RESUMO
Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.
Assuntos
Infecções por HIV , Inibidores da Protease de HIV , HIV-1 , Humanos , Inibidores da Protease de HIV/farmacologia , Inibidores da Protease de HIV/uso terapêutico , Darunavir/farmacologia , Darunavir/uso terapêutico , Sulfato de Atazanavir/farmacologia , Sulfato de Atazanavir/uso terapêutico , Farmacorresistência Viral , HIV-1/genética , Antirretrovirais/uso terapêutico , Infecções por HIV/tratamento farmacológico , Protease de HIV/genética , Protease de HIV/metabolismoRESUMO
Delivery of pharmacologically active nucleoside triphosphate analogs to sites of viral infection is challenging. In prior work we identified a 2'-C-methyl-1'-cyano-7-deaza-adenosine C-nucleotide analog with desirable selectivity and potency for the treatment of hepatitis C virus (HCV) infection. However, the prodrug selected for clinical development, GS-6620, required a high dose for meaningful efficacy and had unacceptable variability due to poor oral absorption as a result of suboptimal solubility, intestinal metabolism, and efflux transport. While obtaining clinical proof of concept for the nucleotide analog, a more effective prodrug strategy would be necessary for clinical utility. Here, we report an alternative prodrug of the same nucleoside analog identified to address liabilities of GS-6620. A phosphoramidate prodrug containing the nonproteinogenic amino acid methylalanine, an isopropyl ester and phenol in the (S) conformation at phosphorous, GS2, was found to have improved solubility, intestinal stability, and hepatic activation. GS2 is a more selective substrate for hepatically expressed carboxyl esterase 1 (CES1) and is resistant to hydrolysis by more widely expressed hydrolases, including cathepsin A (CatA) and CES2. Unlike GS-6620, GS2 was not cleaved by intestinally expressed CES2 and, as a result, was stable in intestinal extracts. Levels of liver triphosphate following oral administration of GS2 in animals were higher than those of GS-6620, even when administered under optimal conditions for GS-6620 absorption. Combined, these properties suggest that GS2 will have better oral absorption in the clinic when administered in a solid dosage form and the potential to extend the clinical proof of concept obtained with GS-6620.
Assuntos
Antivirais/uso terapêutico , Hepacivirus/patogenicidade , Nucleotídeos/uso terapêutico , Pró-Fármacos/uso terapêutico , Triazinas/uso terapêutico , Adenosina/análogos & derivados , Administração Oral , Alanina , Animais , Antivirais/administração & dosagem , Antivirais/farmacocinética , Células CACO-2 , Células Cultivadas , Cães , Hepacivirus/efeitos dos fármacos , Hepatite C/virologia , Humanos , Masculino , Nucleotídeos/administração & dosagem , Nucleotídeos/farmacocinética , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacocinética , Ratos , Triazinas/administração & dosagem , Triazinas/farmacocinética , Replicação Viral/efeitos dos fármacosRESUMO
A series of 2'-fluorinated C-nucleosides were prepared and tested for anti-HCV activity. Among them, the triphosphate of 2'-fluoro-2'-C-methyl adenosine C-nucleoside (15) was a potent and selective inhibitor of the NS5B polymerase and maintained activity against the S282T resistance mutant. A number of phosphoramidate prodrugs were then prepared and evaluated leading to the identification of the 1-aminocyclobutane-1-carboxylic acid isopropyl ester variant (53) with favorable pharmacokinetic properties including efficient liver delivery in animals.
Assuntos
Antivirais/química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Nucleosídeos/química , Nucleosídeos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Amidas/química , Amidas/farmacocinética , Amidas/farmacologia , Animais , Antivirais/farmacocinética , Células CACO-2 , Linhagem Celular , Cricetinae , Descoberta de Drogas , Farmacorresistência Viral , Halogenação , Hepacivirus/genética , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Humanos , Metilação , Simulação de Acoplamento Molecular , Nucleosídeos/farmacocinética , Ácidos Fosfóricos/química , Ácidos Fosfóricos/farmacocinética , Ácidos Fosfóricos/farmacologia , Mutação Puntual , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C-nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC50], 0.048 to 0.68 µM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC50, 1.5 µM). The active 5'-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with Ki/Km values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1'-CN and 2'-C-Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro. Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.
Assuntos
Antivirais/química , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Nucleosídeos/química , Nucleosídeos/farmacologia , Pró-Fármacos/farmacologia , Replicação Viral/efeitos dos fármacos , Antivirais/efeitos adversos , Linhagem Celular Tumoral , Sobrevivência Celular , Células Hep G2 , Humanos , Nucleosídeos/efeitos adversos , Pró-Fármacos/efeitos adversos , Pró-Fármacos/química , Proteínas não Estruturais Virais/antagonistas & inibidoresRESUMO
Hepatitis C virus (HCV) infection presents an unmet medical need requiring more effective treatment options. Nucleoside inhibitors (NI) of HCV polymerase (NS5B) have demonstrated pan-genotypic activity and durable antiviral response in the clinic, and they are likely to become a key component of future treatment regimens. NI candidates that have entered clinical development thus far have all been N-nucleoside derivatives. Herein, we report the discovery of a C-nucleoside class of NS5B inhibitors. Exploration of adenosine analogs in this class identified 1'-cyano-2'-C-methyl 4-aza-7,9-dideaza adenosine as a potent and selective inhibitor of NS5B. A monophosphate prodrug approach afforded a series of compounds showing submicromolar activity in HCV replicon assays. Further pharmacokinetic optimization for sufficient oral absorption and liver triphosphate loading led to identification of a clinical development candidate GS-6620. In a phase I clinical study, the potential for potent activity was demonstrated but with high intra- and interpatient pharmacokinetic and pharmacodynamic variability.
Assuntos
Antivirais/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Hepacivirus/enzimologia , Hepatite C/tratamento farmacológico , Nucleosídeos/farmacologia , Compostos Organofosforados/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Cães , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Hepatite C/enzimologia , Hepatite C/virologia , Humanos , Nucleosídeos/química , Compostos Organofosforados/química , Ratos , Carga ViralRESUMO
Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2'-C-methyl, 4'-methyl and 4'-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.
Assuntos
Antivirais/farmacologia , Núcleo Celular/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Hepacivirus/metabolismo , Mitocôndrias/metabolismo , RNA Polimerase II/metabolismo , Ribonucleosídeos/farmacologia , Transcrição Gênica/efeitos dos fármacos , Animais , Antivirais/efeitos adversos , Bovinos , Linhagem Celular , Hepatite C/tratamento farmacológico , Hepatite C/enzimologia , RNA Viral/biossíntese , Ribonucleosídeos/efeitos adversosRESUMO
A series of 1'-substituted analogs of 4-aza-7,9-dideazaadenosine C-nucleoside were prepared and evaluated for the potential as antiviral agents. These compounds showed a broad range of inhibitory activity against various RNA viruses. In particular, the whole cell potency against HCV when R=CN was attributed to inhibition of HCV NS5B polymerase and intracellular concentration of the corresponding nucleoside triphosphate.