The metabolism and disposition of a potent inhibitor of hepatitis C virus NS3/4A protease

1. Compound A ((1aR,5S,8S,10R,22aR)-5-tert-butyl-N-{(1R,2S)-1-[(cyclopropylsulfonyl)carbamoyl]-2-ethenylcyclopropyl}-14-methoxy-3,6-dioxo-1,1a,3,4,5,6,9,10,18,19,20,21,22,22a-tetradecahydro-8H-7,10-methanocyclopropa[18,19][1,10,3,6]dioxadiazacyclononadecino[12,11-b]quinoline-8-carboxamide) is a prototype of a series of subnanomolar inhibitors of genotypes 1, 2, and 3 hepatitis C virus (HCV) NS3/4A proteases. HCV NS3/4A protease inhibitors have demonstrated high antiviral effects in patients with chronic HCV infection and are likely to form a key component of future HCV therapy.

2. Compound A showed excellent liver exposure in rats, which is essential for compounds intended to treat HCV. The compound was mainly eliminated intact in bile and showed greater than dose proportional systemic exposure in rats. Compound A demonstrated time- and temperature-dependent uptake into rat and human hepatocytes and proved to be a substrate for rat hepatic uptake transporter Oatp1b2 and for human hepatic uptake transporters OATP1B1 and OATP1B3. The liver selectivity observed for this compound is likely to be due to transporter-mediated hepatic uptake together with moderate passive permeability.

3. Metabolism was mainly CYP3A-mediated and generated a reactive epoxide on the vinylcyclopropyl sulfonamide moiety that could be quenched by glutathione. Similar metabolic profiles of Compound A were obtained in liver microsomes of rats and humans. The oral bioavailability at 5?mg/kg was low due to extensive hepatic first-pass effect but clearly the intestinal absorption was enough to deliver a high amount of the compound to the liver. The metabolism and disposition properties of Compound A are particularly attractive to support its evaluation as a drug candidate for the treatment of hepatitis C.

     

High-throughput cell-based screening for hepatitis C virus NS3/4A protease inhibitors

Hepatitis C virus (HCV) encodes a viral protease, nonstructural (NS)3/4A, that is critical for virus maturation. Although NS3/4A has emerged as a promising target for anti-HCV drug discovery, no anti-HCV therapy has succeeded yet based on inhibition of NS3/4A. We have previously shown that EG(delta4AB)SEAP, a reporter consisting of enhanced green fluorescent protein (EG), the NS3-NS4A protease decapeptide recognition sequence (delta4AB), and secreted alkaline phosphatase (SEAP), is an efficient reporter for reflecting NS3/4A proteolytic activity inside cells. In this study, we describe the generation and characterization of a stable cell line, 293EEG(delta4AB)SEAP-NS3/4A, which constitutively expresses EG(delta4AB)SEAP reporter protein and NS3/4A protease. The reporter assay is validated with the compound BILN 2061, a specific and potent peptidomimetic inhibitor of the HCV NS3 protease. Additionally, we show here that this cell line allows screening for NS3/4A protease activity of living cells in 96-well plate format, with a Z factor >0.6. Thus, this cell-based assay may be used for high-throughput screening of chemical libraries.     

Hepatitis C virus NS3/4A protease.

Despite an urgent medical need, a broadly effective anti-viral therapy for the treatment of infections with hepatitis C viruses (HCVs) has yet to be developed. One of the approaches to anti-HCV drug discovery is the design and development of specific small molecule drugs to inhibit the proteolytic processing of the HCV polyprotein. This proteolytic processing is catalyzed by a chymotrypsin-like serine protease which is located in the N-terminal region of non-structural protein 3 (NS3). This protease domain forms a tight, non-covalent complex with NS4A, a 54 amino acid activator of NS3 protease. The C-terminal two-thirds of the NS3 protein contain a helicase and a nucleic acid-stimulated nucleoside triphosphatase (NTPase) activities which are probably involved in viral replication. This review will focus on the structure and function of the serine protease activity of NS3/4A and the development of inhibitors of this activity.     

Hepatitis C virus NS3/4a protease inhibitors

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Biotransformation and mass balance of faldaprevir,a hepatitis C NS3/NS4 protease inhibitor in rats

Abstract

1.?The metabolism, pharmacokinetics, excretion and tissue distribution of a hepatitis C NS3/NS4 protease inhibitor, faldaprevir, were studied in rats following a single 2?mg/kg intravenous or 10?mg/kg oral administration of [¹⁴C]-faldaprevir.

2.?Following intravenous dosing, the terminal elimination t_1/2 of plasma radioactivity was 1.75?h (males) and 1.74?h (females). Corresponding AUC_0–∞, CL and V_ss were 1920 and 1900?ngEq?·?h/mL, 18.3 and 17.7?mL/min/kg and 2.32 and 2.12?mL/kg for males and females, respectively.

3.?After oral dosing, t_1/2 and AUC_0–∞ for plasma radioactivity were 1.67 and 1.77?h and 11?300 and 17?900 ngEq?·?h/mL for males and females, respectively.

4.?In intact rats, ≥90.17% dose was recovered in feces and only ≤1.08% dose was recovered in urine for both iv and oral doses. In bile cannulated rats, 54.95, 34.32 and 0.27% dose was recovered in feces, bile and urine, respectively.

5.?Glucuronidation plays a major role in the metabolism of faldaprevir with minimal Phase I metabolism.

6.?Radioactivity was rapidly distributed into tissues after the oral dose with peak concentrations of radioactivity in most tissues at 6?h post-dose. The highest levels of radioactivity were observed in liver, lung, kidney, small intestine and adrenal gland.     

Telaprevir: a novel NS3/4 protease inhibitor for the treatment of hepatitis C

Hepatitis C virus (HCV) infection affects millions of people worldwide; however, standard therapy with peginterferon and ribavirin has resulted in suboptimal responses. Thus, new anti-HCV drugs with novel mechanisms of action are being studied. In particular, new drugs are being developed that target the NS3/4A protease complex. We evaluated the literature on telaprevir, a new, oral, covalent, reversible NS3/4A HCV protease inhibitor. A MEDLINE search (January 1996-July 2011) was performed to identify relevant clinical trials, and abstracts from hepatology and human immunodeficiency virus (HIV) conferences were reviewed. In large clinical trials, the addition of telaprevir to peginterferon and ribavirin resulted in high sustained virologic response rates in both treatment-na?ve and treatment-experienced patients infected with HCV genotype 1. Clinical data with telaprevir in the HIV-HCV coinfected population are emerging, as well as data on potential drug-drug interactions with this agent. Preliminary data describe the resistance profile of telaprevir; however, more information is needed in this evolving area. Telaprevir's most common adverse events included rash, pruritis, and anemia. Based on available data, this new anti-HCV drug will likely be widely used and may revolutionize the treatment of HCV-infected individuals.     

Boceprevir: a novel NS3/4 protease inhibitor for the treatment of hepatitis C

Hepatitis C virus (HCV) infection affects over 170 million people worldwide and is the most common blood-borne infection in the United States. Standard treatment with peginterferon alfa-ribavirin results in low sustained virologic response (SVR) rates in many patients, especially those who are African-American, are coinfected with human immunodeficiency virus (HIV), or have liver cirrhosis. Because of suboptimal SVR rates, new direct-acting antiviral agents that target HCV viral replication steps are in development. Boceprevir is one of the novel NS3/4A protease inhibitors that was recently approved by the U.S. Food and Drug Administration. We evaluated the literature regarding boceprevir by performing a MEDLINE search (January 1996-July 2011) to identify relevant clinical trials. Abstracts and poster and oral presentations from hepatology and HIV conferences were also reviewed. Potent anti-HCV activity was seen in clinical trials with boceprevir when it was studied in HCV genotype 1-infected patients who were na?ve to or had experience with HCV therapy. Data with boceprevir in HIV-HCV-coinfected patients are currently lacking; however, initial data on drug-drug interactions between boceprevir and antiretrovirals have become available. Resistance to boceprevir has been evaluated in trials as well, although more data are needed in this area. The most common adverse events with boceprevir included anemia and dysgeusia. Based on available data, boceprevir is one of the promising novel direct-acting antiviral agents that will likely reshape the treatment of patients with HCV infection.     

Novel potent hepatitis C virus NS3 serine protease inhibitors derived from proline-based macrocycles

The hepatitis C virus (HCV) NS3 protease is essential for viral replication. It has been a target of choice for intensive drug discovery research. On the basis of an active pentapeptide inhibitor, 1, we envisioned that macrocyclization from the P2 proline to P3 capping could enhance binding to the backbone Ala156 residue and the S4 pocket. Thus, a number of P2 proline-based macrocyclic alpha-ketoamide inhibitors were prepared and investigated in an HCV NS3 serine protease continuous assay (K(i*)). The biological activity varied substantially depending on factors such as the ring size, number of amino acid residues, number of methyl substituents, type of heteroatom in the linker, P3 residue, and configuration at the proline C-4 center. The pentapeptide inhibitors were very potent, with the C-terminal acids and amides being the most active ones (24, K(i*) = 8 nM). The tetrapeptides and tripeptides were less potent. Sixteen- and seventeen-membered macrocyclic compounds were equally potent, while fifteen-membered analogues were slightly less active. gem-Dimethyl substituents at the linker improved the potency of all inhibitors (the best compound was 45, K(i*) = 6 nM). The combination of tert-leucine at P3 and dimethyl substituents at the linker in compound 47 realized a selectivity of 307 against human neutrophil elastase. Compound 45 had an IC(50) of 130 nM in a cellular replicon assay, while IC(50) for 24 was 400 nM. Several compounds had excellent subcutaneous AUC and bioavailability in rats. Although tripeptide compound 40 was 97% orally bioavailable, larger pentapeptides generally had low oral bioavailability. The X-ray crystal structure of compounds 24 and 45 bound to the protease demonstrated the close interaction of the macrocycle with the Ala156 methyl group and S4 pocket. The strategy of macrocyclization has been proved to be successful in improving potency (>20-fold greater than that of 1) and in structural depeptization.     

Preclinical pharmacokinetics and metabolism of a potent non-nucleoside inhibitor of the hepatitis C virus NS5B polymerase

The disposition of compound A, a potent inhibitor of the hepatitis C virus (HCV) NS5B polymerase, was characterized in animals in support of its selection for further development. Compound A exhibited marked species differences in pharmacokinetics. Plasma clearance was 44 ml min-1 kg-1 in rats, 9 ml min-1 kg-1 in dogs and 16 ml min-1 kg-1 in rhesus monkeys. Oral bioavailability was low in rats (10%) but significantly higher in dogs (52%) and monkeys (26%). Compound A was eliminated primarily by metabolism in rats, with biliary excretion accounting for 30% of its clearance. Metabolism was mainly mediated by cyclohexyl hydroxylation, with N-deethylation and acyl glucuronide formation constituting minor metabolic pathways. Qualitatively, the same metabolites were identified using in vitro systems from all species studied, including humans. The low oral bioavailability of compound A in rats was mostly due to poor intestinal absorption. This conclusion was borne out by the findings that hepatic extraction in the rat was only 30%, intraperitoneal bioavailability was good, and compound A was poorly absorbed from the rat isolated intestinal loop, with no detectable intestinal metabolism. Compound A was not an inhibitor of major human cytochrome P450 enzymes, indicating minimal potential for clinical drug-drug interactions. The metabolic clearance of compound A in rat, dog and monkey hepatocytes correlated with the systemic clearance observed in these species. Since compound A was very stable in human hepatocytes, the results suggest that it will be a low clearance drug in humans.     

Preclinical pharmacokinetics and metabolism of a potent non-nucleoside inhibitor of the hepatitis C virus NS5B polymerase

The disposition of compound A, a potent inhibitor of the hepatitis C virus (HCV) NS5B polymerase, was characterized in animals in support of its selection for further development. Compound A exhibited marked species differences in pharmacokinetics. Plasma clearance was 44?ml?min^?1?kg^?1 in rats, 9?ml?min^?1?kg^?1 in dogs and 16?ml?min^?1?kg^?1 in rhesus monkeys. Oral bioavailability was low in rats (10%) but significantly higher in dogs (52%) and monkeys (26%). Compound A was eliminated primarily by metabolism in rats, with biliary excretion accounting for 30% of its clearance. Metabolism was mainly mediated by cyclohexyl hydroxylation, with N-deethylation and acyl glucuronide formation constituting minor metabolic pathways. Qualitatively, the same metabolites were identified using in vitro systems from all species studied, including humans. The low oral bioavailability of compound A in rats was mostly due to poor intestinal absorption. This conclusion was borne out by the findings that hepatic extraction in the rat was only 30%, intraperitoneal bioavailability was good, and compound A was poorly absorbed from the rat isolated intestinal loop, with no detectable intestinal metabolism. Compound A was not an inhibitor of major human cytochrome P450 enzymes, indicating minimal potential for clinical drug–drug interactions. The metabolic clearance of compound A in rat, dog and monkey hepatocytes correlated with the systemic clearance observed in these species. Since compound A was very stable in human hepatocytes, the results suggest that it will be a low clearance drug in humans.     

Discovery of boceprevir, a direct-acting NS3/4A protease inhibitor for treatment of chronic hepatitis C infections

Hepatitis C virus (HCV) infection is the primary cause of liver cirrhosis and hepatocellular carcinoma. HCV is the leading cause of liver transplantation in the USA, and more than 200 million people worldwide are infected with HCV. Before the introduction of NS3 protease inhibitors, the standard of care was treatment with peg-interferon and ribavirin. Recent developments in virology have identified many novel targets in the HCV genome, allowing the development of direct-acting antivirals. In this article, I outline the discovery and development of boceprevir, the first HCV NS3/4A protease inhibitor approved for treatment of genotype 1 HCV infection. Boceprevir greatly improves the sustained virologic response (SVR) and provides new hope for treating genotype 1 infections.     

丙型肝炎病毒NS3/4A丝氨酸蛋白酶活性测定方法研究进展

丙型肝炎病毒（HCV）NS3／4A丝氨酸蛋白酶在病毒多聚蛋白体加工和RNA复制的过程中发挥着重要作用,是治疗HCV的理想靶点。因为NS3／4A丝氨酸蛋白酶在HCV多聚蛋白上负责四个位点的切割,所以可根据此特性设计其活性的测定方法。此文就目前应用于NS3／4A丝氨酸蛋白酶活性测定的方法进行综述,以期为筛选有效的抑制剂提供技术帮助。     

Cross-species absorption, metabolism, distribution and pharmacokinetics of BI 201335, a potent HCV genotype 1 NS3/4A protease inhibitor

The present study describes the cross-species absorption, metabolism, distribution and pharmacokinetics of BI 201335, a potent HCV protease inhibitor currently in phase III clinical trials. BI 201335 showed a good Caco-II permeability (8.7 × 10(-6) cm/sec) and in vitro metabolic stability (predicted hepatic clearence (CL(hep)) <19% Q(h) in all species tested). Single dose PK revealed a clearance of 17, 3.0 and 2.6 mL/min/kg in rat, monkey and dog respectively, with a corresponding oral bioavailability of 29.1, 25.5 and 35.6%. Comparative plasma and liver PK profile in rodents showed a high liver Kp in the rat (42-fold), suggesting high target tissue distribution. Simple allometry based on animal PK predicted a human oral CL/F of 168 mL/min, within two-fold of the observed value (118 mL/min) at 240 mg in healthy volunteers. Allometry of volume of distribution generated a low exponent of 0.59, and a much lower predicted Vss/F (5-fold less than observed). Several different approaches of Vss/F prediction were evaluated and compared with the value observed in human. The averaged Vss/F from preclinical animals provides the best estimation of the observed human value (169 L vs. 175 L). Corresponding human "effective" t(1/2) values were also compared. The predicted human t(1/2) based on the CL from allometry with metabolic corrections and the averaged animal Vss represented the best estimation of the clinical data (12.1 vs. 17.2 hr). The present study demonstrated that the good preclinical ADMEPK profile of BI 201335 is consistent with that observed in the clinic. While preclinical data accurately predicted the human CL, the prediction of human Vss seems to be more challenging. The averaged Vss/F from all tested preclinical animals provided the best prediction of human Vss and the resulting "effective" t(1/2).     

Novel tripeptide inhibitors of hepatitis C virus NS3 serine protease

Available therapies for hepatitis C viral infection, a disease with a growing impact worldwide, have limited success and serious side effects. Among different possibilities to control the infection, the NS3 serine protease inhibitors constitute a promising alternative, based on a similar approach proved successful in the case of HIV antiviral agents. However, the structural particularities of this viral serine protease make the design of small molecule inhibitors a difficult task. In contrast, the peptidic and peptidomimetic approach gave better results, allowing submicromolar levels of inhibition. In this patent, Bristol-Myers Squibb presents new tripeptide inhibitors with improved potency against NS3 protease and a very good selectivity against related serine proteases. The disclosed compounds also exhibited a good inhibitory profile against different viral strain proteases, giving good hopes to the use of NS3 protease inhibitors as a cure to treat hepatitis C viral infections.     

Non-peptidic small-molecule inhibitors of the single-chain hepatitis C virus NS3 protease/NS4A cofactor complex discovered by structure-based NMR screening

NMR-based screening of a customized fragment library identified 16 small-molecule hits that bind weakly (K(D) approximately 100 microM to 10 mM) to substrate binding sites of the NS4A-bound NS3 protease of the hepatitis C virus (HCV). Analogues for five classes of NMR hits were evaluated by a combination of NMR and biochemical data yielding SAR and, in most cases, optimized hits with improved potencies (K(D) approximately K(I) approximately 40 microM to 1 mM). NMR chemical shift perturbation data were used to establish the binding location and orientation of the active site directed scaffolds in these five analogue series. Two of these scaffolds, which bind the enzyme at the proximal S1-S3 and S2' substrate binding sites, were linked together producing competitive inhibitors of the HCV NS3 protease with potencies in the micromolar range. This example illustrates that the low molecular weight scaffolds discovered from structure-based NMR screening can be optimized with focused structure-guided chemistry to produce potent nonpeptidic small-molecule inhibitors of the HCV NS3 protease.     

NS3.4A protease as a target for interfering with hepatitis C virus replication

The hepatitis C virus (HCV) epidemic represents a significant unmet medical need. Current treatment is arduous and less than 50% effective. Indeed, interferon-alpha treatment is so unpleasant that asymptomatic patients will often stop treatment. Heartened by the success of HIV protease inhibitors, researchers have considered inhibition of the HCV NS3 serine protease an attractive mode of intervention. This account discusses the structure and function of NS3 4A and the prospects of its success as a therapeutic target. The review includes a detailed discussion of the role of the NS4A co-factor, requirements for inhibition and some recent examples of peptidomimetic and small-molecule inhibitors.     

Psammaplin A inhibits hepatitis C virus NS3 helicase

Hepatitis C virus (HCV) is the causative agent of hepatitis C, a chronic infectious disease that can lead to development of hepatocellular carcinoma. The NS3 nucleoside triphosphatase (NTPase)/helicase has an essential role in HCV replication, and is therefore an attractive target for direct-acting antiviral strategies. In this study, we employed high-throughput screening using a photo-induced electron transfer (PET) system to identify an inhibitor of NS3 helicase from marine organism extracts. We successfully identified psammaplin A as a novel NS3 inhibitor. The dose–response relationship clearly demonstrates the inhibition of NS3 RNA helicase and ATPase activities by psammaplin A, with IC₅₀ values of 17 and 32 μM, respectively. Psammaplin A has no influence on the apparent K _m value (0.4 mM) of NS3 ATPase activity, and acts as a non-competitive inhibitor. Additionally, it inhibits the binding of NS3 to single-stranded RNA in a dose-dependent manner. Furthermore, psammaplin A shows an inhibitory effect on viral replication, with EC₅₀ values of 6.1 and 6.3 μM in subgenomic replicon cells derived from genotypes 1b and 2a, respectively. We postulate that psammaplin A is a potential anti-viral agent through the inhibition of ATPase, RNA binding and helicase activities of NS3.     

A systematic approach to the optimization of substrate-based inhibitors of the hepatitis C virus NS3 protease: discovery of potent and specific tripeptide inhibitors

The inadequate efficacy and tolerability of current therapies for the infectious liver disease caused by the hepatitis C virus have warranted significant efforts in the development of new therapeutics. We have previously reported competitive peptide inhibitors of the NS3 serine protease based on the N-terminal cleavage products of peptide substrates. A detailed study of the interactions of these substrate-based inhibitors with the different subsites of the serine protease active site led to the discovery of novel residues that increased the affinity of the inhibitors. In this paper, we report the combination of the best binding residues in a tetrapeptide series that resulted in extremely potent inhibitors that bind exquisitely well to this enzyme. A substantial increase in potency was obtained with the simultaneous introduction of a 7-methoxy-2-phenyl-4-quinolinoxy moiety at the gamma-position of the P2 proline and a tert-leucine as a P3 residue. The increase in potency allowed for the further truncation and led to the identification of tripeptide inhibitors. Structure activity relationship studies on this inhibitor series led to the identification of carbamate-containing tripeptides that are able to inhibit replication of subgenomic HCV RNA in cell culture with potencies below 1 microM. This inhibitor series has the potential of becoming antiviral agents for the treatment of HCV infections.     

Approaching a new era for hepatitis C virus therapy: inhibitors of the NS3-4A serine protease and the NS5B RNA-dependent RNA polymerase

The treatment of chronic disease caused by the hepatitis C virus (HCV) is an unmet clinical need, since current therapy is only partially effective and limited by undesirable side effects. The viral serine protease and the RNA-dependent RNA polymerase are the best-studied targets for the development of novel therapeutic agents. These enzymes have been extensively characterized at the biochemical and structural level and thus used to set up screening assays for the identification of selective inhibitors. These efforts lead to the discovery of several classes of compounds with potential antiviral activity. The hepatitis C virus does not replicate in the laboratory. The formidable challenge posed by the difficulty of developing cell-based assays and preclinical animal systems has been partially overcome with several alternative approaches. The development of new assays permitted the optimization of enzyme inhibitors leading eventually to molecules with the desired drug-like properties, the most advanced of which are being considered for clinical trials.     

The design and enzyme-bound crystal structure of indoline based peptidomimetic inhibitors of hepatitis C virus NS3 protease

The design of a series of peptidomimetic inhibitors of the hepatitis C virus NS3 protease is described. These inhibitors feature an indoline-2-carboxamide as a novel heterocyclic replacement for the P3 amino acid residue and N-terminal capping group of tripeptide based inhibitors. The crystal structure of the ternary NS3/NS4A/inhibitor complex for the most active molecule in this series highlights its suitability as an N-terminal capping group of a dipeptide inhibitor of the NS3 protease.