HCV Protease Research Articles

Hepatitis C virus inhibitory hydrolysable tannins from the fruits of Terminalia chebula

Two new hydrolysable tannins, chebumeinin A (1) and chebumeinin B (2), together with eight known related compounds (3-10), were isolated from the fruits of Terminalia chebula. The new compounds were structurally determined by analysis of their spectroscopic data and the known compounds characterized by comparing their spectroscopic data with literature values. All isolates were evaluated by an HCV protease inhibition assay, and some compounds were found to be potently active.

ChemInform Abstract: Synthesis of 2‐(4‐Isopropylthiazol‐2‐yl)‐7‐methoxy‐8‐methylquinolin‐4‐ol; a Quinoline Building Block for Simeprevir Synthesis.

AbstractTwo different approaches to the title compound which is a building block of the drug candidate simeprevir with specific HCV protease inhibitory activity are described.

HCV Triple Therapy is Equally effective in African-Americans and Non-African-Americans

Real-world experience with HCV medications differs from original the index studies. African-Americans (AA) are commonly underrepresented in clinical trials. Therefore, we hypothesized that postmarketing experience with the first generation HCV protease inhibitors (PI) (telaprevir and boceprevir) differs from premarket reports in clinically meaningful ways regarding patient race. We conducted a single-center, retrospective, observational analysis to evaluate the efficacy of HCV triple therapy in a nontrial setting. Clinical response and quantitative laboratory values were compared between AA and non-AA patients throughout the duration of treatment. Patients were included if they started triple therapy treatment after July 2011 and are not a part of any other clinical/pharmaceutical trials. Thirty-five patients with HCV genotype 1 were included in this study—22 non-AA and 13 AAs. AAs had a higher BMI compared to non-AA (33.6 vs 28.8, p = 0.046). “High” baseline HCV RNA values (above 800,000 copies) were more prevalent in AA (69.2 %) vs non-AA (63.6 %). AA had less favorable IL28B genotypes; only 7.7 % had type CC compared to 40.9 % of non-AA. SVR12 was achieved 82 % of the time with each PI but there was no significance in type of PI used (p = 1.00). Patients with cirrhosis were less likely to achieve SVR12 (40 %) compared to those without cirrhosis (88 %, p = 0.013). Thirty-three percent of the patients who received blood transfusions did not achieve SVR12 while every patient who did not require transfusion achieved SVR12 (p = 0.001). All patients with IL28B genotype CC achieved SVR12 (p = 0.007). SVR12 did not differ among AA (77 %) and non-AA (86.4 %) (p = 0.528).

Treatment of HCV infection with the novel NS3/4A protease inhibitors

HCV NS3/4A serine protease inhibitors are the first class of direct acting antivirals (DAA) introduced in clinical practice. The first generation agents, selective against HCV genotype 1, are used in association with pegylated interferons and ribavirin allowing increased cure rates at the price of increased toxicity, significant drug interactions and high risk of selecting mutants conferring cross-resistance to the entire class. A large number of second-wave HCV protease inhibitors are currently in clinical development. Advancements include higher potency, activity against a wider number of genotypes, improved tolerability, easier dosing schedules, although their genetic barrier to resistance remains low, especially for subtype 1a, except for the most recent grazoprevir and ACH-2684. The most relevant progress regards the combination with other classes of DAA allowing construction of interferon-free regimens of short duration, good tolerability with exceptionally high cure rates.

Considerable efficacy of retreatment of chronic Hepatitis C Genotype 1 (G1) Infection with Boceprevir (Victrelis®) in German Real-Life: Interim results from the ongoing Novus study

Background: Triple therapy with the HCV protease inhibitor boceprevir has been approved as new standard of care for patients with chronic HCV G1 infection. The NOVUS observational study was conducted to investigate the efficacy of this novel antiviral therapy in German real-life.

Structure-based design of a novel series of azetidine inhibitors of the hepatitis C virus NS3/4A serine protease

Structural homology between thrombin inhibitors and the early tetrapeptide HCV protease inhibitor led to the bioisosteric replacement of the P2 proline by a 2,4-disubstituted azetidine within the macrocyclic β-strand mimic. Molecular modeling guided the design of the series. This approach was validated by the excellent activity and selectivity in biochemical and cell based assays of this novel series and confirmed by the co-crystal structure of the inhibitor with the NS3/4A protein (PDB code: 4TYD).

ChemInform Abstract: Biomimetic Synthesis of Antrodia Maleimides and Maleic Anhydrides.

AbstractThe short and biomimetic synthesis of the potent HCV protease inhibitor antrodin A, (I), in three steps and 73% overall yield represents a significant improvement over previous routes.

Stereoselective Lithiation and Carboxylation of Boc-Protected Bicyclopyrrolidine: Synthesis of a Key Building Block for HCV Protease Inhibitor Telaprevir

A stereoselective process for the manufacture of bicyclopyrrolidine 7 to 2 has been developed. The process utilizes a stereoselective lithiation/carboxylation sequence. The achiral diamine ligand DPBP induces excellent diastereocontrol, and resolution with (S)-THNA provides the corresponding salt of 8 in high er and dr. Subsequent processing of 8 gives 2 as the oxalate salt in an overall yield of 27% from 7 (based on total molar charge of 7). Compound 2 was obtained with high chemical and chiral purities. The process was successfully demonstrated on >100 kg scale.

Synthesis of HCV Protease Inhibitor MK-1220

Key words MK-1220 - HCV protease inhibitors - macrolactamization - Sonogashira reaction - regioselective Mizoroki–Heck reaction

Managing HCV infection: present and future

The development of direct acting antiviral agents against the hepatitis C virus has revolutionized treatment paradigms for hepatitis C. In 2011 the first HCV protease inhibitors boceprevir and telaprevir were approved, which still needed to be combined with pegylated interferon and ribavirin and were only usable in patients with genotype 1. However, the new triple therapy achieved successful cure rates of hepatitis C in almost 70% of treatment naive patients and immediately became the new gold standard of HCV therapy. With the development of further agents, in particular the recent licensing of sofosbuvir and simeprevir, two further DAA’s have become available. Thereby, for the first time Interferon free regimens are already available for genotype 2 and 3 patients allowing cure rates approaching 90% after 12 weeks of sofosbuvir and ribavirin for genotype 2 treatment naive as well as treatment-experienced patients and 24 weeks of all oral therapy for patients with genotype 3 (again treatment-naive or experienced). For genotype 1 the combination of sofosbuvir with pegylated interferon and ribavirin allows curing over 90% of patients after 12 weeks of triple therapy and therefore has become the preferred HCV therapy for this particular patient group. Also combination of sofosbuvir with other agents have been presented in particular simeprevir or daclatasvir leading to high success rates above 90% even in patients who failed previous triple therapy with one of the first HCV protease inhibitors. Therefore, where available, 2 DAA combinations ± ribavirin are rapidly becoming the new HCV treatment standard for more difficult to treat patient groups. In the near future combinations of antiviral agents with no cross resistance and improved safety profile will further reduce treatment durations and will become the new recommended HCV regimens making HCV the first viral infection to be possibly eradicated worldwide.

Resistance mutations are rare among protease inhibitor treatment-naive hepatitis C genotype-1 patients with or without HIV coinfection.

HCV has a high replication rate and a lack of proofreading activity, leading to a greatly diverse viral population. This diversity may lead to emergence of resistant strains in direct-acting antiviral therapy. The frequency of naturally occurring HCV protease inhibitor (PI) mutations has been addressed in many countries, but there are few data on the prevalence of these mutations in Brazilian patients. We evaluated the sequence of HCV NS3 protease gene in 247 patients (135 HCV-monoinfected and 112 HIV-HCV-coinfected patients). HCV RNA was extracted from plasma and a fragment of 765 base pairs from the NS3 region was amplified and sequenced with Sanger-based technology. HIV-HCV-coinfected patients were more likely to be older than 40 years and have an HCV subtype-1a infection. Overall, 21.9% of patients had at least one amino acid substitution in the NS3 region; 14 patients (5.7%) harboured at least one resistance mutation (T54S, V55A, Q80R) and the Q80K mutation was not found in our case series. There was no difference between monoinfected and coinfected patients regarding the frequency of natural polymorphisms and resistance mutations. Baseline HCV NS3 amino acid substitutions identified herein are considered mostly natural polymorphisms with no clinical impact on PI-based therapy. The identified resistance mutations may be associated with low-level resistance to PIs in vitro. Q80K substitution seems to be a rare event in Brazil. HIV coinfection was not associated with a greater frequency of such substitutions in the studied sample.

Practical Synthesis of A Macrocyclic HCV Protease Inhibitor: A High-Yielding Macrolactam Formation

A practical synthesis of a macrocyclic HCV protease inhibitor, MK-1220, is described. The key features are a new synthesis of the trisubstituted isoquinoline, Sonogashira fragment coupling, and a high-yielding, 18-membered macrolactam formation.

Two-way interaction study between ritonavirboosted danoprevir, a potent HCV protease inhibitor, and ketoconazole in healthy subjects

Danoprevir is a potent, highly selective, macrocyclic, orally bioavailable inhibitor of the hepatitis C virus protease, and a substrate of cytochrome P450 (CYP) 3A. It is co-administered with low-dose ritonavir, a potent CYP3A inhibitor, to enhance danoprevir pharmacokinetics. Ketoconazole is a substrate for and potent selective inhibitor of CYP3A. In this open-label, 3-period study, the 2-way interaction potential between ritonavir-boosted danoprevir (danoprevir/r) and ketoconazole was investigated in 18 healthy subjects. Subjects initially received ketoconazole 200 mg q24h for 4 days (Period 1) followed by a 7-day washout period. Danoprevir/r 100/100 mg q12h was then given for 10 days (Period 2) followed by a further 4 days of danoprevir/r 100/100 mg q12h plus ketoconazole 200 mg q24h (Period 3). Serial blood samples were collected for the determination of danoprevir, ritonavir and/or ketoconazole plasma concentrations, and calculation of pharmacokinetic parameters. Safety and tolerability were monitored throughout the study. Co-administration of ketoconazole with danoprevir/r modestly increased the danoprevir AUCτ by 1.44-fold, with no effect on danoprevir Cmax. Co-administration of danoprevir/r with ketoconazole substantially increased ketoconazole AUCτ and Cmax by 3.71-fold and 1.73-fold, respectively. Danoprevir/r was well tolerated when administered alone or with ketoconazole. These results indicate that the effect of potent CYP3A inhibitors, such as ketoconazole, on danoprevir/r pharmacokinetics is not likely to be clinically relevant.

Successful triple therapy of patients with chronic hepatitis C genotype 1 infection leads to significant improvement of non-invasive liver fibrosis tests

Background and Aim: The new standard of care for patients with chronic hepatitis C genotype 1 (GT1) is triple therapy with pegylated interferon alpha (P), ribavirin (R) and a HCV protease inhibitor (PI). Sustained virological response (SVR) has been shown to lower the incidence of HCC, improve survival and even regression of liver cirrhosis is suggested in some cases. However, data are limited, especially for triple therapy. We aim to evaluate the impact of SVR after triple therapy including PIs on liver fibrosis by non-invasive fibrosis measures in a real-world setting.

PAT Application in the Expedited Development of a Three-Step, One-Stage Synthesis of the Dipeptide Intermediate of HCV Protease Inhibitor Faldaprevir

A concise scalable synthesis of a chiral dipeptide acid, key substructure of the HCV protease inhibitor faldaprevir, has been developed. A green process with an E-factor of 9.2 was achieved utilizing process analytical technology (PAT) to allow effective processing of multiple-steps in a one-stage operation. Mixed anhydride/oxazolone formation, peptide coupling, saponification, and then crystallization of the desired dipeptide acid were completed within 10 h. MultiMaxIR was used to detect the formation and consumption rates of key intermediates and to provide initial safety data which was subsequently confirmed by more comprehensive process safety testing. Further kinetic analysis was performed to determine the range of operability space to ensure conditions for a robust process.

Biomimetic Synthesis of Antrodia Maleimides and Maleic Anhydrides

The short and biomimetic synthesis of the potent HCV protease inhibitor antrodin A, (I), in three steps and 73% overall yield represents a significant improvement over previous routes.

Use of first‐generation HCV protease inhibitors in patients coinfected by HIV and HCV genotype 1

In HCV genotype 1-infected patients with HIV co-infection, tritherapy [HCV protease inhibitors (PIs) plus peg-interferon and ribavirin] has been shown to have an increased rate of sustained virological response. However, complex drug-to-drug interactions and tolerability issues remain a concern. Under the auspices of four French scientific societies of medicine, a committee was charged of establishing guidelines on the use of first-generation HCV PIs in these patients. This scientific committee based its work on preliminary results from tritherapy clinical trials in co-infected patients and, since data on these patients are still scarce, on the statements already made by the French Association for the Study of the Liver (AFEF) on the use of tritherapy in HCV mono-infected patients, written in May 2011 and updated in 2012. Each AFEF guideline concerning HCV monoinfection was examined to determine whether it could be used in the context of HIV/HCV coinfection. These guidelines are addressed for the treatment of coinfected patients with various profiles, including treatment-naïve or patients with failure to previous bitherapy and mention those patients for whom tritherapy should start or those for whom it should be delayed. Preliminary results of triple therapy as well as factors associated to virological response are also discussed. Other issues include virological monitoring, clinical and virological criteria to stop therapy, practical treatment management, treatment adherence and the management of side effects and interactions with antiretroviral drugs. These guidelines were submitted for critical review to independent experts.

Interactions of ketoamide inhibitors on HCV NS3/4A protease target: molecular docking studies

HCV infection in more than 200 million individuals worldwide is a principal health problem. Prior to the development of HCV protease inhibitor combination therapy, HCV infected patients were treated with pegylated interferon-α and ribavirin. The adverse side effects associated with this type of treatment may lead to the discontinuation of treatment in certain number of patients. Currently, the inhibitors of NS3/4A Protease were found promising candidates for the treatment of HCV infection. There are several inhibitors of HCV NS3/4A protease that are passing through clinical improvement showing good potency against HCV infections in a number of patients. To further recognize binding interactions and activity trend, the molecular docking studies were performed on a number of HCV NS3/4A protease ketoamide inhibitors via MOE docking protocol. The docking analysis resulted in the detection of important ligand interactions with respect to binding site of target protein and produced good correlation coefficient (r2 = 0.690) between docking score and biological activities. These molecular docking results should, in our view, contribute for further optimization of ketoamide derivatives as NS3/4A protease inhibitors.

Romiplostim For Thrombocytopenia in Patients with Hepatitis C (HCV)-Associated Liver Disease: Preliminary Report Of An Ongoing Clinical Trial

BackgroundThrombocytopenia (Tp) is frequently observed in individuals with advanced cirrhotic liver disease. Patients with HCV may develop Tp even in the absence of significant liver disease. Decreased thrombopoietin production may also contribute to the Tp. The current management of HCV infection includes the use of the Peg-interferon α (IFN) and ribavirin (RIB), which can induce a sustained viral remission in 40 to 50% of treated patients. However, Peg-INF is a known inhibitor of megakaryocyte growth and maturation and can result in treatment related Tp. Therefore, patients presenting with platelet counts <70,000/mcl are frequently excluded from treatment or often fail treatment due to the development of critically low platelet counts. With this understanding, we initiated a clinical trial of the thrombopoietin receptor agonist, romiplostim, in HCV cirrhotic patients with Tp to determine if platelet count can be increased to >100,000/mcl to allow for HCV treatment with Peg-INF/RIB. MethodsThis is a two phase clinical trial of HCV infected patients with Child-Pugh class A liver disease. All patients were required to have platelet counts <70,000/µl and have liver biopsy confirmed early cirrhosis. Phase I is a double-blind placebo controlled trial of romiplostim given up to 8 weeks to raise platelets to >100,000/mcl before initiating Peg-INF treatment. There are separate 1 to 1 randomizations for patients with platelets 70 to 50,000/µl and patients with platelets<50,000/mcl. Initial treatment dose is 1 µg/kg with weekly progressive increases in dose. For patients who failed to obtain platelet counts >100,000/µl by week 8, blind is terminated and placebo patients can enter the romiplostim arm. Phase II is a dose escalation study of romiplostim during Peg-INF/Rib treatment up to week 24 of HCV treatment. If patients are viral load negative by week 24, romiplostim treatment is held to see if the patients can sustain a safe platelet count with continued HCV therapy. If not, romiplostim is continued until completion of HCV treatment. A protocol amendment allowed the addition of HCV protease inhibitors for genotype 1a patients. ResultsAt the time of this report 21 patients (7F/14M; mean age 54.9 yrs, range 28-72yrs) have been enrolled in this trial; 13 with platelets 70 to 50,000/µl (Mean 62,000/µl; range 55 to 70,000µl) and 8 with platelet counts<50,000/µl (Mean 35,000/µl; range 25-46,000/µl). 17 patients have completed Phase I; 2 patients are ongoing, 1 patient withdrew at wk 5 with no platelet response (Blind remains) and one patient was withdrawn at wk 2 when review of the pre-randomization ultrasound found evidence of an old partial portal thrombosis. Five patients failed to obtain a platelet count of >100,000/ul by wk 8; all on placebo and were rolled over to romiplostim. The mean romiplostim dose for patients completing Phase I with platelets 70 to 50,000/µl was 2.2 mcg/kg and 3.1 mcg/kg for patients with platelets of<50,000/µl. During the 8 wk course of romiplostim there were no SAEs and no change in HCV viral load. Eleven patients have successfully completed Phase II with 24 wks of HCV treatment; with 4 patients on HCV treatment at wks 21, 20, 13 and 9. One patient was withdrawn at wk 14 due to intractable pancytopenia. 7/11 (64%) were HCV viral load negative at wk 24 and continued to completion of their HCV treatment. The mean romiplostim dose for the 11 patients completing Phase II with HCV treatment was 7µg/kg (3 to 10µg/kg). Three major SAEs occurred during Phase II. A 61 y.o. female developed pancytopenia at wk 14 unresponsive to growth factor support with a hypocellular bone marrow. She had a slow recovery of counts over several months. A 63 y.o. female developed portal vein thrombosis at wk 22 of HCV therapy. Her platelet count was 92,000/µl. She was treated with LMW heparin, continued HCV treatment with romiplostim off study and was viral load negative at wk 44 of treatment. A 50 y.o. female had a sudden death at home at wk 20 of HCV therapy. She has a platelet count of 32,000/µl on a clinic visit the day before her death and her romiplostim dose was 10µg/kg. No autopsy was performed. ConclusionIn this interim analysis of ongoing clinical trial, romiplostim appears well tolerated and effective in increasing platelet counts in HCV cirrhotic patients and can maintain a safe platelet count in the majority of patients during HCV antiviral therapy with Peg-INF/RIB.This study was funded by a grant from AMGEN. Disclosures:Liebman:Amgen: Research Funding. Off Label Use: A clinical trial of romiplostim to treat hepatitis C-related thrombocytopenia performed under an FDA IND.

Ligand Bioactive Conformation Plays a Critical Role in the Design of Drugs That Target the Hepatitis C Virus NS3 Protease

A ligand-focused strategy employed NMR, X-ray, modeling, and medicinal chemistry to expose the critical role that bioactive conformation played in the design of a variety of drugs that target the HCV protease. The bioactive conformation (bound states) were determined for key inhibitors identified along our drug discovery pathway from the hit to clinical compounds. All adopt similar bioactive conformations for the common core derived from the hit peptide DDIVPC. A carefully designed SAR analysis, based on the advanced inhibitor 1 in which the P1 to P3 side chains and the N-terminal Boc were sequentially truncated, revealed a correlation between affinity and the relative predominance of the bioactive conformation in the free state. Interestingly, synergistic conformation effects on potency were also noted. Comparisons with clinical and recently marketed drugs from the pharmaceutical industry showed that all have the same core and similar bioactive conformations. This suggested that the variety of appendages discovered for these compounds also properly satisfy the bioactive conformation requirements and allowed for a large variety of HCV protease drug candidates to be designed.