RESUMO
Debio 025 is a potent inhibitor of hepatitis C virus (HCV) replication (J. Paeshuyse et al., Hepatology 43:761-770, 2006). In phase I clinical studies, monotherapy (a Debio 025 dose of 1,200 mg twice a day) resulted in a mean maximal decrease in the viral load of 3.6 log(10) units (R. Flisiak et al., Hepatology 47:817-826, 2008), whereas a reduction of 4.6 log(10) units was obtained in phase II studies when Debio 025 was combined with interferon (R. Flisiak et al., J. Hepatol., 48:S62, 2008). We here report on the particular characteristics of the in vitro anti-HCV activities of Debio 025. The combination of Debio 025 with either ribavirin or specifically targeted antiviral therapy for HCV (STAT-C) inhibitors (NS3 protease or NS5B [nucleoside and nonnucleoside] polymerase inhibitors) resulted in additive antiviral activity in short-term antiviral assays. Debio 025 has the unique ability to clear hepatoma cells from their HCV replicon when it is used alone or in combination with interferon and STAT-C inhibitors. Debio 025, when it was used at concentrations that have been observed in human plasma (0.1 or 0.5 muM), was able to delay or prevent the development of resistance to HCV protease inhibitors as well as to nucleoside and nonnucleoside polymerase inhibitors. Debio 025 forms an attractive drug candidate for the treatment of HCV infections in combination with standard interferon-based treatment and treatments that directly target the HCV polymerase and/or protease.
Assuntos
Antivirais/farmacologia , Ciclosporina/farmacologia , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Proteínas não Estruturais Virais/antagonistas & inibidores , Ensaios Clínicos como Assunto , Ciclofilinas/metabolismo , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Sinergismo Farmacológico , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/genética , Humanos , Replicon/efeitos dos fármacos , Ribavirina/farmacologia , Proteínas não Estruturais Virais/genéticaRESUMO
UNLABELLED: Debio-025 is an oral cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus activity in vitro. Its effect on viral load as well as its influence on intracellular Cyp levels was investigated in a randomized, double-blind, placebo-controlled study. Mean hepatitis C viral load decreased significantly by 3.6 log(10) after a 14-day oral treatment with 1200 mg twice daily (P < 0.0001) with an effect against the 3 genotypes (1, 3, and 4) represented in the study. In addition, the absence of viral rebound during treatment indicates that Debio-025 has a high barrier for the selection of resistance. In Debio-025-treated patients, cyclophilin B (CypB) levels in peripheral blood mononuclear cells decreased from 67 +/- 6 (standard error) ng/mg protein (baseline) to 5 +/- 1 ng/mg protein at day 15 (P < 0.01). CONCLUSION: Debio-025 induced a strong drop in CypB levels, coinciding with the decrease in hepatitis C viral load. These are the first preliminary human data supporting the hypothesis that CypB may play an important role in hepatitis C virus replication and that Cyp inhibition is a valid target for the development of anti-hepatitis C drugs.
Assuntos
Antivirais/uso terapêutico , Ciclofilina A/antagonistas & inibidores , Ciclofilinas/antagonistas & inibidores , Ciclosporina/uso terapêutico , Infecções por HIV/complicações , HIV-1 , Hepatite C/tratamento farmacológico , Peptidilprolil Isomerase/antagonistas & inibidores , Administração Oral , Adulto , Idoso , Antivirais/farmacocinética , Antivirais/farmacologia , Ciclofilina A/análise , Ciclofilinas/análise , Ciclosporina/farmacocinética , Ciclosporina/farmacologia , Método Duplo-Cego , Farmacorresistência Viral , Feminino , Infecções por HIV/imunologia , Hepacivirus/efeitos dos fármacos , Hepatite C/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Peptidilprolil Isomerase/análise , Placebos , Replicação Viral/efeitos dos fármacosRESUMO
DEB025 (alisporivir) is a synthetic cyclosporine with inhibitory activity against human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV). It binds to cyclophilin A (CypA) and blocks essential functions of CypA in the viral replication cycles of both viruses. DEB025 inhibits clinical HIV-1 isolates in vitro and decreases HIV-1 virus load in the majority of patients. HIV-1 isolates being naturally resistant to DEB025 have been detected in vitro and in nonresponder patients. By sequence analysis of their capsid protein (CA) region, two amino acid polymorphisms that correlated with DEB025 resistance were identified: H87Q and I91N, both located in the CypA-binding loop of the CA protein of HIV-1. The H87Q change was by far more abundant than I91N. Additional polymorphisms in the CypA-binding loop (positions 86, 91 and 96), as well as in the N-terminal loop of CA were detected in resistant isolates and are assumed to contribute to the degree of resistance. These amino acid changes may modulate the conformation of the CypA-binding loop of CA in such a way that binding and/or isomerase function of CypA are no longer necessary for virus replication. The resistant HIV-1 isolates thus are CypA-independent.
Assuntos
Fármacos Anti-HIV/farmacologia , Proteínas do Capsídeo/genética , Farmacorresistência Viral , Infecções por HIV/virologia , HIV-1/genética , Polimorfismo Genético , Sequência de Aminoácidos , Capsídeo/efeitos dos fármacos , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Ciclosporina , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/metabolismo , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto/efeitos dos fármacosRESUMO
Cyclophilin A is a peptidyl-propyl isomerase that binds the capsid (p24) protein of HIV-1 and facilitates replication. We report a cyclophilin inhibitor, a non-immunosuppressive cyclosporine analogue, Debio-025, that is about 15-times more potent than cyclosporine A and less toxic resulting in a selectivity index of more than 300. It was equally active against virus strains that were resistant toward inhibitors of the viral entry, fusion, or reverse transcription while it was not inhibitory to HIV-2 or SIV(MAC). Mechanism of action studies demonstrate that Debio-025 inhibits the HIV-1 replication by interfering with an early stage of the viral replication cycle. Indeed, addition of Debio-025 could be postponed for 2h before loosing its antiviral activity. The compound proved inactive against mutant viruses that are independent of cyclophilin A binding suggesting Debio-025 targets the cyclophilin A-capsid interaction.
Assuntos
Fármacos Anti-HIV/farmacologia , Ciclosporina/farmacologia , HIV-1/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Capsídeo/metabolismo , Ciclofilina A/metabolismo , HIV-2/efeitos dos fármacos , Humanos , Ligação Proteica , Vírus da Imunodeficiência Símia/efeitos dos fármacosRESUMO
Debio 025 is a cyclophilin (Cyp) inhibitor without calcineurin-binding properties. The drug inhibits viral replication of genotype 1b and 2a replicons in nanomolar concentrations and shows an additive to synergistic antiviral effect with interferon, ribavirin, and specifically targeted antiviral therapy for hepatitis C (STAT-C) drugs. There is no cross-resistance with protease and polymerase inhibitors. In humans, Debio 025 has shown activity against genotypes 1, 2, 3, and 4, and displays an additive antiviral effect with pegylated interferon (peg-IFN)alpha2a in genotype 1 and 4 patients. The most prominent side effect is reversible hyperbilirubinaemia caused by inhibition of biliary transporters. Debio 025 is a potent anti-HCV drug, with a novel mechanism of action and an efficacy profile that makes it an attractive candidate for combination with current and future HCV treatments.
Assuntos
Antivirais/uso terapêutico , Ciclofilinas/antagonistas & inibidores , Ciclosporina/uso terapêutico , Hepatite C Crônica/tratamento farmacológico , Animais , Antivirais/efeitos adversos , Antivirais/química , Antivirais/farmacocinética , Antivirais/farmacologia , Ciclosporina/efeitos adversos , Ciclosporina/química , Ciclosporina/farmacocinética , Ciclosporina/farmacologia , HumanosRESUMO
Debio-025 is a synthetic cyclosporine with no immunosuppressive capacity but a high inhibitory potency against cyclophilin A (CypA)-associated cis-trans prolyl isomerase (PPIase) activity. A lack of immunosuppressive effects compared to that of cyclosporine was demonstrated both in vitro and in vivo. For three cyclosporines, the inhibitory potential against PPIase activity was quantitatively correlated with that against human immunodeficiency virus type 1 (HIV-1) replication. Debio-025 selectively inhibited the replication of HIV-1 in a CD4+ cell line and in peripheral blood mononuclear cells: potent activity was demonstrated against clinical isolates of various HIV-1 subtypes, including isolates with multidrug resistance to reverse transcriptase and protease inhibitors. Simian immunodeficiency virus and HIV-2 strains were generally resistant to inhibition by Debio-025; however, some notable exceptions of sensitive HIV-2 clinical isolates were detected. In two-drug combination studies, additive inhibitory effects were found between Debio-025 and 19 clinically used drugs of different classes. Clinical HIV-1 isolates that are naturally resistant to Debio-025 and that do not depend on CypA for infection were identified. Comparison of the amino acid sequences of the CypA binding domain of the capsid (CA) protein from Debio-025-sensitive and -resistant HIV-1 isolates indicated that resistance was mostly associated with an H87Q/P exchange. Mechanistically, cyclosporines competitively inhibit the binding of CypA to the HIV-1 CA protein, which is an essential interaction required for early steps in HIV-1 replication. By real-time PCR we demonstrated that early reverse transcription is reduced in the presence of Debio-025 and that late reverse transcription is almost completely blocked. Thus, Debio-025 seems to interfere with the function of CypA during the progression/completion of HIV-1 reverse transcription.
Assuntos
Ciclofilinas/metabolismo , Ciclosporina/farmacologia , HIV-1/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Linhagem Celular , Ciclosporina/síntese química , Ciclosporina/química , Ciclosporina/metabolismo , HIV-1/patogenicidade , HIV-1/fisiologia , Humanos , Células Jurkat , Leucócitos Mononucleares/virologia , Testes de Sensibilidade Microbiana/métodosRESUMO
Cyclophilins (Cyps) are proteins that are ubiquitously present with peptidyl-prolyl cis-trans isomerase activity and play an important role in de novo protein folding and in isomerization of native proteins in several cellular systems. There is growing evidence that indicates CypB is a positive modulator of the HCV RNA-dependent RNA polymerase in the replication complex. Early in vitro and animal data with selective Cyp inhibitors show a potent anti-HCV effect. This anti-HCV effect was confirmed in the first patient study with the selective Cyp inhibitor Debio-025. Preclinical data suggest that Cyp inhibitors may present a higher barrier to the selection of resistance than protease and polymerase inhibitors and that a combination of Cyp inhibitors with either of these drugs or interferon results in additive or synergistic anti-HCV activity. By interfering at the level of host-viral interaction, Cyp inhibition may open the way for a novel approach to anti-HCV treatment that could be complementary, not only to interferon-based treatment, but also to future treatments that directly target HCV replication enzymes such as protease and polymerase inhibitors.
Assuntos
Ciclofilinas/antagonistas & inibidores , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Ciclofilinas/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Hepacivirus/enzimologia , Hepatite C/enzimologia , HumanosRESUMO
Inhibition of mitochondrial permeability transition pore (mPTP) opening by cyclosporin A or ischemic postconditioning attenuates lethal reperfusion injury. Its impact on major post-myocardial infarction events, including worsening of left ventricular (LV) function and death, remains unknown. We sought to determine whether pharmacological or postconditioning-induced inhibition of mPTP opening might improve functional recovery and survival following myocardial infarction in mice. Anesthetized mice underwent 25 min of ischemia and 24 h (protocol 1) or 30 days (protocol 2) of reperfusion. At reperfusion, they received no intervention (control), postconditioning (3 cycles of 1 min ischemia-1 min reperfusion), or intravenous injection of the mPTP inhibitor Debio-025 (10 mg/kg). At 24 h of reperfusion, mitochondria were isolated from the region at risk for assessment of the Ca(2+) retention capacity (CRC). Infarct size was measured by triphenyltetrazolium chloride staining. At 30 days of reperfusion, mortality and LV contractile function (echocardiography) were evaluated. Postconditioning and Debio-025 significantly improved Ca(2+) retention capacity (132 +/- 13 and 153 +/- 31 vs. 53 +/- 16 nmol Ca(2+)/mg protein in control) and reduced infarct size to 35 +/- 4 and 32 +/- 7% of area at risk vs. 61 +/- 6% in control (P < 0.05). At 30 days, ejection fraction averaged 74 +/- 6 and 77 +/- 6% in postconditioned and Debio-025 groups, respectively, vs. 62 +/- 12% in the control group (P < 0.05). At 30 days, survival was improved from 58% in the control group to 92 and 89% in postconditioned and Debio-025 groups, respectively. Inhibition of mitochondrial permeability transition at reperfusion improves functional recovery and mortality in mice.
Assuntos
Mitocôndrias Cardíacas/metabolismo , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão/fisiopatologia , Doença Aguda , Animais , Cálcio/metabolismo , Ciclosporina/farmacologia , Modelos Animais de Doenças , Precondicionamento Isquêmico Miocárdico , Masculino , Camundongos , Mitocôndrias Cardíacas/efeitos dos fármacos , Permeabilidade/efeitos dos fármacos , Análise de Sobrevida , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
UNLABELLED: Cyclosporin A (CsA) inhibits replication of the HCV subgenomic replicon, and this effect is believed to not be mediated by its immunosuppressive action. We found that DEBIO-025, a novel non-immunosuppressive cyclophilin inhibitor derived from CsA, inhibited HCV replication in vitro more potently than CsA. We also examined the inhibitory effect of DEBIO-025 on naive HCV genotypes 1a or 1b in vivo using chimeric mice with human hepatocytes. These mice were treated for 14 days with DEBIO-025, pegylated-interferon alpha-2a (Peg-IFN), a combination of either drugs, or CsA in combination with Peg-IFN. In mice treated with Peg-IFN, serum HCV RNA levels decreased approximately 10-fold whereas DEBIO-025 treatment alone did not induce any significant change. In mice treated with both DEBIO-025 and Peg-IFN, HCV RNA levels decreased more than 100-fold. All mice treated with Peg-IFN combined with CsA died within 4 days. The combination treatment of DEBIO-025 and Peg-IFN reduced HCV RNA levels and core protein expression in liver, indicating that the HCV RNA levels reduction in serum was attributable to intrahepatic inhibition of HCV replication. CONCLUSION: We demonstrated that DEBIO-025 was better tolerated than CsA, and that its anti-HCV effect appeared to be synergistic in combination with Peg-IFN in vivo.
Assuntos
Antivirais/uso terapêutico , Ciclofilinas/antagonistas & inibidores , Ciclosporina/uso terapêutico , Hepatite C/tratamento farmacológico , Interferon-alfa/uso terapêutico , Polietilenoglicóis/uso terapêutico , RNA Viral/efeitos dos fármacos , Animais , Antivirais/farmacologia , Ciclosporina/farmacologia , Genótipo , Hepacivirus/genética , Humanos , Imuno-Histoquímica , Terapia de Imunossupressão , Interferon alfa-2 , Interferon-alfa/farmacologia , Camundongos , Camundongos SCID , Polietilenoglicóis/farmacologia , RNA Viral/metabolismo , Proteínas Recombinantes , Replicon/efeitos dos fármacos , Albumina Sérica , Quimeras de Transplante , Proteínas do Core Viral/metabolismoRESUMO
Cyclosporin A (CsA) inhibits the in vitro replication of HCV subgenomic replicons. We here report on the potent anti-HCV activity of the non-immunosuppressive cyclosporin DEBIO-025. The 50% effective concentration for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by DEBIO-025 was 0.27 +/- 0.03 microg/mL and for CsA 2.8 +/- 0.4 microg/mL. The concentration that reduced the growth of exponentially proliferating Huh 5-2 cells by 50% was greater than 27 microg/mL for DEBIO-025 and 12 +/- 6 microg/mL for CsA, resulting in a selectivity index of approximately 900 for DEBIO-025 and 40 for CsA. The superior activity of DEBIO-025, as compared with CsA, was corroborated by monitoring HCV RNA levels in Huh 5-2, two other HCV subgenomic replicon-containing cell lines, and by monitoring the luciferase signal and viral antigen production in hepatoma cells that had been infected with an infectious full-length chimeric HCV construct. The combination of interferon alpha 2a with either CsA or DEBIO-025 resulted in an additive to slightly synergistic antiviral activity. DEBIO-025, at concentrations of 0.5 and 1 microg/mL, was able to clear cells from their HCV replicon within three to four passages, whereas treatment with CsA at the same concentrations for seven consecutive passages did not result in clearance of the HCV replicon. In conclusion, DEBIO-025, a compound that is also endowed with potent anti-HIV activity and is well tolerated in animals and humans, may form an attractive new option for the therapy of HCV infections, particularly in HCV/HIV co-infected patients.
Assuntos
Antivirais/farmacologia , Ciclosporina/farmacologia , Hepacivirus/fisiologia , Replicação Viral/efeitos dos fármacos , Antivirais/administração & dosagem , Linhagem Celular Tumoral/patologia , Proliferação de Células/efeitos dos fármacos , Ciclosporina/administração & dosagem , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Sinergismo Farmacológico , Hepacivirus/genética , Hepatite C/virologia , Humanos , Concentração Osmolar , RNA Viral/metabolismoRESUMO
Murine primary cells are poorly permissive to human immunodeficiency virus type 1 (HIV-1) vector infection. Retroviral infectivity is influenced by dominant inhibitors such as TRIM5alpha. Sensitivity to TRIM5alpha is altered by interactions between cyclophilin A and the HIV-1 capsid. Here we demonstrate that competitive inhibitors of cyclophilins, cyclosporine or the related Debio-025, stimulate HIV-1 vector transduction of primary murine cells, including bone marrow and macrophages, up to 20-fold. Unexpectedly, the infectivity of an HIV-1 mutant or a simian lentivirus that does not recruit cyclophilin A is also stimulated by these drugs. We propose that cyclosporine and related compounds will be useful tools for experimental infection of murine primary cells. It is possible that HIV-1 infection of murine cells is inhibited by dominant factors related to immunophilins.
Assuntos
Ciclosporina/farmacologia , Vetores Genéticos , HIV-1/metabolismo , Imunossupressores/farmacologia , Transdução Genética , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/virologia , Ciclofilina A/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Feminino , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Humanos , Lentivirus/fisiologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/metabolismo , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
In this study, we asked if a naturally occurring HIV-1 variant exists that circumvents CypA dependence in human cells. To address this issue, we sought viruses for CypA independence using Debio-025, a cyclosporine A (CsA) analog that disrupts CypA-capsid interaction. Surprisingly, viral variants from the Main group replicate even in the presence of the drug. Sequencing analyses revealed that these viruses encode capsid substitutions within the CypA-binding site (V86P/H87Q/I91V/M96I). When we introduced these substitutions into viruses that normally rely on CypA for replication, these mutants no longer depended on CypA, suggesting that naturally occurring capsid substitutions obviate the need for CypA. This is the first demonstration that isolates from the Main group naturally develop CypA-independent strategies to replicate in human cells. Surprisingly, we found that these capsid substitutions render HIV-1 capable of infecting Owl monkey (OMK) cells that highly restrict HIV-1. OMK cell resistance to HIV-1 is mediated via TRIM-Cyp, which arose from a retrotransposition of CypA into the TRIM5 alpha gene. Interestingly, saturation experiments suggest that the Pro86/Gln87/Val91/Ile96 capsid core is "invisible" to TRIM-Cyp. This study demonstrates that specific capsid substitutions can release HIV-1 from both CypA dependence in human cells and TRIM-Cyp restriction in monkey cells.
Assuntos
Proteínas do Capsídeo/química , Capsídeo/química , Ciclofilina A/química , HIV-1/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Aotidae , Sítios de Ligação , Western Blotting , Linhagem Celular , Ciclofilinas/química , Humanos , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Polimorfismo Genético , Retroelementos , Fatores de TempoRESUMO
Cyclosporin A (CsA) is highly neuroprotective in several animal models of acute neurological damage and neurodegenerative disease with inhibition of the mitochondrial permeability transition (mPT) having emerged as a possible mechanism for the observed neuroprotection. In the present study, we have evaluated two new nonimmunosuppressive cyclosporin analogs NIM811 (Novartis) and UNIL025 (Debiopharm) for their ability to inhibit mPT in rat brain-derived mitochondria. Both NIM811 and UNIL025 were found to be powerful inhibitors of calcium-induced mitochondrial swelling under energized and deenergized conditions, and the maximal effects were identical to those of native CsA. The potencies of mPT inhibition by NIM811 and UNIL025 were stronger, with almost one order of magnitude higher potency for UNIL025 compared to CsA, correlating to their respective inhibitory action of cyclophilin activity. These compounds will be instrumental in the evaluation of mPT as a central target for neuroprotection in vivo.