Alisporivir Research Articles

Taming Conformational Heterogeneity on Ion Racetrack to Unveil Principles that Drive Membrane Permeation of Cyclosporines.

Our study reveals the underlying principles governing the passive membrane permeability in three large N-methylated macrocyclic peptides (N-MeMPs): cyclosporine A (CycA), Alisporivir (ALI), and cyclosporine H (CycH). We determine a series of conformers required for robust passive membrane diffusion and those relevant to other functions, such as binding to protein targets or intermediates, in the presence of solvent additives. We investigate the conformational interconversions and establish correlations with the membrane permeability. Nuclear magnetic resonance (NMR) and cyclic ion-mobility spectrometry-mass spectrometry (cIMS-MS) are employed to characterize conformational heterogeneity and identify cis-amides relevant for good membrane permeability. In addition, ion mobility selected cIMS-MS and infrared (IR) multiple-photon dissociation (IRMPD) spectroscopy experiments are conducted to evaluate the energy barriers between conformations. We observe that CycA and ALI, both cyclosporines with favorable membrane permeabilities, display multiple stable and well-defined conformers. In contrast, CycH, an epimer of CycA with limited permeability, exhibits fewer and fewer stable conformers. We demonstrate the essential role of the conformational shift from the aqueous cis MeVal11-MeBmt1 state (A1) to the closed conformation featuring cis MeLeu9-MeLeu10 (C1) in facilitating membrane permeation. Additionally, we highlight that the transition from A1 to the all-trans open conformation (O1) is specifically triggered by the presence of CaCl2. We also capture a set of conformers with cis Sar3-MeLeu4, MeLeu9-MeLeu10, denoted as I. Conformationally selected cIMS-MS and IRMPD data of [CycA+Ca]2+ show immediate repopulation of the original population distribution, suggesting that CaCl2 smooths out the energy barriers. Finally, our work presents an improved sampling molecular dynamics approach based on a refined force field that not only consistently and accurately captures established conformers of cyclosporines but also exhibits strong predictive capabilities for novel conformers.

CyclosporinA Derivative as Therapeutic Candidate for Alport Syndrome by Inducing Mutant Type IV Collagen Secretion.

Type IV collagen α3,4,5 (α345(IV)) is an obligate trimer that is secreted to form a collagen network, which is the structural foundation of basement membrane. Mutation in one of the genes (COL4A3, A4, A5) encoding these proteins underlies the progressive genetic nephropathy Alport syndrome (AS) due to deficiency in trimerization and/or secretion of the α345(IV) trimer. Thus, improving mutant α345(IV) trimerization and secretion could be a good therapeutic approach for AS. Using the nanoluciferase-based platform that we previously developed to detect α345(IV) formation and secretion in HEK293T cells, we screened libraries of natural product extracts and compounds to find a candidate compound capable of increasing mutant α345(IV) secretion. The screening of >13,000 extracts and >600 compounds revealed that cyclosporin A (CsA) increased the secretion of mutant α345(IV)-G1244D. To elucidate the mechanism of the effect of CsA, we evaluated CsA derivatives with different ability to bind to calcineurin (Cn) and cyclophilin (Cyp). Alisporivir (ALV), which binds to Cyp but not to Cn, increased the trimer secretion of mutant α345(IV). Knockdown studies on Cyps showed that PPIF/CypD was involved in the trimer secretion-enhancing activity of CsA and ALV. We confirmed that other α345(IV) mutants are also responsive to CsA and ALV. CsA was previously reported to improve proteinuria in AS patients, but due to its nephrotoxic effect, CsA is not recommended for treatment in AS patients. Our data raise the possibility that ALV could be a safer option than CsA. This study provides a novel therapeutic candidate for AS with an innovative mechanism of action, and reveals an aspect of the intracellular regulatory mechanism of α345(IV) that was previously unexplored.

Effects of immunophilin inhibitors and non-immunosuppressive analogs on coronavirus replication in human infection models.

RationaleHuman coronaviruses (HCoVs) seriously affect human health by causing respiratory diseases ranging from common colds to severe acute respiratory diseases. Immunophilins, including peptidyl-prolyl isomerases of the FK506-binding protein (FKBP) and the cyclophilin family, are promising targets for pharmaceutical inhibition of coronavirus replication, but cell-type specific effects have not been elucidated. FKBPs and cyclophilins bind the immunosuppressive drugs FK506 and cyclosporine A (CsA), respectively.MethodsPrimary human bronchial epithelial cells (phBECs) were treated with CsA, Alisporivir (ALV), FK506, and FK506-derived non-immunosuppressive analogs and infected with HCoV-229E. RNA and protein were assessed by RT-qPCR and immunoblot analysis. Treatment with the same compounds was performed in hepatoma cells (Huh-7.5) infected with HCoV-229E expressing Renilla luciferase (HCoV-229E-RLuc) and the kidney cell line HEK293 transfected with a SARS-CoV-1 replicon expressing Renilla luciferase (SARS-CoV-1-RLuc), followed by quantification of luminescence as a measure of viral replication.ResultsBoth CsA and ALV robustly inhibited viral replication in all models; both compounds decreased HCoV-229E RNA in phBECs and reduced luminescence in HCoV-229E-RLuc-infected Huh7.5 and SARS-CoV-1-RLuc replicon-transfected HEK293. In contrast, FK506 showed inconsistent and less pronounced effects in phBECs while strongly affecting coronavirus replication in Huh-7.5 and HEK293. Two non-immunosuppressive FK506 analogs had no antiviral effect in any infection model.ConclusionThe immunophilin inhibitors CsA and ALV display robust anti-coronaviral properties in multiple infection models, including phBECs, reflecting a primary site of HCoV infection. In contrast, FK506 displayed cell-type specific effects, strongly affecting CoV replication in Huh7.5 and HEK293, but inconsistently and less pronounced in phBECs.

Cyclophilin inhibitors restrict Middle East respiratory syndrome coronavirusviainterferon-λin vitroand in mice

While severe coronavirus infections, including Middle East respiratory syndrome coronavirus (MERS-CoV), cause lung injury with high mortality rates, protective treatment strategies are not approved for clinical use.We elucidated the molecular mechanisms by which the cyclophilin inhibitors cyclosporin A (CsA) and alisporivir (ALV) restrict MERS-CoV to validate their suitability as readily available therapy in MERS-CoV infection.Calu-3 cells and primary human alveolar epithelial cells (hAECs) were infected with MERS-CoV and treated with CsA or ALV or inhibitors targeting cyclophilin inhibitor-regulated molecules including calcineurin, nuclear factor of activated T-cells (NFATs) or mitogen-activated protein kinases. Novel CsA-induced pathways were identified by RNA sequencing and manipulated by gene knockdown or neutralising antibodies. Viral replication was quantified by quantitative real-time PCR and 50% tissue culture infective dose. Data were validated in a murine MERS-CoV infection model.Both CsA and ALV reduced MERS-CoV titres and viral RNA replication in Calu-3 cells and hAECs, improving epithelial integrity. While neither calcineurin nor NFAT inhibition reduced MERS-CoV propagation, blockade of c-Jun N-terminal kinase diminished infectious viral particle release but not RNA accumulation. Importantly, CsA induced interferon regulatory factor 1 (IRF1), a pronounced type III interferon (IFNλ) response and expression of antiviral genes. Downregulation of IRF1 or IFNλ increased MERS-CoV propagation in the presence of CsA. Importantly, oral application of CsA reduced MERS-CoV replication in vivo, correlating with elevated lung IFNλ levels and improved outcome.We provide evidence that cyclophilin inhibitors efficiently decrease MERS-CoV replication in vitro and in vivo via upregulation of inflammatory antiviral cell responses, in particular IFNλ. CsA might therefore represent a promising candidate for treating MERS-CoV infection.

Adaptive Mutations in Replicase Transmembrane Subunits Can Counteract Inhibition of Equine Arteritis Virus RNA Synthesis by Cyclophilin Inhibitors.

Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse RNA viruses, including arteriviruses and coronaviruses, which both belong to the order Nidovirales In this study, we aimed to identify arterivirus proteins involved in the mode of action of cyclophilin inhibitors and to investigate how these compounds inhibit arterivirus RNA synthesis in the infected cell. Repeated passaging of the arterivirus prototype equine arteritis virus (EAV) in the presence of CsA revealed that reduced drug sensitivity is associated with the emergence of adaptive mutations in nonstructural protein 5 (nsp5), one of the transmembrane subunits of the arterivirus replicase polyprotein. Introduction of singular nsp5 mutations (nsp5 Q21R, Y113H, or A134V) led to an ∼2-fold decrease in sensitivity to CsA treatment, whereas combinations of mutations further increased EAV's CsA resistance. The detailed experimental characterization of engineered EAV mutants harboring CsA resistance mutations implicated nsp5 in arterivirus RNA synthesis. Particularly, in an in vitro assay, EAV RNA synthesis was far less sensitive to CsA treatment when nsp5 contained the adaptive mutations mentioned above. Interestingly, for increased sensitivity to the closely related drug ALV, CsA-resistant nsp5 mutants required the incorporation of an additional adaptive mutation, which resided in nsp2 (H114R), another transmembrane subunit of the arterivirus replicase. Our study provides the first evidence for the involvement of nsp2 and nsp5 in the mechanism underlying the inhibition of arterivirus replication by cyclophilin inhibitors.IMPORTANCE Currently, no approved treatments are available to combat infections with nidoviruses, a group of positive-stranded RNA viruses, including important zoonotic and veterinary pathogens. Previously, the cyclophilin inhibitors cyclosporine (CsA) and alisporivir (ALV) were shown to inhibit the replication of diverse nidoviruses (both arteriviruses and coronaviruses), and they may thus represent a class of pan-nidovirus inhibitors. In this study, using the arterivirus prototype equine arteritis virus, we have established that resistance to CsA and ALV treatment is associated with adaptive mutations in two transmembrane subunits of the viral replication machinery, nonstructural proteins 2 and 5. This is the first evidence for the involvement of specific replicase subunits of arteriviruses in the mechanism underlying the inhibition of their replication by cyclophilin inhibitors. Understanding this mechanism of action is of major importance to guide future drug design, both for nidoviruses and for other RNA viruses inhibited by these compounds.

Efficacy and safety of alisporivir for the treatment of hepatitis C infection

ABSTRACTIntroduction: Alisporivir (ALV) (previously known as Debio 025) is a potent, pangenotypic host-targeting antiviral oral agent acting on cyclophilin A, which is necessary for HCV replication.Areas covered: This article reviews the therapeutic efficacy and safety of ALV for the treatment of HCV infection.Expert opinion: Direct-acting antivirals (DAAs) have revolutionized the HCV antiviral treatment paradigm with success rates well above 95% for all HCV genotypes. However, challenges still remain in certain patient populations such as those who have developed resistance and have experienced multi-DAA failure. To cure HCV infection, a treatment regimen must combine antiviral potency and a high barrier to resistance. ALV fulfills this need as shown by the studies evaluating its clinical efficacy. Nevertheless, ALV missed the chance to be included in the HCV treatment armamentarium after the FDA halted clinical studies following reports of serious side effects (three cases of pancreatitis, one lethal). However, it is possible that ALV could still be considered for HCV-infected non-cirrhotic patients that are infected with a multiresistant virus or with HCV genotype 3, although it must be said that the drug industry would be reluctant to invest in new antivirals if the current clinical need is effectively met.

X-ray structure of alisporivir in complex with cyclophilin A at 1.5 Å resolution.

Alisporivir (ALV) is an 11-amino-acid hydrophobic cyclic peptide with N-methyl-D-alanine and N-ethyl-L-valine (NEV) residues at positions 3 and 4, respectively. ALV is a non-immunosuppressive cyclosporin A (CsA) derivative. This inhibitor targets cyclophilins (Cyps), a family of proteins with peptidyl-prolyl cis/trans isomerase enzymatic activity. Cyps act as protein chaperones and are involved in numerous cellular functions. Moreover, Cyps have been shown to be an essential cofactor for the replication of many viruses, including Hepatitis C virus and Human immunodeficiency virus, and have also been shown to be involved in mitochondrial diseases. For these reasons, cyclophilins represent an attractive drug target. The structure of ALV in complex with cyclophilin A (CypA), the most abundant Cyp in humans, has been determined at 1.5 Å resolution. This first structure of the CypA-ALV complex shows that the binding of ALV is highly similar to that of CsA. The high resolution allowed the unambiguous determination of the conformations of residues 3 and 4 in ALV when bound to its target. In particular, the side-chain conformation of NEV4 precludes the interaction of the CypA-ALV complex with calcineurin, a cellular protein phosphatase involved in the immune response, which explains the non-immunosuppressive property of ALV. This study provides detailed molecular insights into the CypA-ALV interaction.

A Cyclophilin Inhibitor for Treating HCV Genotypes 2 and 3 Infections

Hepatitis C virus (HCV) genotype 3 infection is considered difficult to treat without peginterferon and ribavirin; sustained virologic response (SVR) rates are <60% among patients with cirrhosis and previous null response. In an industry-sponsored, phase IIb study, researchers assessed the efficacy of once-daily oral alisporivir (a cyclophilin inhibitor) plus ribavirin (400 mg twice daily) with or without peginterferon (180 µg weekly) for …

The Novel Cyclophilin Inhibitor CPI-431-32 Concurrently Blocks HCV and HIV-1 Infections via a Similar Mechanism of Action.

HCV-related liver disease is the main cause of morbidity and mortality of HCV/HIV-1 co-infected patients. Despite the recent advent of anti-HCV direct acting antivirals (DAAs), the treatment of HCV/HIV-1 co-infected patients remains a challenge, as these patients are refractory to most therapies and develop liver fibrosis, cirrhosis and liver cancer more often than HCV mono-infected patients. Until the present study, there was no suitable in vitro assay to test the inhibitory activity of drugs on HCV/HIV-1 co-infection. Here we developed a novel in vitro “co-infection” model where HCV and HIV-1 concurrently replicate in their respective main host target cells—human hepatocytes and CD4+ T-lymphocytes. Using this co-culture model, we demonstrate that cyclophilin inhibitors (CypI), including a novel cyclosporin A (CsA) analog, CPI-431-32, simultaneously inhibits replication of both HCV and HIV-1 when added pre- and post-infection. In contrast, the HIV-1 protease inhibitor nelfinavir or the HCV NS5A inhibitor daclatasvir only blocks the replication of a single virus in the “co-infection” system. CPI-431-32 efficiently inhibits HCV and HIV-1 variants, which are normally resistant to DAAs. CPI-431-32 is slightly, but consistently more efficacious than the most advanced clinically tested CypI—alisporivir (ALV)—at interrupting an established HCV/HIV-1 co-infection. The superior antiviral efficacy of CPI-431-32 over ALV correlates with its higher potency inhibition of cyclophilin A (CypA) isomerase activity and at preventing HCV NS5A-CypA and HIV-1 capsid-CypA interactions known to be vital for replication of the respective viruses. Moreover, we obtained evidence that CPI-431-32 prevents the cloaking of both the HIV-1 and HCV genomes from cellular sensors. Based on these results, CPI-431-32 has the potential, as a single agent or in combination with DAAs, to inhibit both HCV and HIV-1 infections.

Development and validation of a "capture-fusion" model to study drug sensitivity of patient-derived hepatitis C.

Emerging therapies for chronic hepatitis C viral (HCV) infection involve inhibition of viral enzymes with drug combinations. Natural, or treatment-induced, enzyme polymorphisms reduce efficacy. We developed a phenotyping assay to aid drug selection based on viral transfer from monocytes to hepatocytes. We studied HCV in monocytes from infected patients and developed a model in which patient-derived HCV is "captured" by the cell line THP-1 and replication assessed after fusion to hepatoma cells. We found that monocytes from HCV-infected patients harbor virus that replicates when cells are fused to hepatocytes. THP-1 cells incubated with infected sera capture HCV, which replicates when fused to hepatocytes. Inhibitable replication of all HCV genotypes was achieved (42 of 52 isolates). We measured sensitivity of telaprevir (TVR) and alisporivir (AVR) in different genotypes, and showed differences in 50% inhibitory concentration (IC50 ) correlating with clinical response (TVR IC50 for genotype (G)1 was 0.042 ± 0.003 vs. 0.117 ± 0.015 μM for G3, whereas AVR IC50 for G1 was 0.139 ± 0.013 vs. 0.044 ± 0.007 μM for G3). We tested TVR-resistant viral isolates and identified changes in IC50 . One patient with a poor clinical response to TVR and wild-type viral sequence showed reduced TVR sensitivity in our assay. We studied samples from a 2-week TVR monotherapy study in which 5 of 8 patients with G3 HCV did not respond whereas 3 of 8 patients did. The "capture-fusion" assay correctly identified responders. The capture-fusion model represents a promising new technique that may help identify appropriate treatment strategies for patients with chronic HCV infection.

Alisporivir with peginterferon/ribavirin in patients with chronic hepatitis C genotype 1 infection who failed to respond to or relapsed after prior interferon-based therapy: FUNDAMENTAL, a Phase II trial.

Alisporivir (ALV) is an oral, investigational host-targeting agent, with pangenotypic activity against hepatitis C virus (HCV). This randomized, double-blind, placebo-controlled, Phase II study explored the efficacy and safety of ALV with peginterferon-α2a/ribavirin (PR) in patients with chronic HCV genotype 1 infection in whom prior PR had failed (43% relapsers, 34% null responders and 23% partial responders). Four-hundred-and-fifty-nine patients were randomized (1:1:1:1) to ALV 600 mg once daily (QD), ALV 800 mg QD, ALV 400 twice daily (BID) or placebo plus PR for 48 weeks. When the global ALV trial programme was put on clinical hold, all patients in this study had received ≥31 weeks of randomized treatment; patients completed 48 weeks on PR alone. All ALV groups demonstrated superior rates of complete early virologic response (cEVR; primary endpoint) vs PR alone (P ≤ 0.0131), with highest cEVR rate seen with ALV 400 mg BID (74% vs 36% with PR alone; P < 0.0001). Respective SVR12 rates (key secondary endpoint) were 65% vs 26% in prior relapsers, 63% vs 5% in partial responders and 68% vs 3% in null responders. In patients who received >40 weeks of randomized treatment, the SVR12 rate was 89% for ALV 400 mg BID vs 30% for PR alone (P = 0.0053). Rates of viral breakthrough and relapse were lowest with ALV 400 mg BID. One case of pancreatitis (fully recovered) occurred with ALV/PR. Common AEs were headache, fatigue, anaemia, neutropenia and nausea. Hypertension was infrequent, but more common with ALV. ALV merits further investigation in interferon-free regimens in combination with direct-acting antiviral agents.

Modeling viral kinetics and treatment outcome during alisporivir interferon-free treatment in hepatitis C virus genotype 2 and 3 patients

Alisporivir (ALV) is a cyclophilin inhibitor with pan-genotypic activity against hepatitis C virus (HCV). Here, we characterize the viral kinetics observed in 249 patients infected with HCV genotypes 2 or 3 and treated for 6 weeks with different doses of ALV with or without ribavirin (RBV). We use this model to predict the effects of treatment duration and different doses of ALV plus RBV on sustained virologic response (SVR). Continuous viral decline was observed in 214 (86%) patients that could be well described by the model. All doses led to a high level of antiviral effectiveness equal to 0.98, 0.96, and 0.90 in patients treated with 1,000, 800, and 600 mg of ALV once-daily, respectively. Patients that received RBV had a significantly faster rate of viral decline, which was attributed to an enhanced loss rate of infected cells, δ (mean δ = 0.35 d(-1) vs. 0.21 d(-1) in patients ± RBV, respectively; P = 0.0001). The remaining 35 patients (14%) had a suboptimal response with flat or increasing levels of HCV RNA after 1 week of treatment, which was associated with ALV monotherapy, high body weight, and low RBV levels in patients that received ALV plus RBV. Assuming full compliance and the same proportion of suboptimal responders, the model predicted 71% and 79% SVR after ALV 400 mg with RBV 400 mg twice-daily for 24 and 36 weeks, respectively. The model predicted that response-guided treatment could allow a reduction in mean treatment duration to 25.3 weeks and attain a 78.6% SVR rate. ALV plus RBV may represent an effective IFN-free treatment that is predicted to achieve high SVR rates in patients with HCV genotype 2 or 3 infection.

Current and Future Treatment of Chronic Hepatitis C Genotype 2 and 3

Hepatitis C genotype 2 and 3 accounts for a significant number of HCV infected patients around the world. The combination of peginterferon alpha and ribavirin (PEG-IFN/RBV) demonstrated favourable results in treatment of genotype 2 and 3 infected patients with sustained virological responses rates of up to 80 %. In 2011, the first direct acting antivirals (DAA) have been approved but so far only for genotype 1. Several other antivirals are currently tested in clinical trials. Two compounds demonstrated promising results in patients with genotype 2 and 3. Sofosbuvir (SOF), a NS5B polymerase inhibitor, proved to be effective even without PEG-IFN and approval of an interferon-free regimen is foreseeable. Alisporivir (ALV), a host-targeting antiviral demonstrated encouraging results in phase-I and -II trials in genotype 2 and 3 patients and offers a new mechanism of action. However, further trials are needed to prove long-term safety and the efficacy in difficult-to-treat patients.

Sa1031 Mild to Moderate Hepatic Impairment Has No Effect on the Single Dose Pharmacokinetics of Alisporivir (ALV)

BACKGROUND: VITAL-1 study assessed safety, tolerability and antiviral activity of the cyclophilin inhibitor alisporivir (ALV) as monotherapy or combination with ribavirin (RBV) compared to IFN/RBV and ALV/IFN in treatment-naive GT2/3 HCV patients. We describe genotypic and phenotypic changes in patients from this study who experienced a viral breakthrough (VB). METHODS: VITAL-1 study patients on IFN-free regimens that did not achieve RVR (, 25 U/ml) at week 4 were administered IFN/RBV/600 mg QD ALV starting at week 6 (IFN-add-on). VB defined as an increase of HCV RNA .1 log10 above nadir. The HCV NS5A gene was analyzed by population sequencing. The entire NS5A region isolated at baseline and at VB was cloned into a JFH1subgenomic shuttle vector for phenotypic analyses. Selected mutations were engineered into a wild type replicon and their impact on replication fitness and susceptibility to ALV and direct acting antiviral drugs were determined. RESULTS: The VB rate with IFN-free or IFN-add-on regimens was 2.7% (7/ 260). Low ALV drug level (Cmin , 20% of group mean; , 100 ng/mL) was observed (5/ 7 VB patients). Genotypic analysis of the NS5A gene revealed amino acid changes in these patients at the time of VB compared to baseline. Replicons bearing NS5A from patients at breakthrough conferred 1.2-16.9-fold shift over baseline EC50 values; 5/7 had impaired replication fitness. No cross-resistance to other classes of HCV inhibitors was observed. Most frequently identified mutations were D320E (D316E in G2), R347W and A349V in Domain II of NS5A, which by themselves decreased ALV susceptibility by 5-, 4, and 8-fold, respectively, in vitro when engineered into wild-type replicon. Combination of multiple mutations decreased susceptibility further, but generally at the cost of replication fitness. CONCLUSION: In treatment-naive G2/3 patients with viral breakthrough, mutations in NS5A domain II were selected, which individually conferred ≤ 8-fold reduced susceptibility to ALV in vitro. The combination of multiple mutations was required to further reduce susceptibility which is consistent with the high barrier to resistance for host-targeting ALV. Observed genotypic changes did not confer cross-resistance to other classes of HCV inhibitors, supporting the use of ALV in future drug combination therapies.

Sa1033 Interferon-Free Alisporivir Treatment Down-Regulates Interferon-Stimulated Genes Suggesting a Unique Antiviral Mechanism of Action for the Cyclophilin Inhibitor Alisporivir

BACKGROUND: VITAL-1 study assessed safety, tolerability and antiviral activity of the cyclophilin inhibitor alisporivir (ALV) as monotherapy or combination with ribavirin (RBV) compared to IFN/RBV and ALV/IFN in treatment-naive GT2/3 HCV patients. We describe genotypic and phenotypic changes in patients from this study who experienced a viral breakthrough (VB). METHODS: VITAL-1 study patients on IFN-free regimens that did not achieve RVR (, 25 U/ml) at week 4 were administered IFN/RBV/600 mg QD ALV starting at week 6 (IFN-add-on). VB defined as an increase of HCV RNA .1 log10 above nadir. The HCV NS5A gene was analyzed by population sequencing. The entire NS5A region isolated at baseline and at VB was cloned into a JFH1subgenomic shuttle vector for phenotypic analyses. Selected mutations were engineered into a wild type replicon and their impact on replication fitness and susceptibility to ALV and direct acting antiviral drugs were determined. RESULTS: The VB rate with IFN-free or IFN-add-on regimens was 2.7% (7/ 260). Low ALV drug level (Cmin , 20% of group mean; , 100 ng/mL) was observed (5/ 7 VB patients). Genotypic analysis of the NS5A gene revealed amino acid changes in these patients at the time of VB compared to baseline. Replicons bearing NS5A from patients at breakthrough conferred 1.2-16.9-fold shift over baseline EC50 values; 5/7 had impaired replication fitness. No cross-resistance to other classes of HCV inhibitors was observed. Most frequently identified mutations were D320E (D316E in G2), R347W and A349V in Domain II of NS5A, which by themselves decreased ALV susceptibility by 5-, 4, and 8-fold, respectively, in vitro when engineered into wild-type replicon. Combination of multiple mutations decreased susceptibility further, but generally at the cost of replication fitness. CONCLUSION: In treatment-naive G2/3 patients with viral breakthrough, mutations in NS5A domain II were selected, which individually conferred ≤ 8-fold reduced susceptibility to ALV in vitro. The combination of multiple mutations was required to further reduce susceptibility which is consistent with the high barrier to resistance for host-targeting ALV. Observed genotypic changes did not confer cross-resistance to other classes of HCV inhibitors, supporting the use of ALV in future drug combination therapies.

Sa1030 Alisporivir Interferon-Free G2/3 Vital-1 Study: Low Rate of Viral Breakthrough and Combination of Multiple NS5A Domain II Mutations Required to Reduce Susceptibility to Alisporivir In Vitro

BACKGROUND: VITAL-1 study assessed safety, tolerability and antiviral activity of the cyclophilin inhibitor alisporivir (ALV) as monotherapy or combination with ribavirin (RBV) compared to IFN/RBV and ALV/IFN in treatment-naive GT2/3 HCV patients. We describe genotypic and phenotypic changes in patients from this study who experienced a viral breakthrough (VB). METHODS: VITAL-1 study patients on IFN-free regimens that did not achieve RVR (, 25 U/ml) at week 4 were administered IFN/RBV/600 mg QD ALV starting at week 6 (IFN-add-on). VB defined as an increase of HCV RNA .1 log10 above nadir. The HCV NS5A gene was analyzed by population sequencing. The entire NS5A region isolated at baseline and at VB was cloned into a JFH1subgenomic shuttle vector for phenotypic analyses. Selected mutations were engineered into a wild type replicon and their impact on replication fitness and susceptibility to ALV and direct acting antiviral drugs were determined. RESULTS: The VB rate with IFN-free or IFN-add-on regimens was 2.7% (7/ 260). Low ALV drug level (Cmin , 20% of group mean; , 100 ng/mL) was observed (5/ 7 VB patients). Genotypic analysis of the NS5A gene revealed amino acid changes in these patients at the time of VB compared to baseline. Replicons bearing NS5A from patients at breakthrough conferred 1.2-16.9-fold shift over baseline EC50 values; 5/7 had impaired replication fitness. No cross-resistance to other classes of HCV inhibitors was observed. Most frequently identified mutations were D320E (D316E in G2), R347W and A349V in Domain II of NS5A, which by themselves decreased ALV susceptibility by 5-, 4, and 8-fold, respectively, in vitro when engineered into wild-type replicon. Combination of multiple mutations decreased susceptibility further, but generally at the cost of replication fitness. CONCLUSION: In treatment-naive G2/3 patients with viral breakthrough, mutations in NS5A domain II were selected, which individually conferred ≤ 8-fold reduced susceptibility to ALV in vitro. The combination of multiple mutations was required to further reduce susceptibility which is consistent with the high barrier to resistance for host-targeting ALV. Observed genotypic changes did not confer cross-resistance to other classes of HCV inhibitors, supporting the use of ALV in future drug combination therapies.

821 INTERFERON (IFN)-FREE ALISPORIVIR (ALV) HAS A BETTER OVERALL SAFETY PROFILE COMPARED TO IFN-CONTAINING TREATMENT: A POOLED ANALYSIS OF THE ALV DEVELOPMENT PROGRAM

• Interferon (IFN)-based therapy is known to be associated with significant adverse events, most notably flu-like illness, gastrointestinal symptoms, neuropsychiatric symptoms and bone marrow suppression. By removing IFN from the hepatitis C therapy regimen, the safety profile of the treatment regimen should improve significantly • In this analysis of data from alisporivir (ALV) phase 2 and 3 studies that have been conducted, we evaluated the safety profile of IFN-free ALV therapy compared to IFN combined with pegylated interferon/ribavirin (P/R) and to P/R alone

Sa1081 Therapeutic Dose Effects of the Cyclophilin Inhibitor Alisporivir (ALV) on Circulating Bilirubin Concentrations in Healthy Subjects

Sa1081 Therapeutic Dose Effects of the Cyclophilin Inhibitor Alisporivir (ALV) on Circulating Bilirubin Concentrations in Healthy Subjects

Update on alisporivir in treatment of viral hepatitis C

Introduction: There are two classes of anti-hepatitis C virus (HCV) agents currently in development: direct-acting antivirals (DAA) and host-targeting antivirals (HTA). Cyclophilin inhibitor alisporivir (ALV), previously known as Debio-025 is the most advanced HTA in development.Areas covered: Experimental and clinical studies demonstrated that ALV has high genetic barrier and no cross-resistance to DAA. Pharmacokinetic studies showed a profile suitable for once-daily administration. Phase I and II studies confirmed strong HCV suppression and that addition of ALV to pegylated IFNα (PegIFNα) and ribavirin (RBV) can improve their efficacy significantly. ALV was well tolerated and prevalence of the most frequent clinical and laboratory adverse events was similar to PegIFNα/RBV. Hyperbilirubinemia was the only significant adverse event related to ALV, but it was transient, reversible and not associated with hepatotoxicity or cholestasis.Expert opinion: ALV is pangenotypic, with once-daily administration and safe, therefore medication can be easy and flexible. There is still a need of data in difficult-to-treat populations and genetic studies allowing selection of possible non-responders. Registration of ALV for IFN-based treatment is expected within 3 years, but ALV is also a good candidate for IFN-sparing combinations with DAA.