1,200 mg ribavirin daily, for 24-48 weeks depending on viral genotype. Response was evaluated at weeks 12, 24, 48 (end of treatment response) and 72 had sustained virological response). Overall, 22/50 patients (43.8%) had end of treatment response and 20/50 (40%) sustained virological response. HIV- patients responded similarly to the general population (58.1%), while HIV+ patients had very low response rates (10.5%). The high rate of discontinuation (36.9%) as a result of side effects contributed to the observed low response rate in the HIV+ group. The only factor strongly associated with sustained virological response in the HIV- patients was the reduction of HCV RNA at 12 weeks (p = 0.001). Patients with viral genotypes other than 1 had higher SVR rates, but this was not found to be statistically significant. Peg-INF plus ribavirin is safe for the treatment of CHC monoinfected patients with inherited bleeding disorders, with similar response rates to nonhemophiliacs. On the contrary, in HIV coinfected hemophilic patients under highly active antiretroviral therapy it is associated with severe toxicity and very poor sustained virological response rates. Careful evaluation and several considerations are needed before starting treatment in this population.

The ability to control plant viral diseases with chemicals has great potential value for agriculture, but few chemicals are available to date due to the difficulty in obtaining effective drugs. IMP dehydrogenase is an enzyme which catalyzes the conversion of inosine 5'-monophosphate to xanthosine 5'-monophosphate in the de novo purine nucleotide synthetic pathway, and is considered a sensitive target for antiviral drugs. Two IMPDH inhibitors, tiazofurin (TR) and mycophenolic acid (MPA), were tested for their inhibitory effect on Grapevine leafroll-associated virus 3 (GLRaV-3) in in vitro grapevine explants. TR administration produced plantlets characterized by negative ELISA readings. No PCR products were obtained from these samples. This was confirmed by the absence of viral particles. MPA was essentially ineffective against GLRaV-3 replication in Sangiovese explants. This is the first report of GLRaV-3 eradication in grapevine explants following TR administration.

The antiviral activity versus flaviviruses of artemisinin, a safe drug obtained from Artemisia annua and commonly used to treat malaria, has been investigated using as an IN VITRO model bovine epithelial cells from embryonic trachea (EBTr) infected with the cytopathic strain Oregon C24V, of bovine viral diarrhoea virus (BVDV), which is a member of the Flaviviridae family. Antiviral activity was estimated by the degree of protection against the cytopathic effect of BVDV on host cells and by the reduction in BVDV-RNA release to the culture medium. To induce an intermediate cytopathic effect in non-treated cells, EBTr cells were first exposed to BVDV for 48 h and then incubated with virus-free medium for 72 h. Ribavirin and artemisinin (up to 100 microM) induced no toxicity in host cells, whereas a slight degree of toxicity was observed for IFN-alpha at concentrations above 10 U/mL up to 100 U/mL. Treatment of infected cells with IFN-alpha, ribavirin and artemisinin markedly reduced BVDV-induced cell death. A combination of these drugs resulted in an additive protective effect. These drugs induced a significant reduction in the production/release of BVDV virions by infected EBTr cells; there was also an additive effect when combinations of them were assayed. These results suggest a potential usefulness of artemisinin in combination with current pharmacological therapy for the treatment of human and veterinary infections by flaviviruses.

Ribavirin, an N-glycosyl nucleoside (1-beta-D-ribofuranosyl-1, 2, 4-triazole-3 carboxamide), is a synthetic purine nucleoside analogue with a broad spectrum of antiviral activity, however, its high toxicity poses a major disadvantage of its use as a therapeutic. Various studies have shown that vitamin B12 plays a significant role in maintaining the stability of the human genome. We therefore investigated the potential beneficial effect of vitamin B12 in reducing ribavirin-induced genotoxicity. To test this, we used the cytokinesis-block micronucleus (CBMN) assay. Human blood cells were treated in vitro with increasing doses of ribavirin (0.05, 0.17, 0.32, 0.47 and 0.65 micromol/ml) for three different periods of time (2, 4 and 17 hrs). Duplicate cultures were supplemented with 50 mul of vitamin B12 during the drug treatment (final concentration of 13.5 microg/ml). Micronuclei formation and cell proliferation potential were then scored in both sets of samples and the corresponding controls. The results showed that supplementation with vitamin B12 lowered the frequency of micronuclei (Z = 2.02, p < 0.04) and recovered the proliferation potential of the treated cells for each treatment period, except for the conditions with the highest concentration of ribavirin and the shortest time. These observations underscore the unique beneficial effects of vitamin B12 in reducing genotoxicity, particularly by recovering the proliferation potential of treated cells, as demonstrated by the decrease in mononucleated cells and enhancement of binucleated and polynucleated cells. The mechanism by which vitamin B12 reduces ribavirin-induced genotoxicity is related to de novo synthesis of nucleotides, and is worthy of further investigation.

Ribavirin has been reported to cause error-prone replication and viral extinction in RNA viruses. The antiviral activity of ribavirin against West Nile virus (WNV) was evaluated in various cell lines to select a model in which mutagenic effects could be studied. The antiviral activity was greatest in HeLa cells as compared to CV-1, L929, Vero, or MA-104 cells. WNV was also passaged sequentially in cell monolayers treated with ribavirin to determine whether cumulative mutations could lead to viral extinction in these cell lines. The virus was abrogated in HeLa cells after 4 passages, while high viral titers persisted after many passages in other cells. A molecular clone of WNV was propagated in HeLa cells treated with 15 microg/mL ribavirin, and sequencing of viral genome segments revealed significant increases in transition mutations, demonstrating that ribavirin induced error-prone replication. The relative infectivity of viral RNA synthesized in the presence of ribavirin was shown to be reduced compared with untreated controls. These data support the hypothesis that error catastrophe is one of the modes of action for ribavirin against WNV.

Interferon (IFN)-alpha monotherapy, as well as the more effective combination therapy of IFN-alpha and ribavirin, are currently used for patients with chronic hepatitis C caused by hepatitis C virus (HCV) infection, although the mechanisms of the antiviral effects of these reagents on HCV remain ambiguous, and side effects such as anemia due to the administration of ribavirin present a problem for patients who are advanced in years. Using a recently developed reporter assay system in which genome-length dicistronic HCV RNA encoding Renilla luciferase gene was found to replicate efficiently, we found that mizoribine, an imidazole nucleoside, inhibited HCV RNA replication. The anti-HCV activity of mizoribine (IC50: approximately 100 microM) was similar to that of ribavirin. Using this genome-length HCV RNA replication monitor system, we were the first to demonstrate that the combination of IFN-alpha and ribavirin exhibited more effective anti-HCV activity than the use of IFN-alpha alone. Moreover, we found that the anti-HCV activity of mizoribine in co-treatment with IFN-alpha was at least equivalent to that of ribavirin. This effect was apparent in the presence of at least 5 microM mizoribine. Since mizoribine is currently used in several clinical applications and has not been associated with severe side effects, mizoribine is considered to be of potential use as a new anti-HCV reagent in combination with IFN-alpha.

The present study was planned to evaluate the toxic effects of ribavirin on the reproductive parameters in the male Wistar rat. Rats (11--13 weeks old) were treated with 5 injections (i.p.) of 20, 100 or 200 mg/kg/day ribavirin at intervals of 24 h. The testes were processed for histopathological analysis on days 14, 35, 70 and 105 after the last exposure. The parameters studied were body weight, the weights of the testis, epididymis, seminal vesicle and prostate, seminiferous tubular diameter (STD), epithelial height (SE), epithelial sloughing, incidence of stage XIV tubules, sperm abnormality and total serum level of testosterone. Data were analysed by ANOVA and the Bonferroni post hoc test for significances between different groups. There was a decrease in body weight and organ weights, excluding those of the testis and epididymis, against control at higher dose-levels. Ribavirin induced the formation of vacuoles, gaps and sloughing of the seminiferous epithelium. The STD, SE and the incidences of stage XIV tubules decreased on days 14 and 35. Ribavirin also induced the formation of sperm with microcephaly and cephalocaudal junction defects, with or without fibrils jetting out. All these morphological defects recovered to control limit by day 105. The serum level of testosterone was decreased at all dose-levels and time points, although recovery had started by day 105. In conclusion, ribavirin is gonadotoxic in male rats but the effects are reversible after a period of 105 days. However, the endocrine-disrupting properties of ribavirin persist beyond this period.

Background/aims: Current therapies for patients with chronic hepatitis C virus (HCV) do not achieve sustained viral clearance in most patients, and are associated with severe toxic effects. Our aim was to investigate the efficacy and safety of etanercept as adjuvant to interferon and ribavirin in treatment-naive patients with HCV., Methods: Double-blind, randomized, placebo controlled trial. Fifty patients with chronic HCV were randomly assigned to receive interferon alfa-2b and ribavirin with either etanercept or placebo for 24 weeks. The main outcome measure was the absence of HCV RNA at 24 weeks, the on treatment response at the end of the etanercept randomization period., Results: At 24 weeks, HCV RNA was absent in 63% (12/19) etanercept patients compared to 32% (8/25) placebo patients (P=0.04). In addition, patients receiving etanercept had lower frequency of most adverse events categories compared to placebo., Conclusions: Etanercept given for 24 weeks as adjuvant therapy to interferon and ribavirin significantly improved virologic response at the end of the etanercept randomization period among patients with HCV, and was associated with decreased incidence of most adverse effects associated with interferon and ribavirin.

Adenovirus infections are a frequent and serious complication following allogeneic haematopoietic stem cell transplantation (HSCT). The antiviral drugs cidofovir and ribavirin have been used as first-line therapy for disseminated infections with variable results. In the present study, in vitro susceptibility to these two drugs was evaluated on HEp-2 cells in adenovirus reference strains representing serotypes of each of the six species and in clinical isolates. Susceptibility to cidofovir was comparable between species with inhibition of replication of all tested serotypes in a narrow dose range (IC50=17-81 microM). However, susceptibility to ribavirin was highly dependent on the species. Serotypes from species A, B, D, E and F were all resistant to ribavirin (IC50=396 to >500 microM). Only replication of serotypes from species C was inhibited by ribavirin (IC50=48-108 microM). This species-dependent susceptibility of adenovirus to ribavirin was confirmed in clinical isolates. When tested on other cell lines (PLC, A549 and 293), all species were revealed to be resistant to ribavirin. If our in vitro findings are predictive of virological responses in vivo, these results suggest that ribavirin would not be effective for management of non-C species adenovirus infections after HSCT.

Introduction: Chronic hepatitis C-therapy is limited to the combined use of PEG-ylated interferons and ribavirin. Side effects of this therapy include retinal changes that manifest with bleeding, cotton wool spots and/or thrombosis., Patient: A 51-year-old man presented with a sudden decrease of visual acuity. Chronic hepatitis C was known for the duration of 6 months and treated for 3 months with a combined therapy of PEG-interferon alpha2B and ribavirin. A sudden visual loss occurred in the right eye and bilateral visual field defects were detected. Ophthalmoscopic examination revealed bilateral papilloedema. After withdrawal of PEG-interferon alpha2B and ribavirin, a standard haemodilution therapy was started without functional improvement. After papilloedema regression, a beginning secondary atrophy of the optic nerve was diagnosed bilaterally. Vision did not improve., Conclusion: The ocular side effects of combined hepatitis C therapy with PEG-interferon alpha2B and ribavirin range from mild retinal changes to severely impaired vision. Treatment should be carried out in co-operation with an ophthalmologist. With respect to the uncertain effectiveness of hepatitis C therapy with interferon alpha2B and ribavirin, therapy should be stopped as soon as severe ophthalmological complications occur.

The safety of viramidine was evaluated and compared with ribavirin in one-month and six-month studies in rats and one-month and nine-month studies in monkeys. Viramidine administration produced hemolytic anemia, characterized by decreases in hemoglobin (Hgb) concentrations, which was accompanied by erythroid hyperplasia of the bone marrow or increase in reticulocyte counts. In the 1-month study in rats, viramidine or ribavirin dosing at 120 mg/kg/day reduced Hgb concentrations (12-16% and 13-20% compared to controls, respectively) and caused slight erythroid hyperplasia in the bone marrow. In the 6-month study in rats, viramidine or ribavirin dosing at 90 mg/kg/day reduced Hgb concentrations (16-18% and 18% compared to controls, respectively) and increased reticulocyte counts (>25%). Although toxicity effects in monkeys were similar to rats, they occurred at higher doses of viramidine compared to ribavirin. In the 1-month monkey study, viramidine at 600 mg/kg/day caused slight or no reduction (9%-0% in males and females) in Hgb concentrations compared to moderate reduction for ribavirin at 300 mg/kg/day (14%-20% in males and females). There were no signs of erythroid hyperplasia in bone marrow or significant increase in reticulocytes detected after viramidine or ribavirin dosing in monkeys. In the 9-month study in monkeys, viramidine at 180 mg/kg/day slightly reduced Hgb concentrations (5-11%) as compared to ribavirin at 60 mg/kg/day (9-12%). Both treatments had an increase in reticulocytes (49-53% vs. 43-59% compared to controls, respectively). Moderate decrease in Hgb levels (26-29%) together with large increase in reticulocyte counts (203-224%) were seen for viramidine at 600 mg/kg/day. All the changes were reversible after a recovery phase in both rats and monkeys. It is concluded that ribavirin and viramidine produced similar toxicity in rats. However, viramidine is safer than ribavirin in monkeys.

Hepatitis C treatment with interferon alpha-2b (IFN-alpha) and ribavirin has been related to decreased bone mineral density. The aim of this study was to investigate the in vitro effects of different concentrations of ribavirin and IFN-alpha on osteoblast-like cells. Human osteoblast-like cells obtained by the outgrowth of cells from bone chips were exposed to ribavirin (0.1-10 microg/mL) or IFN-alpha (0.1-1000 UI/mL). At regular time-points, cultures were harvested for posterior analysis. Alkaline phosphatase (ALP) activity was determined on days 7 and 14, and cell growth was accessed by C3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell count on days 1, 3, 5, and 7. Flow cytometry analysis was used for investigating cell death on days 1, 3, 5, and 7. IFN-alpha affected ALP expression only at the higher concentration (1000 UI/mL) after 7 days (P < 0.05). No effects were detected in cell growth. In ribavirin treated cultures, concentrations higher than 2.5 microg/mL were associated with a decrease in ALP activity within 7 and 14 days (P < 0.01 and P < 0.001, respectively). Furthermore, the reduction in cell growth was dose-dependent and was detected after the fifth day. This decrease can be explained by an increase in the number of dead cells and a decrease in cell proliferation. In conclusion, our experiments demonstrated that ribavirin reduced, in a time- and dose-dependent manner, the number of metabolically active cells through a decrease in proliferation and an increase in cell death, and induced an impairment in osteoblast differentiation. These negative effects of ribavirin on osteblast-like cells might contribute to the bone loss reported in vivo.

Tiazofurin (TZF) is a cytostatic drug that leads to depletion of the GTP pool in tumor and normal cells via its active metabolite tiazofurin-adenine dinucleotide (TAD). TAD was detected in different cell lines, but not in erythrocytes, so the mechanism of erythrocytotoxicity of TZF remains unclear. The purpose of this study was to evaluate in vitro and in vivo action of tiazofurin on rat erythrocytes (RBC). After two decades of clinical trials the question of erythrocytotoxicity of TZF had remained unexplained making this study justified. Since we have previously demonstrated early erythrocytotoxic effects in male Wistar rats, we extend this finding on isolated RBC. Isolated erythrocytes from untreated animals were treated in buffered solution or plasma containing TZF. In addition, groups of 10 rats were treated with 200 and 1000 mg/kg of TZF and hematologic parameters were analyzed by flowcytometry and by the analysis of the peripheral blood smears. Early signs of hemolysis or aberrant structures were monitored by scanning probe microscopy (SPM). We suggest that correlation exists between early erythrocytotoxicity and irregularities in erythrocyte morphology and membrane integrity. We also found that TZF affects responsiveness to oxidative stress. This is in concordance with flowcytometric findings describing anisocytosis and anisochromosis of RBC.

In a retrospective analysis, we identified 38 evaluable patients who received intravenous ribavirin after adenovirus or RSV detection in the respiratory and/or gastrointestinal tract throughout the years 1998 and 2001. A total of 43 treatment cycles are analyzed. Intravenous ribavirin was combined with cidofovir in about half of the patients. In six out of eight patients treated because of RSV isolates from the respiratory tract, the virus was no longer detectable after treatment. In case of adenovirus isolates, the treatment was efficacious in eradicating the virus from the respiratory tract in more than 60% and from the gastrointestinal tract in 75% of treatment cycles. The addition of cidofovir was not beneficial in eradicating RSV isolates, but somewhat improved the virus clearance of adenovirus. Virus clearance was associated with a trend to a better median survival after virus detection. There were some episodes of suspected hemolysis and a trend towards lower leukocyte counts, reaching grade 3 toxicity in about 15% of treatment cycles. However, in general, intravenous ribavirin was well tolerated. In conclusion, the possible efficacy of intravenous Ribavirin in controlling RSV or adenovirus infections after allogeneic stem cell transplantations should be evaluated in prospective clinical trials.

The ribavirin/interferon-alpha combination is currently the standard therapy for patients with chronic hepatitis C. However, ribavirin causes hemolytic anemia as a significant side-effect. Levovirin, an L-enantiomer of ribavirin, possesses similar immunomodulatory activity to ribavirin but lacks direct antiviral activity or hemolytic anemia. Viramidine is a liver-targeting prodrug of ribavirin with much less potential for hemolytic anemia. The aim of the present study is to profile the hepatic toxicological gene response to ribavirin, levovirin and viramidine. Rats were dosed orally with 120 mg kg(-1) day(-1) of ribavirin and viramidine and 2000 mg kg(-1) day(-1) of levovirin for 8 days. Ribavirin did not cause any significant change (>threefold) in gene expression as analyzed by the Affymetrix GeneChip technique. Levovirin decreased the mRNA level of CYP7A1 by fourfold but did not affect the expression of CYP27/CYP7B1 that functions as an alternative pathway for cholesterol metabolism. Viramidine down-regulated both expressed sequence tag 233569 and heat shock protein 86 genes threefold. The changes at mRNA level of these genes were confirmed by the reverse transcription competitive polymerase chain reaction technique. None of the compounds changed the liver/body weight ratio, the major cytochrome P-450 protein levels or enzyme activities. The data indicated that a high dose of ribavirin, levovirin or viramidine did not cause significant change at the transcription level of most of the liver toxicological genes., (Copyright 2003 John Wiley & Sons, Ltd.)

A divergent synthesis of two novel tiazofurin analogues, 2-(3-deoxy-3-fluoro-beta-D-xylofuranosyl)thiazole-4-carboxamide (2) and 2-(3-acetamido-3-deoxy-beta-D-xylofuranosyl)thiazole-4-carboxamide (3), has been achieved starting from D-glucose. Both nucleoside analogues were evaluated for their in vitro cytotoxicity against several human leukaemia and solid tumour cell lines.

Ribavirin, part of the current first line combination therapy for the treatment of chronic hepatitis C, may cause haemolytic anaemia and poses a significant challenge to the clinical management of the disease. Viramidine, a prodrug of ribavirin, is currently under development. In-vitro partition demonstrated that viramidine had less association with RBCs than ribavirin in rat, monkey and man, and thus has less liability for haemolytic anaemia than ribavirin. In a whole body autoradiography study in rats following oral dosing (30 mg/kg) of [14C]ribavirin or [14C]viramidine to monkeys, viramidine produced 32% higher radioactivity in the liver than ribavirin, indicating a better liver-targeting properties. In portal vein-cannulated cynomolgus monkeys following single oral dosing (30 mg/kg) of [3H]viramidine or [3H]ribavirin, viramidine retained 3X higher radioactivity in the liver than ribavirin. Viramidine dosing also produced a higher viramidine to ribavirin ratio in portal plasma than in systemic plasma, indicating that the liver was the main site for the viramidine conversion to ribavirin and subsequent trapping of the drug. After multiple oral dosing (10 mg/kg) of [14C]ribavirin or [14C]viramidine to monkey, viramidine yielded three times the drug level in the liver but only half in RBCs compared to ribavirin. Viramidine and ribavirin had comparable toxicity profiles in a 28-day toxicity study in rats. In contrast, viramidine had much better safety profiles than ribavirin in a 28-day toxicity study in monkeys. In conclusion, viramidine has better liver-targeting properties and safety profiles than ribavirin in animals.

The genotoxic and cytotoxic effects of the antiviral drug, ribavirin, was studied in rat bone marrow by employing the micronucleus assay. Ribavirin in doses of 10, 15, 20, 30, 50, 75, 100 and 200 mg/kg, and cyclophosphamide (CP) 40 mg/kg (only for sex-difference study) were injected intraperitoneally. Bone marrow was collected at 24 h and 48 h following the injection. To evaluate the recovery, the bone marrow was also sampled at 72 h from 20, 100 and 200 mg/kg treated rats. The micronucleus assay was conducted according to the standard procedure. Ribavirin elevated the incidence of micronuclei (except 10 mg/kg) in erythrocytes (P<0.01). The micronucleated polychromatic erythrocytes showed the initial steep increase at 15 and 20 mg/kg dose level, then with the gradual increase, possibly due to the limited metabolism and action of higher doses. The incidence of micronucleated normochromatic erythrocytes was not dose dependent. The effect was more at 48 h than 24 h due to prolonged toxicity of the drug or its metabolites, and by 72 h, recovery was observed even though the genotoxicity was significant. The PCE% decreased as the dose was increased up to 75 mg/kg, then without much difference between two higher doses. Only 100 mg/kg ribavirin and CP showed more toxicity on male rats. Cytotoxicity was seen due to hindered erythropoiesis or cell destruction. Our findings suggest that ribavirin is genotoxic and cytotoxic agent for rat bone marrow.

Mechanisms of cytotoxicity of an antiviral drug, ribavirin was studied in the rat bone marrow and testis. Ribavirin at the dose levels of 20, 100 and 200 mg/kg was treated (i.p.) either as single (for bone marrow) or 5 (for testis) treatments. Bone marrow smears were obtained at 24, 48 and 72 h following the exposure and stained with the May-Gruenwald-Giemsa combination. Smears were screened for the incidence of dead cells, and at 24 h, a total of 2000 erythrocytes were counted to obtain the ratio of polychromatic erythrocytes (PCEs) to normochromatic erythrocytes (NCEs) (P/N). Step 19 spermatids/stage VII tubule, dividing cells (meiotic figures)/stage XIV tubule and the incidence of tubules with dead cells were counted in periodic acid--Schiff's reaction and haematoxylin (PAS-H) stained testicular sections on days 14, 35, 70 and 105. Significant decrease in the step 19 spermatids and meiotic figures, and increase in the incidence of tubules with dead cells (P < 0.05-0.01) were observed mainly on days 14 and 35. The cell death was observed in the bone marrow mainly at the two higher dose levels and significant decrease (P < 0.001) in P/N ratio was observed. This present study concludes that the cytotoxicity of ribavirin in these two target cell-lines in due to the induction of cell death and prevention of the cell division.

Thirty-nine hemophiliac patients, negative for human immunodeficiency virus, with chronic hepatitis C who failed to respond to interferon (IFN) at 3 million units (MU) given subcutaneously thrice weekly for at least 3 months were retreated with 5 MU IFN for 6 months followed by 3 MU IFN in combination with daily oral doses of 1 or 1.2 g ribavirin. Thirty-four patients (87%) completed the study. In 4 patients treatment was discontinued because of treatment-related symptoms; 1 patient dropped out. Dosage reduction was required in 10 patients (26%) because of ribavirin-related anemia or IFN-related side effects. By intention-to-treat analysis, 14 (37%) had a sustained virologic response with preference for those infected by genotypes other than type 1 (43% versus 12%) and with high transaminases levels (168 U/L versus 116 U/L). Thus, IFN and ribavirin combination therapy led to a sustained suppression of hepatitis in one third of hemophiliac patients resistant to conventional monotherapy.

Ribavirin (1-beta-D-ribofuranosyl-1,2,4, triazole-3 carboxamide) is a broad-spectrum antiviral drug. This study was aimed to investigate the mutagenicity of ribavirin on germ cells by employing sperm morphology assay. Male Wistar rats were treated with water, cyclophosphamide (CP) 40 mg/kg, and ribavirin 20, 100 and 200 mg/kg (i.p.) for 5 consecutive days at intervals of 24h. Following the last exposure, at 14, 28, 35, 42 and 70 days, the epididymal sperm smears were obtained and stained according to the standard procedure. One thousand sperms per animal were classified into normal and different abnormal types. Both CP and ribavirin-induced anomalies of head and tail of sperm except at 70 days. In CP groups, maximum incidence was observed at 28, 35 and 42 days. Ribavirin 20 mg/kg induced maximum incidence at 14 and 42 days, 100 mg/kg at 28 and 42 days and 200 mg/kg at 28-42 days. These results show that ribavirin is mutagenic to rat germ cells in a transient fashion.

Tiazofurine is a nucleoside analog with oncolytic activity being developed by Ribapharm (formerly ICN Pharmaceuticals) as a potential treatment for leukemia. It is metabolized to TAD (thiazole-4-carboxamide adenine dinucleotide), an inhibitor of IMP dehydrogenase. This inhibition results in the reduction of guanylate levels and the halting of neoplastic proliferation. The compound is in phase II/III trials [215553]. It is expected that Ribapharm will file an orphan drug application for tiazofurine, as a treatment for myelogenous leukemia, following the drug's completion of phase III trials by the end of 2002. The company has reported that phase III trials will begin by the end of 2000. Preliminary studies involving 21 patients have been carried out and the results reported by the company. During these studies, seven patients with chronic myelogenous leukemia had a complete hematologic response and two patients had a partial response. Of the patients with a complete response, six had marrow and peripheral responses. Ribapharm, through a Russian subsidiary of ICN, is also planning to conduct phase II studies of tiazofurine involving patients suffering from advanced ovarian cancer or multiple myeloma which is resistant to conventional therapy. The company has reported that the multiple myeloma limited phase II study is still undergoing planning, with an intended start date in late 2000 [381453]. In March 2000, Chase Hambrecht & Quist predicted that first approval could be towards the end of 2001 [384894].

(1) Ribavirin is licensed for patients with hepatitis C who have not been treated previously or who have relapsed. According to the licence, ribavirin must be combined with interferon alfa-2b. (2) The clinical file is bulky and of acceptable methodological quality. (3) Some trials have shown that ribavirin monotherapy has no effect on viral load. Two trials involving patients who had relapsed after transaminase normalisation on interferon alfa-2b monotherapy show that the ribavirin + interferon alfa-2b combination is more effective than interferon alfa-2b alone in rendering viraemia undetectable and normalising transaminase activity for at least 6 months after treatment cessation. (4) A trial of patients resistant to interferon alfa monotherapy showed that it was more beneficial to combine ribavirin + interferon alfa-2b than to double the dose of interferon alfa-2b alone, in terms of driving viraemia below the detection limit and persistently normalising transaminase activity. (5) Three trials involving patients who had not been treated previously showed that the ribavirin + interferon alfa-2b combination was more effective than interferon alfa-2b monotherapy in driving viraemia below the detection limit for at least 6 months after treatment cessation, and in reducing the liver inflammation score. No effect on fibrosis or on the risk of cirrhosis has yet been demonstrated. (6) The best duration of treatment is not known, but seems to depend on prognostic factors. The following factors might permit shorter treatment: relatively low viral load, early-stage fibrosis, female sex, age below 40 years, and infection by genotype 2 or 3 strains. (7) The ribavirin + interferon alfa-2b combination causes haemolytic anaemia in most patients, and this can be severe. (8) Ribavirin is teratogenic and causes sperm defects.

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.

In this study hematological toxicity was analyzed after the single and repeated applications of tiazofurin (TZF). Cellularity of bone marrow, spleen and peripheral blood was examined, spanning the period of fifty days after the initial application. Analysis of hematological parameters was performed by slightly modified conventional techniques. The fraction of erythroid series was monitored during the experiment. Presented data describe kinetics of damage and recovery of hemopoietic tissue. Our results indicate that the effect of tiazofurin on cellularity of bone marrow and spleen and on erythropoiesis is reversible and dose dependent within tested dose range and therapeutic regimes. Twenty days after the application normal function of hemopoietic tissues was restored. This approach and results can be useful in defining the timing for sequencing and combination therapy with tiazofurin.

Intravenous ribavirin was given to nine patients who had developed severe adenovirus-induced hemorrhagic cystitis (AD-HC) which was resistant to conventional therapy or where there was involvement of other organs after allogeneic BMT. Three patients recovered completely from AD-HC, two of whom had been resistant to vidarabine. All three had received sibling BMTs (2 HLA matched, 1 HLA mismatched). Five patients who received BMTs from related (2 HLA mismatched) or unrelated (1 HLA matched, 2 HLA mismatched) showed an improvement in symptoms but had recurrent AD-HC after discontinuation of ribavirin. Improvement in clinical symptoms and termination of virus excretion were well correlated. The last patient who received a mismatched unrelated BMT died during ribavirin therapy. Ribavirin was notably more effective among patients receiving BMTs from siblings in contrast to patients receiving BMTs from alternative donors (<0.05). One patient experienced severe pancytopenia during the second treatment with ribavirin after HC recurrence and recovered after ceasing ribavirin. Thus, ribavirin seems to be very effective for severe AD-HC for some recipients who receive transplants from a genetically close donor. Bone Marrow Transplantation (2000) 25, 545-548.