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Antiviral Research (v.83, #2)
In vitro and in vivo pharmacokinetic characterization of two novel prodrugs of zidovudine by Mario Alfredo Quevedo; Margarita Cristina Briñón (pp. 103-111).
This work deals with the in vitro and in vivo pharmacokinetic characterization of 3′-azido-2′,3′-dideoxy-5′- O-oxalatoylthymidine (AZT-Ac) and 3′-azido-2′,3′-dideoxy-5′- O-isonicotinoyl-thymidine (AZT-Iso), two novel prodrugs of the anti-HIV agent zidovudine [3′-azido-2′,3′-dideoxythymidine (AZT)]. AZT, AZT-Ac and AZT-Iso intestinal permeation properties and plasma concentration profiles in rats after intravenous administration were studied. Using the everted gut sac intestinal permeation assay, it was observed that AZT was subjected to saturable transport mechanisms in the jejunum and the proximal ileum, while no saturation was found in the distal ileum. AZT-Ac was able to permeate the intestinal segment at a lower rate than AZT but resisting enzymatic hydrolysis, while no evidence of saturation was found. On the other hand, AZT-Iso was completely hydrolyzed in the intestinal tissue, with AZT being found in the permeated samples. In vivo studies demonstrated that AZT plasma half-life ( t1/2) is extended after administration of AZT-Ac compared to AZT (2.16 and 0.96h, respectively), while after administering AZT-Iso the t1/2 of the regenerated AZT was shorter (0.38h). A relationship is proposed between these observed in vivo pharmacokinetic features and previous studies of protein-binding properties, concluding that AZT-Ac is a very promising prodrug of AZT in the search for more effective and safer anti-HIV agents.
Keywords: AZT prodrugs; Intestinal permeation; Plasma profiles; Plasma stability
Identification of bisindolylmaleimides and indolocarbazoles as inhibitors of HCV replication by tube-capture-RT-PCR by Yuko Murakami; Kohji Noguchi; Satoshi Yamagoe; Tetsuro Suzuki; Takaji Wakita; Hidesuke Fukazawa (pp. 112-117).
We devised a screening method for hepatitis C virus (HCV) inhibitors by exploiting the JFH1 viral culture system. The viral RNA released in the medium was adsorbed onto PCR plates, and real-time RT-PCR was performed by directly adding the one-step RT-PCR reaction mixture to the wells. The “tube-capture-RT-PCR” method obviates the need for labor-intensive RNA isolation and should allow high-throughput screening of HCV inhibitors. To substantiate the validity of the assay for drug screening, a pilot screen of an inhibitor library composed of 95 compounds was performed. In addition to the known inhibitors of HCV replication included in the library, the assay identified the PKC inhibitor bisindolylmaleimide I (BIM I) as an HCV replication inhibitor. BIM I was also effective in reducing the viral protein level in genotype 1b and 2a subgenomic replicon cells, indicating inhibition of HCV replication. Further assays revealed that a broad range of bisindolylmaleimides and indolocarbazoles inhibit HCV, but no correlation was found between the PKC inhibition pattern and anti-HCV activity. These series of compounds represent new classes of inhibitors that may warrant further development.
Keywords: HCV; Bisindolylmaleimide; Tube-capture-RT-PCR; High-throughput
Genotypic analysis of the protease and reverse transcriptase of non-B HIV type 1 clinical isolates from naïve and treated subjects by Laura Monno; Luigia Scudeller; Gaetano Brindicci; Annalisa Saracino; Grazia Punzi; Antonio Chirianni; Antonella Lagioia; Nicoletta Ladisa; Sergio Lo Caputo; Gioacchino Angarano (pp. 118-126).
One hundred and ninety-two pol sequences of drug-naïve and drug-experienced subjects infected with non-B HIV-1 subtypes were analyzed to identify treatment-related amino acid changes which might be relevant for drug-resistance and possibly not included in the accepted mutation list for the B subtype. The correspondence analysis identified non-B-specific and subtype-specific polymorphisms which should not be mistaken for mutations. Multiple χ2 were performed to detect the differences between naïve vs treated subjects and between different subtypes. To verify the contribution of each single mutation to the resistance levels as predicted by the Virtual Phenotype™—LM, simple univariate linear regression was used with fold resistance as a dependent variable and individual mutations as predictors. Commonly accepted protease (PR) and reverse transcriptase (RT) positions along with mutants at RT positions 118 and 90 were significantly associated with treatment. Two unusual PR (K14R and I66F) and five RT positions (E28K, S68G, H221Y, L228R/H and P294A) were also associated with treatment ( p<0.01). Only minimal variations were observed with respect to commonly accepted amino acid changes. All amino acid changes correlated with treatment influenced the resistance levels to each single drug. Our findings demonstrate that there are no substantial differences regarding known resistance-associated mutations and the newly emergent substitutions between non-B and B subtype strains.
Keywords: HIV-1; Non-B subtypes; Resistance
The longitudinal quantitative assessment by transient elastography of chronic hepatitis C patients treated with pegylated interferon alpha-2b and ribavirin by Eiichi Ogawa; Norihiro Furusyo; Kazuhiro Toyoda; Hiroaki Takeoka; Shinji Maeda; Jun Hayashi (pp. 127-134).
The aim of this study was to assess the association between liver stiffness measured by transient elastography (FibroScan®) and the efficacy of pegylated interferon alpha-2b plus ribavirin combination treatment for patients with chronic hepatitis C virus (HCV) infection. We prospectively studied 145 Japanese patients with chronic HCV infection. FibroScan was done at baseline, at the end of treatment, and at 48 and 96 weeks after the end of treatment. The FibroScan values were significantly decreased for sustained virological response (SVR) patients (the mean rate of change; −16.2%, −32.2% and −43.5%) in comparison with non-SVR patients (−7.2%, −2.1% and +17.3%) at the end of treatment ( P=0.0127), and 48 weeks ( P<0.0001) and 96 weeks ( P<0.0001) after the end of treatment. Among the non-SVR patients, the FibroScan values were significantly decreased for patients with biochemical response (BR) (−17.9%, −30.0% and −27.1%) in comparison with non-BR (−4.1%, +6.4% and +30.6%) at the end of treatment ( P=0.0270), and 48 weeks ( P<0.0001) and 96 weeks ( P<0.0001) after the end of treatment. The FibroScan values may predict a progressively better clinical outcome for patients with successful virological and biochemical responses.
Keywords: Transient elastography; Hepatitis C virus; Pegylated interferon alpha; Ribavirin
Comparison of HCV NS3 protease and NS5B polymerase inhibitor activity in 1a, 1b and 2a replicons and 2a infectious virus by Matthew S. Paulson; Huiling Yang; I-hung Shih; Joy Y. Feng; Eric M. Mabery; Margaret F. Robinson; Weidong Zhong; William E. Delaney IV (pp. 135-142).
The hepatitis C virus infection system represents an important new tool for drug discovery. In this study, we compared the in vitro antiviral efficacy of several NS3 and NS5B inhibitors in genotype 1a, 1b, and 2a replicons and in the 2a infectious virus system. The nucleoside inhibitor 2′-C-methyl adenosine showed similar efficacy in each system tested. Three non-nucleoside inhibitors had small differences in potency between genotype 1a and 1b. In contrast, there was a dramatic loss of potency for these non-nucleoside inhibitors in the genotype 2a replicon, 2a infectious virus, and 2a NS5B biochemical assays. The protease inhibitor BILN-2061 had similar efficacy against 1a and 1b replicons but was 61–109-fold less potent against the 2a replicon and virus, respectively. VX-950, a covalent protease inhibitor, had similar efficacy (<3-fold changes in EC50) regardless of genotype or subtype. Importantly, we observed a significant correlation ( p<0.0001) in antiviral potency between the 2a replicon and 2a infectious virus for all classes of compounds tested.
Keywords: Hepatitis C virus; Infection; Antiviral; Replicon
A biotechnological product and its potential as a new immunomodulator for treatment of animal phlebovirus infection: Punta Toro virus by Nelson Durán; Brian B. Gowen; Fabio T.M. Costa; Giselle Z. Justo; Marcelo Brocchi; Odilon S. Nunes; Iseu S. Nunes (pp. 143-147).
Intracellular pathogens with widespread drug-resistance contribute substantially to the increasing rates in morbidity and mortality due to emerging and reemerging diseases. Thus, the development of new drugs, including those that can enhance the immune response, is urgently needed. The immunomodulator, P-MAPA, a proteinaceous aggregate of ammonium and magnesium phospholinoleate-palmitoleate anhydride derived from Aspergillus oryzae, have been shown to induce antitumor activities. The ability of this compound to elicit protective immunity against viral infections has not been fully explored. Here, we report findings on the use of P-MAPA as an antiviral agent in a mouse model of acute phleboviral (Punta Toro virus) disease. A dose administered i.p. 24h post-infectious challenge (100mg/kg dose of P-MAPA) was remarkably effective at preventing death due to Punta Toro virus infection. This dose also reduced systemic viral burden and liver discoloration assayed on day 3 of infection. Taken together, our findings indicate that non-specific immunotherapy with P-MAPA appears to be an effective treatment for blocking Punta Toro virus-induced disease and suggest that further exploration with other viral disease models is warranted.
Keywords: Immunomodulator; Virus; Punta Toro virus; P-MAPA
A reporter cell line for rapid and sensitive evaluation of hepatitis C virus infectivity and replication by Michaela Iro; Jeroen Witteveldt; Allan G.N. Angus; Ilka Woerz; Artur Kaul; Ralf Bartenschlager; Arvind H. Patel (pp. 148-155).
The human pathogen hepatitis C virus (HCV) is associated with chronic liver disease. The recent development of the cell culture infectious HCV (HCVcc) system has opened up avenues for detailed studies on the life cycle of the virus and its interaction with the host cell. Current methods to quantitate virus infectivity in cell culture are time-consuming and labor-intensive. This study describes the generation of a cell-based secreted alkaline phosphatase (SEAP) reporter assay to facilitate in vitro studies of HCV infection and replication. This assay is based on a novel reporter cell line stably expressing the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted alkaline phosphatase via a recognition sequence of the viral NS3/4A serine protease. The SEAP reporter from a similar construct has previously been shown to be released from the fusion protein and be secreted into the extracellular culture medium following cleavage by the viral NS3/4A protease. The reporter cell line enabled rapid and sensitive quantification of HCV infection and viral replication in cell culture. The utility of this system for investigating virus entry, and for high throughput screening of entry inhibitors and other antiviral compounds was demonstrated using several inter- and intra-genotypic chimeras of HCV.
Keywords: Abbreviations; FCS; fetal calf serum; FFU; focus forming units; HCV; hepatitis C virus; HCVcc; cell-cultured HCV; IFN-α; interferon-alpha; IgG; immunoglobulin G; IRES; internal ribosome entry site; IU; international unit; MAb; monoclonal antibody; MLV; murine leukemia virus; NS; non-structural; PBS; phosphate-buffered saline; RLU; relative light units; RT-qPCR; real-time-quantitative polymerase chain reaction; SEAP; secreted alkaline phosphatase; CCID; 50; 50% cell culture infective dose; WT; wild type; Pur; R; puromycin resistance geneHepatitis C virus; HCV; Chimera; Reporter cell line; SEAP; NS3/4A protease
Inhibition of HIV-1 replication by long-term treatment with a chimeric RNA containing shRNA and TAR decoy RNA by Jacob S. Barnor; Yuichiro Habu; Norio Yamamoto; Naoko Miyano-Kurosaki; Koichi Ishikawa; Naoki Yamamoto; Hiroshi Takaku (pp. 156-164).
Combinatorial therapies for the treatment of HIV-1 infection are effective for reducing patient viral loads and slowing the progression to AIDS. Our strategy was based on an anti-HIV-1 shRNA vector system in which HIV-1 vif-shRNA was fused to a decoy TAR RNA (mini-TAR RNA) to generate vif-shRNA-decoy TAR RNA under the control of the human U6 Pol III promoter. Upon expression in human cells, the RNA molecule was cleaved into its component parts, which inhibited HIV-1 replication in a synergistic manner. This chimeric RNA expressed a dual RNA moiety and greatly enhanced the inhibition of HIV-1 replication under the production of resistant virus by short interference RNA (siRNA) in long-term culture assays. We suggest that this technique provides a practical basis for the application of siRNA-based gene therapy in the treatment of HIV/AIDS.
Keywords: Chimeric RNA; siRNA-escape variants; Virus breakthrough; Long-term inhibition; Lentiviral vector
Induction of multiple pro-inflammatory cytokines by respiratory viruses and reversal by standardized Echinacea, a potent antiviral herbal extract by Manju Sharma; Shawn A. Anderson; Roland Schoop; James B. Hudson (pp. 165-170).
Several viruses associated with upper respiratory diseases have been shown to stimulate the secretion of pro-inflammatory cytokines, including chemokines, sometimes in the absence of viral cytopathology. We evaluated the ability of a standardized preparation of the popular herbal medicine Echinacea (Echinaforce®, an ethanol extract of herb and roots of E. purpurea, and containing known concentrations of marker compounds) to inhibit the viral induction of various cytokines in a line of human bronchial epithelial cells (BEAS-2B), and in two other human cell lines. All of the viruses tested, rhinoviruses 1A and 14, influenza virus, respiratory syncytial virus, adenovirus types 3 and 11, and herpes simplex virus type 1, induced substantial secretion of IL-6 and IL-8 (CXCL8), in addition to several other chemokines, depending on the virus, although only viable viruses were able to do this. In every case however Echinacea inhibited this induction. The Echinacea preparation also showed potent virucidal activity against viruses with membranes, indicating the multi-functional potential of the herb. These results support the concept that certain Echinacea preparations can alleviate “cold and flu” symptoms, and possibly other respiratory disorders, by inhibiting viral growth and the secretion of pro-inflammatory cytokines.
Keywords: Echinacea; Pro-inflammatory cytokines; Chemokines; Rhinovirus; Influenza virus; Respiratory syncytial virus
Glycyrrhizin inhibits influenza A virus uptake into the cell by Andrea Wolkerstorfer; Harald Kurz; Nicole Bachhofner; Oliver H.J. Szolar (pp. 171-178).
We investigated the mechanism by which glycyrrhizin (GL), the main active component of licorice roots, protects cells from infection with influenza A virus (IAV). We found that GL treatment leads to a clear reduction in the number of IAV-infected human lung cells as well as a reduction in the CCID50 titer by 90%. The antiviral effect, however, was limited to one or two virus replication cycles. Analysis of different GL treatment protocols suggested that the antiviral effect of GL was limited to an early step in the virus replication cycle. A direct inhibitory action of GL on IAV particles could be excluded and GL did not interact with virus receptor binding either. The antiviral effect of GL was abolished by treatment 1h after virus infection, whereas pre-treatment and treatment during and after virus adsorption led to a reduction in the cytopathic effect, reduced viral RNA within the cells and in the cell supernatants, and reduced viral hemagglutination titers. Detailed virus uptake analyses unambiguously demonstrated reduced virus uptake in various GL-treated cells. These observations lead to the conclusion, that the antiviral activity of GL is mediated by an interaction with the cell membrane which most likely results in reduced endocytotic activity and hence reduced virus uptake. These insights might help in the design of structurally related compounds leading to potent anti-influenza therapeutics.
Keywords: Abbreviations; GL; glycyrrhizin; IAV; influenza A virusInfluenza A virus; Glycyrrhizin; Antiviral activity; Virus uptake
Antiviral activity of indole derivatives by Michele Giampieri; Alessandro Balbi; Mauro Mazzei; Paolo La Colla; Cristina Ibba; Roberta Loddo (pp. 179-185).
Unsymmetrical methylene derivatives5 were prepared following a known method, by reaction of the Mannich bases of 2-naphthols4 with indoles. All synthesized compounds were tested against a wide panel of viruses, since previous work showed that Mannich bases on 7-hydroxycoumarin1 and unsymmetrical methylene derivatives2 were endowed with some antiviral activities. The symmetrical Mannich bases4 were completely inactive, whereas the unsymmetrical methylene derivatives5, although possessing a certain degree of toxicity, showed a significant activity against RSV. Some of compounds5 showed a moderate antiviral activity against HIV-1, BVDV, YFV and CVB-2. The lack of activity of Mannich bases4 demonstrates the crucial importance for antiviral activity of coumarin moiety present in Mannich bases1.
Keywords: RSV; HIV-1; BVDV; YFV; CVB-2; Mannich bases; Indole derivatives
Anti-hepatitis B virus activity of chlorogenic acid, quinic acid and caffeic acid in vivo and in vitro by Gui-Feng Wang; Li-Ping Shi; Yu-Dan Ren; Qun-Fang Liu; Hou-Fu Liu; Ru-Jun Zhang; Zhuang Li; Feng-Hua Zhu; Pei-Lan He; Wei Tang; Pei-Zhen Tao; Chuan Li; Wei-Min Zhao; Jian-Ping Zuo (pp. 186-190).
Chlorogenic acid and its related compounds are abundant plant polyphenols that have a diverse antiviral activity. In this study, HepG2.2.15 cells and duck hepatitis B virus infection model were used as in vitro and in vivo models to evaluate their anti-HBV activity. In the cell model, all the three compounds inhibited HBV-DNA replication as well as HBsAg production. Chlorogenic acid and caffeic acid also reduced serum DHBV level in DHBV-infected duckling model. Moreover, the anti-HBV activity of crude extracts of coffee beans, which have a high content of chlorogenic acid, was studied. Both the extracts of regular coffee and that of decaffeinated coffee showed inhibitory effect on HBV replication.
Keywords: Abbreviations; HBV; hepatitis B virus; HBsAg; hepatitis B virus surface antigen; HBeAg; hepatitis B e antigen; ELISA; enzyme-linked immuno ab sorbent assay; DHBV; duck hepatitis B virus; HIV; human immunodeficiency virus; HSV; herpes simplex virus; MEM; minimal essential medium; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; RSV; respiratory syncytial virus; SARS; severe acute respiratory syndrome; SDS; sodium dodecyl sulfate; SRM; selected reaction monitoring; TPA; 12-O-tetradecanoylphorbol-13-acetateChlorogenic acid; Quinic acid; Caffeic acid; Coffee crude extracts; HBV
Macrolide antibiotics inhibit respiratory syncytial virus infection in human airway epithelial cells by Masanori Asada; Motoki Yoshida; Tomoko Suzuki; Yukimasa Hatachi; Takahiko Sasaki; Hiroyasu Yasuda; Katsutoshi Nakayama; Hidekazu Nishimura; Ryoichi Nagatomi; Hiroshi Kubo; Mutsuo Yamaya (pp. 191-200).
To examine the effects of macrolide antibiotics on RS virus infection in airways, human tracheal epithelial cells were pre-treated with bafilomycin A1 and clarithromycin, and infected with RS virus. Viral titers in supernatant fluids and RNA of RS virus, and concentrations of cytokines in supernatant fluids, including interleukin-6 increased with time after infection. Bafilomycin A1 and clarithromycin reduced viral titers in supernatant fluids of RS virus, RNA of RS virus, the susceptibility to RS virus infection, and concentrations of cytokines induced by virus infection.N-acetyl-S-geranylgeranyl-l -cysteine, an inhibitor for a small GTP binding protein of RhoA, isoform A of the Ras-homologus (Rho) family, an active form of which is associated with RS virus infection via binding to its fusion protein (F protein), reduced viral titers in supernatant fluids and RNA of RS virus. Bafilomycin A1 and clarithromycin inhibited RhoA activation induced by lysophosphatidic acid in the cells. Fasudil, an inhibitor of Rho kinase, also reduced viral titers in supernatant fluids and RNA of RS virus. These findings suggest that macrolide antibiotics may inhibit RS virus infection, partly through the reduced expression of F protein receptor, activated RhoA, and the inhibition of subsequent Rho kinase activation in human airway epithelial cells.
Keywords: Respiratory syncytial virus; RhoA; Bronchial asthma; COPD; Macrolide
Inhibition of porcine endogenous retrovirus (PERV) replication by HIV-1 gene expression inhibitors by Minyi Shi; Xin Wang; Mika Okamoto; Sonshin Takao; Masanori Baba (pp. 201-204).
Porcine endogenous retrovirus (PERV) is persistently integrated into the host genomic DNA as a provirus and released from a variety of porcine cells. PERV infects a certain range of human cells, which is a major concern in xenotransplantation. Therefore, the use of viral gene expression inhibitors could be envisaged, if they reduce PERV production from porcine organs and minimize viral transmission to human recipients. In the present study, four HIV-1 gene expression inhibitors were examined for their inhibitory effect on PERV replication in porcine cells constitutively producing the virus. Among the compounds, the fluoroquinolone derivative K-37 and the bacterial product EM2487 displayed potent and selective inhibition of PERV replication in the cells mediated by the suppression of viral mRNA synthesis. Thus, retroviral gene expression inhibitors may be able to reduce the risk of PERV transmission.
Keywords: Xenotransplantation; PERV; HIV-1; Gene expression; Chemotherapy