RESUMO
The development of new antiviral compounds active against hepatitis C virus (HCV) has surged in recent years. In order for these new compounds to be efficacious in humans, optimal dosage regimens for each compound must be elucidated. We have developed a novel in vitro pharmacokinetic/pharmacodynamic system, the BelloCell system, to identify optimal dosage regimens for anti-HCV compounds. In these experiments, genotype 1b HCV replicon-bearing cells (2209-23 cells) were inoculated onto carrier flakes in BelloCell bottles and treated with MK-4519, a serine protease inhibitor. Our dose-ranging studies illustrated that MK-4519 inhibited replicon replication in a dose-dependent manner, yielding a 50% effective concentration (EC(50)) of 1.8 nM. Dose-fractionation studies showed that shorter dosing intervals resulted in greater replicon suppression, indicating that the time that the concentration is greater than the EC(50) is the pharmacodynamic parameter for MK-4519 linked with inhibition of replicon replication. Mutations associated with resistance to serine protease inhibitors were detected in replicons harvested from all treatment arms. These data suggest that MK-4519 is highly active against genotype 1b HCV, but monotherapy is not sufficient to prevent the amplification of resistant replicons. In summary, our findings show that the BelloCell system is a useful and clinically relevant tool for predicting optimal dosage regimens for anti-HCV compounds.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , RNA Viral/antagonistas & inibidores , Serina Proteases/genética , Inibidores de Serina Proteinase/farmacologia , Proteínas não Estruturais Virais/genética , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Cálculos da Dosagem de Medicamento , Farmacorresistência Viral/genética , Genes Reporter , Genótipo , Hepacivirus/fisiologia , Hepatite C Crônica/virologia , Humanos , Concentração Inibidora 50 , Luciferases , Modelos Biológicos , Mutação , Replicon , Serina Proteases/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Intravenous zanamivir is recommended for the treatment of hospitalized patients with complicated oseltamivir-resistant influenza virus infections. In a companion paper, we show that the time above the 50% effective concentration (time>EC(50)) is the pharmacodynamic (PD) index predicting the inhibition of viral replication by intravenous zanamivir. However, for other neuraminidase inhibitors, the ratio of the area under the concentration-time curve to the EC(50) (AUC/EC(50)) is the most predictive index. Our objectives are (i) to explain the dynamically linked variable of intravenous zanamivir by using different half-lives and (ii) to develop a new, mechanism-based population pharmacokinetic (PK)/PD model for the time course of viral load. We conducted dose fractionation studies in the hollow-fiber infection model (HFIM) system with zanamivir against an oseltamivir-resistant influenza virus. A clinical 2.5-h half-life and an artificially prolonged 8-h half-life were simulated for zanamivir. The values for the AUC from 0 to 24 h (AUC(0-24)) of zanamivir were equivalent for the two half-lives. Viral loads and zanamivir pharmacokinetics were comodeled using data from the present study and a previous dose range experiment via population PK/PD modeling in S-ADAPT. Dosing every 8 h (Q8h) suppressed the viral load better than dosing Q12h or Q24h at the 2.5-h half-life, whereas all regimens suppressed viral growth similarly at the 8-h half-life. The model provided unbiased and precise individual (Bayesian) (r(2), >0.96) and population (pre-Bayesian) (r(2), >0.87) fits for log(10) viral load. Zanamivir inhibited viral release (50% inhibitory concentration [IC(50)], 0.0168 mg/liter; maximum extent of inhibition, 0.990). We identified AUC/EC(50) as the pharmacodynamic index for zanamivir at the 8-h half-life, whereas time>EC(50) best predicted viral suppression at the 2.5-h half-life, since the trough concentrations approached the IC(50) for the 2.5-h but not for the 8-h half-life. The model explained data at both half-lives and holds promise for optimizing clinical zanamivir dosage regimens.
Assuntos
Modelos Teóricos , Orthomyxoviridae/efeitos dos fármacos , Zanamivir/farmacologia , Animais , Área Sob a Curva , Linhagem Celular , CãesRESUMO
In 2009, a novel H1N1 influenza A virus emerged and spread worldwide, initiating a pandemic. Various isolates obtained from disparate parts of the world were shown to be uniformly resistant to the adamantanes but sensitive to the neuraminidase inhibitors oseltamivir and zanamivir. Over time, resistance to oseltamivir became more prevalent among pandemic H1N1 virus isolates, while most remained susceptible to zanamivir. The government has proposed the use of intravenous (i.v.) zanamivir to treat serious influenza virus infections among hospitalized patients. To use zanamivir effectively for patients with severe influenza, it is necessary to know the optimal dose and schedule of administration of zanamivir that will inhibit the replication of oseltamivir-sensitive and -resistant influenza viruses. Therefore, we performed studies using the in vitro hollow-fiber infection model system to predict optimal dosing regimens for zanamivir against an oseltamivir-sensitive and an oseltamivir-resistant virus. Our results demonstrated that zanamivir, at a dose of 600 mg given twice a day (Q12h), inhibited the replication of oseltamivir-sensitive and oseltamivir-resistant influenza viruses throughout the course of the experiment. Thus, our findings suggest that intravenous zanamivir, at a dose of 600 mg Q12h, could be used to treat hospitalized patients suffering from serious infections with oseltamivir-sensitive or -resistant influenza viruses.
Assuntos
Antivirais/farmacologia , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Oseltamivir/farmacologia , Zanamivir/farmacologia , Ácidos Carbocíclicos , Animais , Linhagem Celular , Ciclopentanos/farmacologia , Cães , Farmacorresistência Viral , Guanidinas/farmacologiaRESUMO
One of the biggest challenges in the effort to treat and contain influenza A virus infections is the emergence of resistance during treatment. It is well documented that resistance to amantadine arises rapidly during the course of treatment due to mutations in the gene coding for the M2 protein. To address this problem, it is critical to develop experimental systems that can accurately model the selection of resistance under drug pressure as seen in humans. We used the hollow-fiber infection model (HFIM) system to examine the effect of amantadine on the replication of influenza virus, A/Albany/1/98 (H3N2), grown in MDCK cells. At 24 and 48 h postinfection, virus replication was inhibited in a dose-dependent fashion. At 72 and 96 h postinfection, virus replication was no longer inhibited, suggesting the emergence of amantadine-resistant virus. Sequencing of the M2 gene revealed that mutations appeared at between 48 and 72 h of drug treatment and that the mutations were identical to those identified in the clinic for amantadine-resistant viruses (e.g., V27A, A30T, and S31N). Interestingly, we found that the type of mutation was strongly affected by the dose of the drug. The data suggest that the HFIM is a good model for influenza virus infection and resistance generation in humans. The HFIM has the advantage of being a highly controlled system where multiplicity parameters can be directly and accurately controlled and measured.
Assuntos
Amantadina/farmacologia , Antivirais/farmacologia , Farmacorresistência Viral/genética , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Modelos Biológicos , Proteínas da Matriz Viral/genética , Animais , Linhagem Celular , Cães , Humanos , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/fisiologia , Rim/virologia , Mutação , Replicação Viral/efeitos dos fármacosRESUMO
Variola major virus remains a potent weapon of bioterror. There is currently an investigational-new-drug application for cidofovir for the therapy of variola major virus infections. Stittelaar and colleagues compared the levels of effectiveness of postexposure smallpox vaccination (Elstree-RIVM) and antiviral treatment with cidofovir or an acyclic nucleoside phosphonate analogue 6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidine (HPMPO-DAPy) after lethal intratracheal infection of cynomolgus monkeys with monkeypox virus, a variola virus surrogate. Their results demonstrated that either compound was more effective than vaccination with the Ellstree vaccine (K. J. Stittelaar et al., Nature 439:745-748, 2006). An unanswered question is how to translate this information into therapy for poxvirus infections in people. In a proof-of-principle study, we used a novel in vitro hollow-fiber infection model system to determine the pharmacodynamics of vaccinia virus infection of HeLa-S3 cells treated with cidofovir. Our results demonstrate that the currently licensed dose of cidofovir of 5 mg/kg of body weight weekly with probenecid (which ameliorates nephrotoxicity) is unlikely to provide protection for patients intentionally exposed to Variola major virus. We further demonstrate that the antiviral effect is independent of the schedule of drug administration. Exposures (area under the concentration-time curve) to cidofovir that will have a robust protective effect will require doses that are 5 to 10 times that currently administered to humans. Such doses may cause nephrotoxicity, and therefore, approaches that include probenecid administration as well as schedules of administration that will help ameliorate the uptake of cidofovir into renal tubular epithelial cells need to be considered when addressing such treatment for people.
Assuntos
Antivirais/farmacologia , Citosina/análogos & derivados , Monkeypox virus/efeitos dos fármacos , Monkeypox virus/imunologia , Organofosfonatos/farmacologia , Infecções por Poxviridae/tratamento farmacológico , Vacina Antivariólica/farmacologia , Cidofovir , Citosina/farmacologia , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Modelos TeóricosRESUMO
MDCK cells transfected with the human beta-galactoside alpha-2,6-sialyltransferase 1 gene (AX-4 cells) were used to determine the drug susceptibility and pharmacodynamically linked variable of oseltamivir for influenza virus. For dose-ranging studies, five hollow-fiber units were charged with 10(2) A/Sydney/5/97 (H3N2) influenza virus-infected AX-4 cells and 10(8) uninfected AX-4 cells. Each unit was treated continuously with different oseltamivir carboxylate concentrations in virus growth medium for 6 days. For dose fractionation studies, one hollow-fiber unit received no drug, one unit received a 1x 50% effective concentration (EC(50)) exposure to oseltamivir by continuous infusion, one unit received the same AUC(0-24) (area under the concentration-time curve from 0 to 24 h) by 1-h infusion every 24 h, one unit received the same total exposure in two equal fractions every 12 h, and one unit received the same total exposure in three equal fractions every 8 h. Each infusion dose was followed by a no-drug washout, producing the appropriate half-life for this drug. The effect of the drug on virus replication was determined by sampling the units daily, measuring the amount of released virus by plaque assay, and performing a hemagglutination assay. The drug concentration in the hollow-fiber infection model systems was determined at various times by liquid chromatography-tandem mass spectrometry. The dose-ranging study showed that the EC(50)s for oseltamivir carboxylate for the A/Sydney/5/97 strain of influenza virus was about 1.0 ng/ml. The dose fractionation study showed that all treatment arms suppressed virus replication to the same extent, indicating that the pharmacodynamically linked variable was the AUC(0-24)/EC(50) ratio. This implies that it may be possible to treat influenza virus infection once daily with a dose of 150 mg/day.
Assuntos
Antivirais/farmacologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Oseltamivir/análogos & derivados , Animais , Área Sob a Curva , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Vírus da Influenza A Subtipo H3N2/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Modelos Biológicos , Oseltamivir/farmacocinética , Oseltamivir/farmacologiaRESUMO
Sargassum fusiforme (Harvey) Setchell has been shown to be a highly effective inhibitor of HIV-1 infection. To identify its mechanism of action, we performed bioactivity-guided fractionation on Sargassum fusiforme mixture. Here, we report isolation of a bioactive fraction SP4-2 (S. fusiforme), which at 8 mug/ml inhibited HIV-1 infection by 86.9%, with IC50 value of 3.7 mug. That represents 230-fold enhancement of antiretroviral potency as compared to the whole extract. Inhibition was mediated against both CXCR4 (X4) and CCR5 (R5) tropic HIV-1. Specifically, 10 mug/ml SP4-2 blocked HIV-1 fusion and entry by 53%. This effect was reversed by interaction of SP4-2 with sCD4, suggesting that S. fusiforme inhibits HIV-1 infection by blocking CD4 receptor, which also explained observed inhibition of both X4 and R5-tropic HIV-1. SP4-2 also inhibited HIV-1 replication after virus entry, by directly inhibiting HIV-1 reverse transcriptase (RT) in a dose dependent manner by up to 79%. We conclude that the SP4-2 fraction contains at least two distinct and biologically active molecules, one that inhibits HIV-1 fusion by interacting with CD4 receptor, and another that directly inhibits HIV-1 RT. We propose that S. fusiforme is a lead candidate for anti-HIV-1 drug development.
Assuntos
Produtos Biológicos/farmacologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Sargassum , Fármacos Anti-HIV/farmacologia , Antígenos CD4/efeitos dos fármacos , Antígenos CD4/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Inibidores Enzimáticos , Infecções por HIV/metabolismo , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/fisiologia , Humanos , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Receptores Virais/metabolismo , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacosRESUMO
We analyzed the dynamics of an influenza A/Albany/1/98 (H3N2) viral infection, using a set of mathematical models highlighting the differences between in vivo and in vitro infection. For example, we found that including virion loss due to cell entry was critical for the in vitro model but not for the in vivo model. Experiments were performed on influenza virus-infected MDCK cells in vitro inside a hollow-fiber (HF) system, which was used to continuously deliver the drug amantadine. The HF system captures the dynamics of an influenza infection, and is a controlled environment for producing experimental data which lend themselves well to mathematical modeling. The parameter estimates obtained from fitting our mathematical models to the HF experimental data are consistent with those obtained earlier for a primary infection in a human model. We found that influenza A/Albany/1/98 (H3N2) virions under normal experimental conditions at 37 degrees C rapidly lose infectivity with a half-life of approximately 6.6+/-0.2 h, and that the lifespan of productively infected MDCK cells is approximately 13 h. Finally, using our models we estimated that the maximum efficacy of amantadine in blocking viral infection is approximately 74%, and showed that this low maximum efficacy is likely due to the rapid development of drug resistance.
Assuntos
Amantadina/uso terapêutico , Vírus da Influenza A , Rim/virologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Animais , Morte Celular , Linhagem Celular , Cães , Farmacorresistência Viral , Vírus da Influenza A Subtipo H3N2 , Virus da Influenza A Subtipo H5N1 , Modelos Biológicos , Infecções por Orthomyxoviridae/virologia , Fatores de Tempo , Vírion , Virologia/instrumentação , Virologia/métodos , VirulênciaRESUMO
Pharmacodynamic investigation of antiviral compounds studies the relationship between drug exposure and the virological response. These studies are usually performed in animals and, eventually, in humans and are a very expensive proposition. To find a more efficient and less expensive method for determining pharmacodynamics of antiviral and antimicrobial compounds, the hollow fibre infection model (HFIM) system was developed to perform pharmacodynamic studies in vitro. This review covers the authors' studies on the use of in vitro hollow fibre bioreactor technologies for determining the pharmacodynamics of antiviral compounds for viruses grown in cultured cells, including HIV grown in CD4+ lymphoblastoid cells, vaccinia viruses grown in HeLa-S3 cells and influenza viruses grown in Madin-Darby canine kidney cells. Where possible, correlations between the pharmacodynamic index derived from the in vitro HFIM systems and clinical pharmacodynamic studies are made.
Assuntos
Antivirais/farmacocinética , Reatores Biológicos , Membranas Artificiais , Vírus/efeitos dos fármacos , Animais , Humanos , Vírus/crescimento & desenvolvimentoRESUMO
Monitoring antiviral resistance in influenza is critical to public health epidemiology and pandemic preparedness activities. Effective monitoring requires methods to detect low-level resistance and to monitor the change in resistance as a function of time and drug treatment. Resistance-conferring single-nucleotide mutations in influenza virus are ideal targets for such methods. In the present study, fives sets of paired TaqMan allele-specific PCR (ASPCR) assays were developed and validated for quantitative single-nucleotide polymorphism (SNP) analysis. This novel method using Delta Ct is termed allele-specific mixture analysis (ASMA) or FluASMA. The FluASMA assays target L26F, V27A, A30T, and S31N mutations in the A/Albany/1/98 (H3N2) M2 gene and H275Y mutation in the A/New Caledonia/20/99 (H1N1) NA gene and have a limit of quantification of 0.25-0.50% mutant. The error for % mutant estimation was less than 10% in all FluASMA assays, with intra-run Delta Ct coefficient of variance (CoV) at Assuntos
Farmacorresistência Viral/genética
, Vírus da Influenza A Subtipo H1N1/isolamento & purificação
, Vírus da Influenza A Subtipo H3N2/isolamento & purificação
, Influenza Humana/virologia
, Polimorfismo de Nucleotídeo Único
, Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
, Alelos
, Humanos
, Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos
, Vírus da Influenza A Subtipo H1N1/genética
, Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos
, Vírus da Influenza A Subtipo H3N2/genética
, Mutação Puntual
RESUMO
This report describes a flow cytometry drug susceptibility assay that uses a single fluorochrome-labeled monoclonal antibody to determine the acyclovir susceptibilities of herpes simplex virus (HSV) type 1 or type 2 clinical isolates. This assay yields 50% effective doses (drug concentrations that reduce the number of antigen-positive cells by 50%) for HSV clinical isolates that are equivalent to those obtained with the plaque reduction assay.
Assuntos
Aciclovir/farmacologia , Anticorpos Monoclonais/imunologia , Antígenos Virais/análise , Antivirais/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Citometria de Fluxo , Corantes Fluorescentes , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 2/imunologia , HumanosRESUMO
A flow cytometric (fluorescence-activated cell sorter [FACS]) assay was developed for analysis of the drug susceptibilities of wild-type and drug-resistant influenza A and B virus laboratory strains and clinical isolates for the neuraminidase (NA) inhibitors oseltamivir carboxylate, zanamivir, and peramivir. The drug susceptibilities of wild-type influenza viruses and those with mutations in the hemagglutinin (HA) and/or NA genes rendering them resistant to one or more of the NA inhibitors were easily determined with the FACS assay. The drug concentrations that reduced the number of virus-infected cells or the number of PFU by 50% as determined by the FACS assay were similar to those obtained with the more time-consuming and labor-intensive virus yield reduction assay. The NA inhibition (NAI) assay confirmed the resistance patterns demonstrated by the FACS and virus yield assays for drug-resistant influenza viruses with mutations in the NA gene. However, only the FACS and virus yield assays detected NA inhibitor-resistant influenza viruses with mutations in the HA gene but not in the NA gene. The FACS assay is more rapid and less labor-intensive than the virus yield assay and just as quantitative. The FACS assay determines the drug susceptibilities of influenza viruses with mutations in either the HA or NA genes, making the assay more broadly useful than the NAI assay for measuring the in vitro susceptibilities of influenza viruses for NA inhibitors. However, since only viruses with mutations in the NA gene that lead to resistance to the NA inhibitors correlate with clinical resistance, this in vitro assay should not be used in the clinical setting to determine resistance to NA inhibitors. The assay may be useful for determining the in vivo susceptibilities of other compounds effective against influenza A and B viruses.