Safety, tolerability, and antiviral effect of RG-101 in patients with chronic hepatitis C: a phase 1B, double-blind, randomised controlled trial.

BACKGROUND: miR-122 is an important host factor for hepatitis C virus (HCV) replication. The aim of this study was to assess the safety and tolerability, pharmacokinetics, and antiviral effect of a single dose of RG-101, a hepatocyte targeted N-acetylgalactosamine conjugated oligonucleotide that antagonises miR-122, in patients with chronic HCV infection with various genotypes. METHODS: In this randomised, double-blind, placebo-controlled, multicentre, phase 1B study, patients were randomly assigned to RG-101 or placebo (7:1). We enrolled men and postmenopausal or hysterectomised women (aged 18-65 years) with chronic HCV genotype 1, 3, or 4 infection diagnosed at least 24 weeks before screening who were either treatment naive to or relapsed after interferon-α based therapy. Patients with co-infection (hepatitis B virus or HIV infection), evidence of decompensated liver disease, or a history of hepatocellular carcinoma were excluded. Randomisation was done by an independent, unblinded, statistician using the SAS procedure Proc Plan. The first cohort received one subcutaneous injection of 2 mg/kg RG-101 or placebo; the second cohort received one subcutaneous injection of 4 mg/kg or placebo. Patients were followed up for 8 weeks (all patients) and up to 76 weeks (patients with no viral rebound and excluding those who were randomised to the placebo group) after randomisation. The primary objective was safety and tolerability of RG-101. This trial was registered with EudraCT, number 2013-002978-49. FINDINGS: Between June 4, 2014, and Oct 27, 2014, we enrolled 32 patients with chronic HCV genotype 1 (n=16), 3 (n=10), or 4 (n=6) infections. In the first cohort, 14 patients were randomly assigned to receive 2 mg/kg RG-101 and two patients were randomly assigned to receive placebo, and in the second cohort, 14 patients were randomly assigned to receive 4 mg/kg RG-101 and two patients were randomly assigned to receive placebo. Overall, 26 of the 28 patients dosed with RG-101 reported at least one treatment-related adverse event. At week 4, the median viral load reduction from baseline was 4·42 (IQR 3·23-5·00) and 5·07 (4·19-5·35) log10 IU/mL in patients dosed with 2 mg/kg RG-101 or 4 mg/kg RG-101. Three patients had undetectable HCV RNA levels 76 weeks after a single dose of RG-101. Viral rebound at or before week 12 was associated with the appearance of resistance associated substitutions in miR-122 binding regions in the 5' UTR of the HCV genome. INTERPRETATION: This study showed that one administration of 2 mg/kg or 4 mg/kg RG-101, a hepatocyte targeted N-acetylgalactosamine conjugated anti-miR-122 oligonucleotide, was well tolerated and resulted in substantial viral load reduction in all treated patients within 4 weeks, and sustained virological response in three patients for 76 weeks. FUNDING: Regulus Therapeutics, Inc.

In vitro antiviral activity and preclinical and clinical resistance profile of miravirsen, a novel anti-hepatitis C virus therapeutic targeting the human factor miR-122.

Miravirsen is a ß-D-oxy-locked nucleic acid-modified phosphorothioate antisense oligonucleotide targeting the liver-specific microRNA-122 (miR-122). Miravirsen demonstrated antiviral activity against hepatitis C virus (HCV) genotype 1b replicons with a mean 50% effective concentration (EC50) of 0.67 µM. No cytotoxicity was observed up to the highest concentration tested (>320 µM) in different cell culture models, yielding a therapeutic index of ≥ 297. Combination studies of miravirsen with interferon α2b, ribavirin, and nonnucleoside (VX-222) and nucleoside (2'-methylcytidine) inhibitors of NS5B, NS5A (BMS-790052), or NS3 (telaprevir) indicated additive interactions. Miravirsen demonstrated broad antiviral activity when tested against HCV replicons resistant to NS3, NS5A, and NS5B inhibitors with less than 2-fold reductions in susceptibility. In serial passage studies, an A4C nucleotide change was observed in the HCV 5' untranslated region (UTR) from cells passaged in the presence of up to 20 µM (40-fold the miravirsen EC50 concentration) at day 72 of passage but not at earlier time points (up to 39 days of passage). Likewise, a C3U nucleotide change was observed in the HCV 5'UTR from subjects with viral rebound after the completion of therapy in a miravirsen phase 2 clinical trial. An HCV variant constructed to contain the A4C change was fully susceptible to miravirsen. A C3U HCV variant demonstrated overall reductions in susceptibility to miravirsen but was fully susceptible to all other anti-HCV agents tested. In summary, miravirsen has demonstrated broad antiviral activity and a relatively high genetic barrier to resistance. The identification of nucleotide changes associated with miravirsen resistance should help further elucidate the biology of miR-122 interactions with HCV. (The clinical trial study has been registered at ClinicalTrials.gov under registration no. NCT01200420).

Treatment of HCV infection by targeting microRNA.

BACKGROUND: The stability and propagation of hepatitis C virus (HCV) is dependent on a functional interaction between the HCV genome and liver-expressed microRNA-122 (miR-122). Miravirsen is a locked nucleic acid-modified DNA phosphorothioate antisense oligonucleotide that sequesters mature miR-122 in a highly stable heteroduplex, thereby inhibiting its function. METHODS: In this phase 2a study at seven international sites, we evaluated the safety and efficacy of miravirsen in 36 patients with chronic HCV genotype 1 infection. The patients were randomly assigned to receive five weekly subcutaneous injections of miravirsen at doses of 3 mg, 5 mg, or 7 mg per kilogram of body weight or placebo over a 29-day period. They were followed until 18 weeks after randomization. RESULTS: Miravirsen resulted in a dose-dependent reduction in HCV RNA levels that endured beyond the end of active therapy. In the miravirsen groups, the mean maximum reduction in HCV RNA level (log10 IU per milliliter) from baseline was 1.2 (P=0.01) for patients receiving 3 mg per kilogram, 2.9 (P=0.003) for those receiving 5 mg per kilogram, and 3.0 (P=0.002) for those receiving 7 mg per kilogram, as compared with a reduction of 0.4 in the placebo group. During 14 weeks of follow-up after treatment, HCV RNA was not detected in one patient in the 5-mg group and in four patients in the 7-mg group. We observed no dose-limiting adverse events and no escape mutations in the miR-122 binding sites of the HCV genome. CONCLUSIONS: The use of miravirsen in patients with chronic HCV genotype 1 infection showed prolonged dose-dependent reductions in HCV RNA levels without evidence of viral resistance. (Funded by Santaris Pharma; ClinicalTrials.gov number, NCT01200420.).

Development of hepatitis C virus chimeric replicons for identifying broad spectrum NS3 protease inhibitors.

Several potent inhibitors of hepatitis C virus (HCV) NS3/4A protease have been identified that show great clinical potential against genotype 1. Due to the tremendous genetic diversity that exists among HCV isolates, development of broad spectrum inhibitors is challenging. With a limited number of lab strains available for preclinical testing, new tools are required for assessing protease inhibitor activity. We developed a chimeric replicon system for evaluating NS3 protease inhibitor activity against naturally occurring isolates. NS3/4A genes were cloned from the plasma of HCV-infected individuals and inserted into lab strain replicons, replacing the native sequences. The chimeric reporter replicons were transfected into Huh 7.5 cells, their replication monitored by luciferase assays, and their susceptibilities to inhibitors determined. Viable chimeras expressing heterologous genotypes 1, 2, 3, and 4 protease domains were identified that exhibited varying susceptibilities to inhibitors. Protease inhibitor spectrums observed against the chimeric replicon panel strongly correlated with published enzymatic and clinical results. This cell-based chimeric replicon system can be used to characterize the activities of protease inhibitors against diverse natural isolates and may improve the ability to predict dose and clinical efficacy.

HIV capsid is a tractable target for small molecule therapeutic intervention.

Despite a high current standard of care in antiretroviral therapy for HIV, multidrug-resistant strains continue to emerge, underscoring the need for additional novel mechanism inhibitors that will offer expanded therapeutic options in the clinic. We report a new class of small molecule antiretroviral compounds that directly target HIV-1 capsid (CA) via a novel mechanism of action. The compounds exhibit potent antiviral activity against HIV-1 laboratory strains, clinical isolates, and HIV-2, and inhibit both early and late events in the viral replication cycle. We present mechanistic studies indicating that these early and late activities result from the compound affecting viral uncoating and assembly, respectively. We show that amino acid substitutions in the N-terminal domain of HIV-1 CA are sufficient to confer resistance to this class of compounds, identifying CA as the target in infected cells. A high-resolution co-crystal structure of the compound bound to HIV-1 CA reveals a novel binding pocket in the N-terminal domain of the protein. Our data demonstrate that broad-spectrum antiviral activity can be achieved by targeting this new binding site and reveal HIV CA as a tractable drug target for HIV therapy.

Triple combination of amantadine, ribavirin, and oseltamivir is highly active and synergistic against drug resistant influenza virus strains in vitro.

The rapid emergence and subsequent spread of the novel 2009 Influenza A/H1N1 virus (2009 H1N1) has prompted the World Health Organization to declare the first pandemic of the 21st century, highlighting the threat of influenza to public health and healthcare systems. Widespread resistance to both classes of influenza antivirals (adamantanes and neuraminidase inhibitors) occurs in both pandemic and seasonal viruses, rendering these drugs to be of marginal utility in the treatment modality. Worldwide, virtually all 2009 H1N1 and seasonal H3N2 strains are resistant to the adamantanes (rimantadine and amantadine), and the majority of seasonal H1N1 strains are resistant to oseltamivir, the most widely prescribed neuraminidase inhibitor (NAI). To address the need for more effective therapy, we evaluated the in vitro activity of a triple combination antiviral drug (TCAD) regimen composed of drugs with different mechanisms of action against drug-resistant seasonal and 2009 H1N1 influenza viruses. Amantadine, ribavirin, and oseltamivir, alone and in combination, were tested against amantadine- and oseltamivir-resistant influenza A viruses using an in vitro infection model in MDCK cells. Our data show that the triple combination was highly synergistic against drug-resistant viruses, and the synergy of the triple combination was significantly greater than the synergy of any double combination tested (P<0.05), including the combination of two NAIs. Surprisingly, amantadine and oseltamivir contributed to the antiviral activity of the TCAD regimen against amantadine- and oseltamivir-resistant viruses, respectively, at concentrations where they had no activity as single agents, and at concentrations that were clinically achievable. Our data demonstrate that the TCAD regimen composed of amantadine, ribavirin, and oseltamivir is highly synergistic against resistant viruses, including 2009 H1N1. The TCAD regimen overcomes baseline drug resistance to both classes of approved influenza antivirals, and thus may represent a highly active antiviral therapy for seasonal and pandemic influenza.

New small-molecule inhibitor class targeting human immunodeficiency virus type 1 virion maturation.

A new small-molecule inhibitor class that targets virion maturation was identified from a human immunodeficiency virus type 1 (HIV-1) antiviral screen. PF-46396, a representative molecule, exhibits antiviral activity against HIV-1 laboratory strains and clinical isolates in T-cell lines and peripheral blood mononuclear cells (PBMCs). PF-46396 specifically inhibits the processing of capsid (CA)/spacer peptide 1 (SP1) (p25), resulting in the accumulation of CA/SP1 (p25) precursor proteins and blocked maturation of the viral core particle. Viral variants resistant to PF-46396 contain a single amino acid substitution in HIV-1 CA sequences (CAI201V), distal to the CA/SP1 cleavage site in the primary structure, which we demonstrate is sufficient to confer significant resistance to PF-46396 and 3-O-(3',3'-dimethylsuccinyl) betulinic acid (DSB), a previously described maturation inhibitor. Conversely, a single amino substitution in SP1 (SP1A1V), which was previously associated with DSB in vitro resistance, was sufficient to confer resistance to DSB and PF-46396. Further, the CAI201V substitution restored CA/SP1 processing in HIV-1-infected cells treated with PF-46396 or DSB. Our results demonstrate that PF-46396 acts through a mechanism that is similar to DSB to inhibit the maturation of HIV-1 virions. To our knowledge, PF-46396 represents the first small-molecule HIV-1 maturation inhibitor that is distinct in chemical class from betulinic acid-derived maturation inhibitors (e.g., DSB), demonstrating that molecules of diverse chemical classes can inhibit this mechanism.

Preclinical characterization of PF-00868554, a potent nonnucleoside inhibitor of the hepatitis C virus RNA-dependent RNA polymerase.

PF-00868554 is a nonnucleoside inhibitor of the hepatitis C virus (HCV) RNA polymerase, which exerts its inhibitory effect by binding to the thumb base domain of the protein. It is a potent and selective inhibitor, with a mean 50% inhibitory concentration of 0.019 microM against genotype 1 polymerases and a mean 50% effective concentration (EC(50)) of 0.075 microM against the genotype 1b-Con1 replicon. To determine the in vitro antiviral activity of PF-00868554 against various HCV strains, a panel of chimeric replicons was generated, in which polymerase sequences derived from genotype 1a and 1b clinical isolates were cloned into the 1b-Con1 subgenomic reporter replicon. Our results indicate that PF-00868554 has potent in vitro antiviral activity against a majority (95.8%) of genotype 1a and 1b replicons, with an overall mean EC(50) of 0.059 microM. PF-00868554 showed no cytotoxic effect in several human cell lines, up to the highest concentration evaluated (320 microM). Furthermore, the antiviral activity of PF-00868554 was retained in the presence of human serum proteins. An in vitro resistance study of PF-00868554 identified M423T as the predominant resistance mutation, resulting in a 761-fold reduction in susceptibility to PF-00868554 but no change in susceptibility to alpha interferon and a polymerase inhibitor that binds to a different region. PF-00868554 also showed good pharmacokinetic properties in preclinical animal species. Our results demonstrate that PF-00868554 has potent and broad-spectrum antiviral activity against genotype 1 HCV strains, supporting its use as an oral antiviral agent in HCV-infected patients.

Development of intergenotypic chimeric replicons to determine the broad-spectrum antiviral activities of hepatitis C virus polymerase inhibitors.

To address the need for broad-spectrum antiviral activity characterization of hepatitis C virus (HCV) polymerase inhibitors, we created a panel of intergenotypic chimeric replicons containing nonstructural (NS) protein NS5B sequences from genotype 2b (GT2b), GT3a, GT4a, GT5a, and GT6a HCV isolates. Viral RNA extracted from non-GT1 HCV patient plasma was subjected to reverse transcription. The NS5B region was amplified by nested PCR and introduced into the corresponding region of the GT1b (Con-1) subgenomic reporter replicon by Splicing by Overlap Extension (SOEing) PCR. Stable cell lines were generated with replication-competent chimeras for in vitro antiviral activity determination of HCV nonnucleoside polymerase inhibitors (NNIs) that target different regions of the protein. Compounds that bind to the NNI2 (thiophene carboxylic acid) or NNI3 (benzothiadiazine) allosteric sites showed 8- to >1,280-fold reductions in antiviral activity against non-GT1 NS5B chimeric replicons compared to that against the GT1b subgenomic replicon. Smaller reductions in susceptibility, ranging from 0.2- to 33-fold, were observed for the inhibitor binding to the NNI1 (benzimidazole) site. The inhibitor binding to the NNI4 (benzofuran) site showed broad-spectrum antiviral activity against all chimeric replicons evaluated in this study. In conclusion, evaluation of HCV NNIs against intergenotypic chimeric replicons showed differences in activity spectrum for inhibitors that target different regions of the enzyme, some of which could be associated with specific residues that differ between GT1 and non-GT1 polymerases. Our study demonstrates the utility of chimeric replicons for broad-spectrum activity determination of HCV inhibitors.

Potential use of antiviral agents in polio eradication.

In 1988, the World Health Assembly launched the Global Polio Eradication Initiative, which aimed to use large-scale vaccination with the oral vaccine to eradicate polio worldwide by the year 2000. Although important progress has been made, polio remains endemic in several countries. Also, the current control measures will likely be inadequate to deal with problems that may arise in the postpolio era. A panel convoked by the National Research Council concluded that the use of antiviral drugs may be essential in the polio eradication strategy. We here report on a comparative study of the antipoliovirus activity of a selection of molecules that have previously been reported to be inhibitors of picornavirus replication and discuss their potential use, alone or in combination, for the treatment or prophylaxis of poliovirus infection.

In vitro resistance study of AG-021541, a novel nonnucleoside inhibitor of the hepatitis C virus RNA-dependent RNA polymerase.

A novel class of nonnucleoside hepatitis C virus (HCV) polymerase inhibitors characterized by a dihydropyrone core was identified by high-throughput screening. Crystallographic studies of these compounds in complex with the polymerase identified an allosteric binding site close to the junction of the thumb and finger domains, approximately 30 A away from the catalytic center. AG-021541, a representative compound from this series, displayed measurable in vitro antiviral activity against the HCV genotype 1b subgenomic replicon with a mean 50% effective concentration of 2.9 muM. To identify mutations conferring in vitro resistance to AG-021541, resistance selection was carried out using HCV replicon cells either by serial passages in increasing concentrations of AG-021541 or by direct colony formation at fixed concentrations of the compound. We identified several amino acid substitutions in the AG-021541-binding region of the polymerase, including M423(T/V/I), M426T, I482(S/T), and V494A, with M423T as the predominant change observed. These mutants conferred various levels of resistance to AG-021541 and structurally related compounds but remained sensitive to interferon and HCV polymerase inhibitors known to interact with the active site or other allosteric sites of the protein. In addition, dihydropyrone polymerase inhibitors retained activity against replicons that contain signature resistance changes to other polymerase inhibitors, including S282T, C316N, M414T, and P495(S/L), indicating their potential to be used in combination therapies with these polymerase inhibitors. AG-021541-resistant replicon cell lines provide a valuable tool for mechanism-of-action studies of dihydropyrone polymerase inhibitors. The clinical relevance of in vitro resistance to HCV polymerase inhibitors remains to be investigated.

Identification and characterization of UK-201844, a novel inhibitor that interferes with human immunodeficiency virus type 1 gp160 processing.

More than 10(6) compounds were evaluated in a human immunodeficiency virus type 1 (HIV-1) high-throughput antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (UK-201844). UK-201844 exhibited antiviral activity against HIV-1 NL4-3 in MT-2 and PM1 cells, with 50% effective concentrations of 1.3 and 2.7 microM, respectively, but did not exhibit measurable antiviral activity against the closely related HIV-1 IIIB laboratory strain. UK-201844 specifically inhibited the production of infectious virions packaged with an HIV-1 envelope (Env), but not HIV virions packaged with a heterologous Env (i.e., the vesicular stomatitis virus glycoprotein), suggesting that the compound targets HIV-1 Env late in infection. Subsequent antiviral assays using HIV-1 NL4-3/IIIB chimeric viruses showed that HIV-1 Env sequences were critical determinants of UK-201844 susceptibility. Consistent with this, in vitro resistant-virus studies revealed that amino acid substitutions in HIV-1 Env are sufficient to confer resistance to UK-201844. Western analysis of HIV Env proteins expressed in transfected cells or in isolated virions showed that UK-201844 inhibited HIV-1 gp160 processing, resulting in the production of virions with nonfunctional Env glycoproteins. Our results demonstrate that UK-201844 represents the prototype for a unique HIV-1 inhibitor class that directly or indirectly interferes with HIV-1 gp160 processing.

Development of a novel dicistronic reporter-selectable hepatitis C virus replicon suitable for high-throughput inhibitor screening.

Hepatitis C virus (HCV) research and drug discovery have been facilitated by the introduction of cell lines with self-replicating subgenomic HCV replicons. Early attempts to carry out robust, high-throughput screens (HTS) using HCV replicons have met with limited success. Specifically, selectable replicons have required laborious reverse transcription-PCR quantitation, and reporter replicons have generated low signal-to-noise ratios. In this study, we constructed a dicistronic single reporter (DSR)-selectable HCV replicon that contained a humanized Renilla luciferase (hRLuc) gene separated from the selectable Neo(r) marker by a short peptide cleavage site. The mutations E1202G, T1280I, and S2197P were introduced to enhance replicative capability. A dicistronic dual-reporter HCV replicon cell line (DDR) was subsequently created by transfection of Huh-7 cells with the DSR replicon to monitor antiviral activity and by the introduction of the firefly luciferase (FLuc) reporter gene into the host cell genome to monitor cytotoxicity. The DDR cell line demonstrated low signal variation within the HTS format, with a calculated Z' value of 0.8. A pilot HTS consisting of 20 96-well plates with a single concentration (10 microM) of 1,760 different compounds was executed. Hits were defined as compounds that reduced hRLuc and FLuc signals > or =50 and < or =40%, respectively, relative to those in a compound-free control. Good reproducibility was demonstrated, with a calculated confirmation rate of >75%. The development of a robust, high-throughput HCV replicon assay where the effects of inhibitors can be monitored for antiviral activity and cytotoxicity should greatly facilitate HCV drug discovery.

Rhinovirus chemotherapy.

Human rhinoviruses (HRV), members of the Picornaviridae family, are comprised of over 100 different virus serotypes. HRV represent the single most important etiological agents of the common cold [Arruda, E., Pitkaranta, A., Witek Jr., T.J., Doyle, C.A., Hayden, F.G., 1997. Frequency and natural history of rhinovirus infections in adults during autumn. J. Clin. Microbiol. 35, 2864-2868; Couch, R.B., 1990. Rhinoviruses. In: Fields, B.N., Knipe, D.M. (Eds.), Virology. Raven Press, New York, pp. 607-629; Turner, R.B., 2001. The treatment of rhinovirus infections: progress and potential. Antivir. Res. 49 (1), 1-14]. Although HRV-induced upper respiratory illness is often mild and self-limiting, the socioeconomic impact caused by missed school or work is enormous and the degree of inappropriate antibiotic use is significant. It has been estimated that upper respiratory disease accounts for at least 25 million absences from work and 23 million absences of school annually in the United States [Anzueto, A., Niederman, M.S., 2003. Diagnosis and treatment of rhinovirus respiratory infections. Chest 123 (5), 1664-1672; Rotbart, H.A., 2002. Treatment of picornavirus infections. Antivir. Res. 53, 83-98]. Increasing evidences also describe the link between HRV infection and more serious medical complications. HRV-induced colds are the important predisposing factors to acute otitis media, sinusitis, and are the major factors in the induction of exacerbations of asthma in adults and children. HRV infections are also associated with lower respiratory tract syndromes in individuals with cystic fibrosis, bronchitis, and other underlying respiratory disorders [Anzueto, A., Niederman, M.S., 2003. Diagnosis and treatment of rhinovirus respiratory infections. Chest 123 (5), 1664-1672; Gern, J.E., Busse, W.W., 1999. Association of rhinovirus infections with asthma. Clin. Microbiol. Rev. 12 (1), 9-18; Pitkaranta, A., Arruda, E., Malmberg, H., Hayden, F.G., 1997. Detection of rhinovirus in sinus brushings of patients with acute community-acquired sinusitis by reverse transcription-PCR. J. Clin. Microbiol. 35, 1791-1793; Pitkaranta, A., Virolainen, A., Jero, J., Arruda, E., Hayden, F.G., 1998. Detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction. Pediatrics 102, 291-295; Rotbart, H.A., 2002. Treatment of picornavirus infections. Antivir. Res. 53, 83-98]. To date, no effective antiviral therapies have been approved for either the prevention or treatment of diseases caused by HRV infection. Thus, there still exists a significant unmet medical need to find agents that can shorten the duration of HRV-induced illness, lessen the severity of symptoms, minimize secondary bacterial infections and exacerbations of underlying disease and reduce virus transmission. Although effective over-the-counter products have been described that alleviate symptoms associated with the common cold [Anzueto, A., Niederman, M.S., 2003. Diagnosis and treatment of rhinovirus respiratory infections. Chest 123 (5), 1664-1672; Gwaltney, J.M., 2002a. Viral respiratory infection therapy: historical perspectives and current trials. Am. J. Med. 22 (112 Suppl. 6A), 33S-41S; Turner, R.B., 2001. The treatment of rhinovirus infections: progress and potential. Antivir. Res. 49 (1), 1-14; Sperber, S.J., Hayden, F.G., 1988. Chemotherapy of rhinovirus colds. Antimicrob. Agents Chemother. 32, 409-419], this review will primarily focus on the discovery and development of those agents that directly or indirectly impact virus replication specifically highlighting new advances and/or specific challenges with their development.

In vitro selection of mutations in human immunodeficiency virus type 1 reverse transcriptase that confer resistance to capravirine, a novel nonnucleoside reverse transcriptase inhibitor.

Capravirine (CPV; formerly AG1549 and S-1153) is a novel, nonnucleoside reverse transcriptase inhibitor (NNRTI) of human immunodeficiency virus type 1 (HIV-1) that has demonstrated potent in vitro antiviral activity against several HIV-1 laboratory strains and clinical isolates with EC50 values ranging from 0.7 to 10.3 nM. In this study, we evaluated the resistance and cross-resistance profiles of CPV through selection of resistant HIV-1 variants from in vitro serial passage of HIV-1 NL4-3 and HIV-1 IIIB and by performing susceptibility assays on HIV-1 variants constructed to contain CPV-specific amino acid substitutions in reverse transcriptase (RT). Results demonstrate that HIV-1 variants selected at increasing CPV concentrations contained multiple substitutions in diverse patterns including L100I, Y181C, G190E and/or L234I in various combinations with K101R/E, K103T, V106A/I, V108I, E138K, T139K, A158T, V179D/I/G, Y188D, V189I, G190A, F227C, W229R, L234F, M230I/L and P236H/T. Interestingly, HIV-1 variants constructed to contain the T215Y zidovudine (AZT)-resistance associated substitution with CPV-resistance associated substitutions V106A, Y181C, F227C, F227L, L234I or V106A/F227L demonstrated 2.4-5.4-fold increased susceptibility to CPV. Results also demonstrate that the CPV-resistance associated substitutions Y181C, F227C, F227L and L234I reverse the phenotypic resistance to AZT conferred by the T215Y substitution.

High-throughput human immunodeficiency virus type 1 (HIV-1) full replication assay that includes HIV-1 Vif as an antiviral target.

Antiviral screens have proved useful for the identification of novel human immunodeficiency virus type 1 (HIV-1) inhibitors. In this study, we describe an HIV-1 full replication (HIV-1 Rep) assay that incorporates all of the targets required for replication in T-cell lines, including the HIV-1 Vif gene. The HIV-1 Rep assay was designed to exhibit optimal sensitivity to late-stage as well as early-stage inhibitors to maximize the likelihood of identification of novel target antiviral compounds in a screen. In addition, the flexibility of the HIV-1 Rep assay allows the rapid evaluation of antiviral compounds against different virus strains in different T-cell lines without significant modification of the assay format. We demonstrate that the HIV-1 Rep assay exhibits characteristics (e.g., a favorable Z' value) compatible with high-throughput screening in a 384-well format. The utility of the HIV-1 Rep assay was demonstrated in a high-throughput screen of >10(6) compounds. To our knowledge, this study represents the first example of an HIV-1 antiviral screen that includes Vif as a functional target and was executed on an industrial scale.

In vitro antiviral activity and single-dose pharmacokinetics in humans of a novel, orally bioavailable inhibitor of human rhinovirus 3C protease.

(E)-(S)-4-((S)-2-{3-[(5-methyl-isoxazole-3-carbonyl)-amino]-2-oxo-2H-pyridin-1-yl}-pent-4-ynoylamino)-5-((S)-2-oxo-pyrrolidin-3-yl)-pent-2-enoic acid ethyl ester (Compound 1) is a novel, irreversible inhibitor of human rhinovirus (HRV) 3C protease {inactivation rate constant (Kobs/[I]) of 223,000 M-1s-1}. In cell-based assays, Compound 1 was active against all HRV serotypes (35 of 35), HRV clinical isolates (5 of 5), and related picornaviruses (8 of 8) tested with mean 50% effective concentration (EC50) values of 50 nM (range, 14 to 122 nM), 77 nM (range, 72 to 89 nM), and 75 nM (range, 7 to 249 nM), respectively. Compound 1 inhibited HRV 3C-mediated polyprotein processing in infected cells in a concentration-dependent manner, providing direct confirmation that the cell-based antiviral activity is due to inhibition of 3C protease. In vitro and in vivo nonclinical safety studies showed Compound 1 to be without adverse effects at maximum achievable doses. Single oral doses of Compound 1 up to 2,000 mg in healthy volunteers were found to be safe and well tolerated in a phase I-ascending, single-dose study. Compound 1 estimated free observed maximum concentration in plasma (Cmax) for 500-, 1,000-, and 2,000-mg doses were higher than the protein binding-corrected EC50 required to inhibit 80% of the HRV serotypes tested. Treatment of HRV 52-infected cells with one to five 2-h pulses of 150 nM Compound 1 (corresponding to the Cmax at the 500-mg dose) was sufficient to effect a significant reduction in viral replication. These experiments highlight Compound 1 as a potent, orally bioavailable, irreversible inhibitor of HRV 3C protease and provide data that suggest that Cmax rather than the Cmin might be the key variable predicting clinical efficacy.

A novel HIV-1 antiviral high throughput screening approach for the discovery of HIV-1 inhibitors.

Antiviral high throughput screens remain a viable option for identifying novel target inhibitors. However, few antiviral screens have been reduced to practice on an industrial scale. In this study, we describe an HIV-1 dual reporter assay that allows for the simultaneous evaluation of the potential antiviral activities and cytotoxicities of compounds in a high throughput screen (HTS) format. We validate the assay with known HIV-1 inhibitors and show that the antiviral and cytotoxic activities of compounds are reproducibly measured under screening conditions. In addition, we show that the assay exhibits parameters (e.g., signal-to-background ratios and Z' coefficients) suitable for high throughout screening. In a pilot screen, we demonstrate that non-specific or cytotoxic compounds represent a significant fraction of the hits identified in an antiviral screen and that these false positives are identified and deprioritized by the HIV-1 dual reporter assay at the primary screening step. We propose that the HIV-1 dual reporter assay represents a novel approach to HIV-1 antiviral screening that allows for the effective execution of industrial scale HTS campaigns with significantly greater returns on resource investment when compared to previous methods.

An optical thin film assay incorporating rhinovirus protease inhibitors as detector reagents.

Human rhinoviruses (HRV) are the main cause of the common cold. Viral replication utilizes the activity of the HRV3C protease (3CP) enzyme [Antimicrob. Agents Chemother. 43 (1999) 2444; Antimicrob. Agents Chemother. 44 (2000) 1236]. Therefore, 3CP is an attractive target for antiviral drug development, and a new class of orally bioavailable irreversible 3CP inhibitors has been designed [J. Med. Chem. 45 (2002) 1607]. We have used related inhibitors to develop a rapid test for rhinovirus. The optical immuno assay (OIA) thin film detection technology utilizes an optically coated silicon surface to convert specific molecular binding events into visual color changes by altering the reflective properties of light through molecular thin films. The purpose of this study was to develop a rapid assay for the determination of 3CP combining the Thermo Electron Bio Star OIA technology and the newly designed inhibitor compounds. The advantage of this assay was in its approach, in which therapeutic and diagnostic targets are the same thus allowing patients with detected rhinoviruses to receive optimal treatment. Three different biotinylated inhibitor compounds were synthesized. The length of the spacer between the inhibitor and biotin core was 5, 10, and 15 atoms. These compounds were incorporated into the OIA format for the HRV assay development. A rapid (20 min) OIA test was developed using a 15 atom spacer biotinylated inhibitor (4). Forty different HRV serotypes were studied and thirty three serotypes of these 40 were detected (80%).

Phase II, randomized, double-blind, placebo-controlled studies of ruprintrivir nasal spray 2-percent suspension for prevention and treatment of experimentally induced rhinovirus colds in healthy volunteers.

Human rhinovirus (HRV) infections are usually self-limited but may be associated with serious consequences, particularly in those with asthma and chronic respiratory disease. Effective antiviral agents are needed for preventing and treating HRV illnesses. Ruprintrivir (Agouron Pharmaceuticals, Inc., San Diego, Calif.) selectively inhibits HRV 3C protease and shows potent, broad-spectrum anti-HRV activity in vitro. We conducted three double-blind, placebo-controlled clinical trials in 202 healthy volunteers to assess the activity of ruprintrivir in experimental HRV infection. Subjects were randomized to receive intranasal ruprintrivir (8 mg) or placebo sprays as prophylaxis (two or five times daily [2x/day or 5x/day] for 5 days) starting 6 h before infection or as treatment (5x/day for 4 days) starting 24 h after infection. Ruprintrivir prophylaxis reduced the proportion of subjects with positive viral cultures (for 5x/day dosing groups, 44% for ruprintrivir treatment group versus 70% for placebo treatment group [P=0.03]; for 2x/day dosing groups, 60% for ruprintrivir group versus 92% for placebo group [P=0.004]) and viral titers but did not decrease the frequency of colds. Ruprintrivir treatment reduced the mean total daily symptom score (2.2 for ruprintrivir treatment group and 3.3 for the placebo treatment group [P=0.014]) by 33%. Secondary endpoints, including viral titers, individual symptom scores, and nasal discharge weights, were also reduced by ruprintrivir treatment. Overall, ruprintrivir was well tolerated; blood-tinged mucus and nasal passage irritation were the most common adverse effects reported. Pharmacokinetic analysis of plasma and nasal ruprintrivir concentrations revealed intranasal drug residence with minimal systemic absorption. Results from these studies in experimental rhinoviral infection support continued investigation of intranasal ruprintrivir in the setting of natural HRV infection.