gt1b (Ki=0.01±<0.01 nM);gt1a (Ki=0.01±0.01 nM);gt2a (Ki=0.08±0.02 nM);gt2b (Ki=0.15±0.06 nM);gt3a (Ki=0.90±0.2 nM)

MK-5172 (Grazoprevir) is effective in biochemical assays against major genotypes and variants engineered with common resistant mutations, with Ki of 0.01±<0.01 nM (gt1b), 0.01±0.01 nM (gt1a), 0.08±0.02 nM (gt2a), 0.15±0.06 nM (gt2b), 0.90±0.2 nM (gt3a), 0.07±0.01 nM (gt1bR155K), 0.14±0.03 nM (gt1bD168V), 0.30±0.04 nM (gt1bD168Y), 5.3±0.9 nM (gt1bA156T), and 12±2 nM (gt1bA156V), respectively. In the replicon assay, MK-5172 demonstrates subnanomolar to low-nanomolar EC50s against genotypes 1a, 1b, and 2a, with EC50s of 0.5±0.1 nM, 2±1 nM, and 2±1 nM for gt1bcon1, gt1a, and gt2a, respectively. MK-5172 is potent against a panel of HCV replication mutants NS5A (Y93H) (EC50=0.7±0.3 nM), NS5B nucleosides (S282T) (EC50=0.3±0.1 nM), and NS5B (C316Y) (EC50=0.4±0.2). MK-5172 maintains the excellent potency against the gt 3a enzyme as well as a broad panel of mutant enzymes, has excellent potency in the replicon system [gt1b IC50(50% NHS)=7.4 nM; gt1a IC50(40% NHS)=7 nM], and shows excellent rat liver exposure.

MK-5172 (Grazoprevir) demonstrates high in vivo efficacy against chronic-HCV-infected chimpanzees. When dosed to dogs, MK-5172 shows low clearance of 5 mL/min/kg and a 3 h half-life after iv dosing and has good plasma exposure (AUC=0.4 μM h) after a 1 mg/kg oral dose. Dog liver biopsy studies showed that the liver concentration of MK-5172 after the 1 mg/kg oral dose is 1.4 μM at the 24 h time point. Similar to its behavior in rats, MK-5172 demonstrates effective partitioning into liver tissue and maintains high liver concentration, relative to potency, 24 h after oral dosing in dogs.

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In vivo efficacy. [1]
To demonstrate in vivo efficacy, Grazoprevir/MK-5172 was administered orally to three chronically HCV-infected chimpanzees at a dose of 1 mg per kg twice daily for 7 days. Two of the chimpanzees had wild-type (WT) gt1a or gt1b infections with high viral titers (∼106 IU/ml). A third chimpanzee had a modest viral titer (∼104 IU/ml) that was gt1a NS3 R155K virus. This chimpanzee maintained a chronic R155K viral infection in the absence of prior experimental treatment with an HCV small molecule inhibitor (J. Fontenot, personal communication). Pharmacodynamic responses to MK-5172 are shown in Fig. 4A.

All animals experienced an immediate, profound reduction in viral titer. The gt1a (WT) infection was suppressed ∼4 logs within 2 days to ∼100 IU/ml, and viral suppression was maintained throughout dosing. The gt1b infection was suppressed more than 5 logs to the level of quantification (20 IU/ml); there was no genetic evidence for the emergence of resistance either during dosing or postdosing.

The gt1a NS3 R155K-infected chimp experienced a rapid ∼2-log reduction in viral titer. Viral load gradually drifted higher during the remainder of the dosing period and returned to baseline levels only following cessation of dosing. The virus was homogenous for the R155K mutation throughout the study. There were no additional mutations elicited by dosing and no genetic evidence to suggest that fluctuations in viral titer either during or postdosing were due to newly emerging resistant variants.

MK-5172/Grazoprevir concentrations were determined from matched plasma and liver biopsy samples collected 12 h after administration of the final dose (Table 7). Drug concentrations were significantly higher in the liver, ranging from 0.85 to 1.99 μM, compared to the low-nanomolar concentrations in plasma. This yields liver-to-plasma ratios of 425 to 785. Viral load reductions at this time point were greater than 4 logs for the gt1a and gt1b infections and 0.8 logs for the gt1a NS3 R155K infection. Although a pharmacokinetic-pharmacodynamic relationship cannot be determined from a single drug dose, the viral load reductions are more reflective of drug concentrations in liver.

The in vivo efficacy of Grazoprevir/MK-5172 is illustrated further by comparing responses of the gt1b-infected chimpanzee to either MK-5172 or vaniprevir under identical dosing regimens (Fig. 4B). The viral titer was suppressed an additional log with MK-5172. Liver drug concentrations 12 h after final doses were also ∼4-fold higher with MK-5172, at 1.97 μM compared to 0.54 μM with vaniprevir, indicating better drug exposure at the site of HCV replication.

On the basis of the greater potency across both genotypes and clinically relevant resistant mutants, the improved pharmacokinetics, the excellent 24-h liver concentrations in preclinical species, and the in vivo efficacy in HCV-infected chimpanzees, Grazoprevir/MK-5172 was selected for clinical development.

recombinant HCV NS3/4A enzymes are expressed and purified from E. Coli. Enzyme sequences are derived from genotype 1a (gt1a) H77, gt1b con1, gt2a JFH1, gt2b HCJ8, and gt3a NZL1. Inhibition of HCV NS3/4A protease activity in reaction mixtures containing MK-5172 (Grazoprevir), Vaniprevir, or the reference compounds Danoprevir and TMC435 is determined in a time-resolved fluorescence assay. Cell-based HCV replicon assays are conducted in genotype 1b (con1) stable cell line HB1 or a gt2a cell line (JFH) in the presence of either 10% fetal bovine serum (FBS) or 40% normal human serum (NHS). Determinations of 50% effective concentrations (EC50s) against the panel of genotype or mutant replicon cell lines are conducted using a TaqMan-based assay. The 50% cytotoxic concentration (CC50) is determined in the HCV replicon cell line with the use of an MTS assay. Potency determinations against clinical genotype 1 NS3/4A sequences are made using a transient cell-based phenotype assay. The NS3/4A patient isolates are cloned from human plasma infected with HCV. Broad counterscreening, in which MK-5172 is evaluated for its inhibitory potency at a concentration of 10 μM, is conducted at MDS Pharma Services.

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Enzymatic Assays. [2]
Compound inhibitory potencies were determined with use of a time-resolved fluorescence assay for NS3/4A protease activity. The NS3 protease assay was performed in a final volume of 100 µL in assay buffer containing 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt (HEPES), pH 7.5, 150 mM NaCl, 15 % glycerol, 0.15 % Triton X-100, 10 mM dithiothreitol (DTT), and 0.1 % PEG8000. The NS3 protease was pre-incubated with various concentrations of inhibitors in dimethylsulfoxide (DMSO) for 30 minutes. The reaction was initiated by adding the time-resolved fluorescence (TRF) peptide substrate (final concentration 100 nM). NS3 mediated hydrolysis of the substrate was quenched after 1 h at RT with 100 µL of 500 mM 2-(Nmorpholino)ethanesulfonic acid (MES), pH 5.5. Product fluorescence was detected using either a Victor V2 or Fusion fluorophotometer with excitation at 340 nm and emission at 615 nm with a 400 µs delay. The inhibition constants were derived using a standard four-parameter fit to the data. Full length NS3/4A protease sequences from gt 1b (BK), gt 3a (NZL1), or gt1b encoding amino acid mutations R155K, A156T, A156V, or D168V were expressed and purified from E. coli. as his-tagged fusion proteins using a previously described protocol.26 Protease mutations were engineered into the gt1b expression construct using standard molecular biology techniques.

HB1 cells (30,000 per well) are seeded of a 6-well tissue culture plate per drug concentration. The next day (day 0), the medium is replenished with fresh medium and MK-5172 at the appropriate drug concentration. Cells from a single well per drug concentration are harvested on days 0, 1, and 2, washed, and stored frozen until evaluation. The fourth well is similarly harvested on day 3.5 except that 30,000 cells are reseeded with fresh medium and MK-5172 at the appropriate drug concentration. For additional time points, cells are passaged and harvested every one-half week for 2 weeks. For the third week, cells are similarly treated except that cells received replenishing medium which contained 0.5 mg/ml G418 without protease inhibitor.

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In vitro assays. [1]
Recombinant HCV NS3/4A enzymes were expressed and purified from Escherichia coli as previously described. Enzyme sequences were derived from genotype 1a (gt1a) H77 (GenBank accession no. AF09606), gt1b con1 (GenBank accession no. AJ238799), gt2a JFH1 (GenBank accession no. AB047639), gt2b HCJ8 (GenBank accession no. D10988), and gt3a NZL1 (GenBank accession no. D17763). Inhibition of HCV NS3/4A protease activity in reaction mixtures containing Grazoprevir/MK-5172, vaniprevir, or the reference compounds danoprevir and TMC435 (Fig. 1) was determined in a time-resolved fluorescence assay. Cell-based HCV replicon assays were conducted in genotype 1b (con1) stable cell line HB1 or a gt2a cell line (JFH) in the presence of either 10% fetal bovine serum (FBS) or 40% normal human serum (NHS). Determinations of 50% effective concentrations (EC50s) against the panel of genotype or mutant replicon cell lines were conducted using a TaqMan-based assay. The 50% cytotoxic concentration (CC50) was determined in the HCV replicon cell line with the use of an MTS assay according to the manufacturer's protocol. Potency determinations against clinical genotype 1 NS3/4A sequences were made using a transient cell-based phenotype assay. The NS3/4A patient isolates were cloned from human plasma infected with HCV. Broad counterscreening, in which MK-5172 was evaluated for its inhibitory potency at a concentration of 10 μM, was conducted at MDS Pharma Services.

For in vitro resistance selections, 100,000 HB1 cells were seeded into a T162 Z-top flask and cultured in the presence of 0.5 mg/ml G418 and the desired concentration of Grazoprevir/MK-5172. Cells were cultured for approximately 3 weeks with regular exchanges of medium until sufficient cell death had occurred to enable distinct colonies to form. After expansion, total RNA was isolated, used as a template to generate NS3/4a cDNA, and sequenced using conventional molecular biology techniques. Mutations were identified through comparison with the sequence generated from untreated cells.

For the 2-week potency evaluations, 30,000 HB1 cells were seeded per well of a 6-well tissue culture plate per drug concentration. The next day (day 0), the medium was replenished with fresh medium and Grazoprevir/MK-5172 at the appropriate drug concentration. Cells from a single well per drug concentration were harvested on days 0, 1, and 2, washed, and stored frozen until evaluation. The fourth well was similarly harvested on day 3.5 except that 30,000 cells were reseeded with fresh medium and MK-5172 at the appropriate drug concentration. For additional time points, cells were passaged and harvested every one-half week for 2 weeks. For the third week, cells were similarly treated except that cells received replenishing medium which contained 0.5 mg/ml G418 without protease inhibitor.

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Replicon Assay. [2]
Inhibition of viral replication was determined with use of the HCV bicistronic replicon assay27 adapted for quantitative analysis using in situ hybridization.28 Huh-7 cells that were stably transfected with HCV replicon RNA (gt 1b con1 sequence;28 gt 2a JFH sequence29) were seeded into 96 well plates impregnated with scintillant at a density of 20,000 cells per well and incubated at 37 °C/5%CO2 for 24 h in the presence of Dulbecco’s modified eagle’s medium (DMEM) supplemented with 50% NHS. Compound in DMSO was added to 1%, and incubated for a further 24 h. Cells were fixed by treatment with 10% formaldehyde and permeabilized by treatment with 0.25% Triton X100. A radiolabeled RNA probe that hybridizes to the neomycin resistance gene of the replicon was added, and hybridized at 50 °C for 18 h, followed by RNase A treatment to remove unhybridized probe and washing. The plate was then counted in a Topcount NXT. The inhibition constants were derived using a standard four-parameter fit to the data.

\n\n\nRats and Dogs
Research is conducted on rats and dogs. Grazoprevir is formulated in polyethylene glycol 200 (PEG200) and given as a bolus at either 2 mg/kg of body weight (for rats) or 0.5 mg/kg (for dogs) in studies where the drug is dosed intravenously. The compound's crystalline potassium salt is dosed as a solution in PEG400 at 5 mg/kg (for rats) or 1 mg/kg (for dogs) for oral studies.In every study, blood samples are taken at the appropriate times in tubes containing EDTA, and the plasma is separated by centrifugation and kept at -70°C until analysis. After protein precipitation, Grazoprevir (MK-5172) levels are quantified using high-performance liquid chromatography/mass spectroscopy (LC/MS/MS). At the end of the experiment, liver samples are taken from rat studies. After sedation, liver biopsy samples (20 μL) are taken from the dog. Following protein precipitation, tissue samples are homogenized in four volumes of deionized water, and drug concentrations are assessed using LC/MS/MS.\n

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\n\nPharmacokinetic studies. [1]
\nFor studies in which Grazoprevir/MK-5172 was dosed intravenously to rats or dogs, the compound was formulated in polyethylene glycol 200 (PEG200) and administered as a bolus at either 2 mg/kg of body weight (rat) or 0.5 mg/kg (dog). For oral studies, the crystalline potassium salt of the compound was dosed as a solution in PEG400 at 5 mg/kg (rat) or 1 mg/kg (dog). For all studies, blood samples were collected in EDTA-containing tubes at appropriate times and plasma was separated by centrifugation and stored at −70°C until analysis. Quantitation of Grazoprevir/MK-5172 levels was conducted by high-performance liquid chromatography/mass spectroscopy (LC/MS/MS) following protein precipitation. Liver samples were obtained from rat studies at the termination of the experiment. For dog, liver biopsy samples (∼20 μl) were collected following sedation. Tissue samples were homogenized in four volumes of deionized water, and drug concentrations were determined by LC/MS/MS after protein precipitation.\n

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\n\nStudies in HCV-infected chimpanzees. [1]
\nThe HCV genotype was determined by a line probe assay and confirmed by reverse transcription-PCR (RT-PCR) rescue and sequencing of HCV genetic materia. HCV-infected chimpanzees were dosed orally at 1 mg per kg twice daily (b.i.d.) for 7 days by the voluntary ingestion of Grazoprevir/MK-5172 (in a Tang vehicle) or vaniprevir (in a milk vehicle). Viral load determinations were performed on plasma samples using the HCV TaqMan assay. Grazoprevir/MK-5172 drug concentrations in plasma or liver biopsy specimens were conducted as described above (under “Pharmacokinetic studies”). Viral resistance analysis was conducted according to a previously published protocol.\n

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\n\nPharmacokinetics. [2]
\nPharmacokinetic characterization of test agents was conducted in conscious male Sprague-Dawley rats (300-500 g; n = 2-3/study) or male and female beagle dogs (13-15 kg; n = 3/study). Compounds (e.g.Grazoprevir) were dosed intravenously to fasted rats and dogs. Compounds in DMSO were administered as a bolus (1.0, 0.1 mL/kg respectively) in DMSO. For oral studies in rat and dog, compounds were dosed as a solution in polyethyleneglycol 400 (PEG400) (2.0 mL/kg). Typical doses were 2 mg/kg IV and 5 mg/kg P.O. to rats and 0.5 mg/kg IV and 1 mg/kg P.O. to dogs. Blood samples for the determination of test agent plasma concentration were obtained at multiple time points up to 24 h after single dose test agent administration. Liver samples were taken at terminal time points for rats and as liver biopsies in dog. Liver samples were homogenized in buffer prior to analysis. Plasma and liver samples were analyzed using liquid-liquid extraction and LC/MS with appropriate standards and QCs. Pharmacokinetic parameters were calculated using Watson software. \n\n

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\n\n

Formulated in polyethylene glycol 200 (PEG200); administered as a bolus at either 2 mg/kg of body weight (Rats) or 0.5 mg/kg (dog). For oral studies, the crystalline potassium salt of the compound is dosed as a solution in PEG400 at 5 mg/kg (Rats) or 1 mg/kg (dog).

Rat and dog

Absorption, Distribution and Excretion
Glazoprovir reaches peak plasma concentrations 0.5–3 hours after administration. The absolute bioavailability of glazoprovir is 27%. Peak concentrations increase 2.8-fold when administered with food, but this increase in exposure is considered clinically insignificant. Glazoprovir is primarily excreted in feces (90%), with very little excretion in urine (<1%). The apparent volume of distribution of glazoprovir is estimated to be 1250 liters. It is believed to be primarily distributed in the liver, and its absorption is facilitated by the organic anion transport polypeptide 1B1/3. Clearance of glazoprovir has not been determined. Metabolism/Metabolites Glazoprovir is partially eliminated via CYP3A-mediated oxidative metabolism. Circulating metabolites have not been detected in human plasma. Biological Half-Life In HCV-infected individuals, the geometric mean apparent terminal half-life of glazoprovir is 31 hours. The pharmacokinetic properties of compound 15/grazoprevir potassium were evaluated in several animal models (Table 3). In rats, compound 15 was cleared from plasma at a rate of 28 mL/min/kg with a plasma half-life of 1.4 h. Following an oral dose of 5 mg/kg, plasma exposure of compound 15 was good, with an AUC of 0.7 μM·h. Hepatic exposure was also quite good (23 μM at 4 h), with compound 15 still present in the liver 24 hours after a single oral dose of 5 mg/kg. After 24 hours, the hepatic concentration of compound 15 was 0.2 μM, more than 25 times higher than the IC50 value obtained using a replicon assay with 50% NHS. In dogs, compound 15/grazoprevir was cleared from intravenous administration at a rate as low as 5 mL/min/kg with a half-life of 3 h, and showed good plasma exposure after an oral dose of 1 mg/kg (AUC = 0.4 μM·h). Liver biopsy studies in dogs showed that 24 hours after oral administration of 1 mg/kg, the liver concentration of compound 15 was 1.4 μM. Similar to its performance in rats, compound 15 was efficiently distributed to the liver tissue in dogs and maintained a high liver concentration (relative to its potency) 24 hours after oral administration. [2]

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Effects During Pregnancy and Lactation
◉ Overview of Medications Used During Lactation
Grazoprevir has not been studied in breastfeeding women receiving treatment for hepatitis C. Because it binds to maternal plasma proteins at a rate exceeding 98.9%, its levels in breast milk are likely to be very low. Some sources suggest that breastfeeding should be avoided when grazoprevir is used in combination with ribavirin.
Hepatitis C is not transmitted through breast milk, and breast milk has been shown to inactivate the hepatitis C virus (HCV). However, the U.S. Centers for Disease Control and Prevention (CDC) recommends that breastfeeding should be considered if the mother has cracked or bleeding nipples. It is unclear whether this warning applies to mothers receiving treatment for hepatitis C.
Infants born to HCV-infected mothers should be tested for HCV infection; nucleic acid testing is recommended because maternal antibodies are present in the infant for the first 18 months after birth and until the infant's autoimmune response develops.
◉ Impact on breastfed infants
No published information found as of the revision date.
◉ Impact on lactation and breast milk
No published information found as of the revision date.

[1]. Antimicrob Agents Chemother.2012 Aug;56(8):4161-7.

[2]. ACS Med Chem Lett.2012 Mar 2;3(4):332-6.

[3]. Signal Transduct Target Ther. 2021 May 29;6(1):212.

HCV NS3/4a protease inhibitors are effective drugs for treating chronic hepatitis C virus infection. Bosavirin and terabhivir have recently been approved as adjunctive therapy to pegylated interferon/ribavirin combination therapy for patients with genotype 1 infection. Overcoming antiviral resistance, broad genotype coverage, and convenient dosing regimens are important characteristics for future drugs that can be used in combination without interferon. This article reports preclinical results of the novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor MK-5172, which is currently in clinical development. This compound exhibits sub-nanomolar activity against a wide range of enzyme profiles covering major hepatitis C virus (HCV) genotypes and early protease inhibitor-resistant variants. In replicon selection assays, MK-5172 showed high selective pressure, producing only a very small number of resistant colonies. In rats and dogs, MK-5172 showed good plasma and liver exposure, with 24-hour liver concentrations suggesting once-daily dosing is sufficient. When MK-5172 was administered to chimpanzees infected with chronic gt1a or gt1b HCV at a dose of 1 mg/kg body weight twice daily (bid) for 7 days, viral load was reduced by 4 to 5 log units. Based on its preclinical results, MK-5172 is expected to have broad activity against multiple HCV genotypes and clinically significant drug-resistant variants, making it ideal for inclusion in novel all-oral treatment regimens. [1]

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We designed a novel class of HCV NS3/4a protease inhibitors using a molecular modeling strategy, which contain P2 to P4 macrocyclic structures. Based on the properties of previous clinical compounds and exploring the P2 and linker regions of this series of compounds, we optimized their potency against multiple genotypes and mutant enzymes, cellular activity, and exposure to rat liver after oral administration. These studies ultimately identified clinical candidate compound 15 (MK-5172), which is active against NS3/4a types 1–3 and clinically relevant mutant enzymes, and has good plasma and excellent liver exposure in a variety of animals. [2]

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Grazoprevir is an aza-macrocyclic compound belonging to the hepatitis C protease inhibitor class, used in combination with elbasvir (trade name Zepatier) for the treatment of chronic hepatitis C virus type 1 or 4 infection in adults. It has a dual role as an antiviral drug, a hepatoprotective agent, and a hepatitis C protease inhibitor. It is an aza-macrocyclic compound, carbamate, lactam, aromatic ether, cyclopropane compound, N-sulfonylformamide, and quinoxaline derivative.
Grazoprevir is a direct-acting antiviral drug used in combination therapy for chronic hepatitis C. Chronic hepatitis C is an infectious liver disease caused by hepatitis C virus (HCV) infection. HCV is a single-stranded RNA virus with nine different genotypes, of which genotype 1 is the most common in the United States, affecting 72% of chronic HCV patients. Since 2011, significant progress has been made in treatment options for chronic hepatitis C with the development of direct-acting antiviral agents (DAAs) such as granzopvir. Granzopvir is an inhibitor of NS3/4A, which are serine proteases encoded [synthesized] by HCV genotypes 1 and 4. These enzymes are crucial for viral replication, cleaving viral-encoded polyproteins into mature proteins such as NS3, NS4A, NS4B, NS5A, and NS5B. The resistance barrier for NS3/4A inhibitors is lower than that for NS5B inhibitors (another class of direct-acting antiviral agents). Substitutions at amino acid positions 155, 156, or 168 are known to lead to resistance. Replacement of the enzyme catalytic triad composed of H58, D82, and S139 may also alter the drug's affinity for NS3/4A or the activity of the enzyme itself. Despite this drawback, grazoprevir remains effective against hepatitis C virus, especially when used in combination with [DB11574]. In 2016, the American Association for the Study of Liver Diseases (AASLD) and the Infectious Diseases Society of America (IDSA) jointly published guidelines recommending the combination of grazoprevir and [DB11574] for the treatment of hepatitis C virus infections of types 1a, 1b, and 4. Grazoprevir and [DB11574] can be used in combination with or without [DB00811] to achieve a cure or sustained virological response (SVR), with therapeutic effects typically achieved after 12 weeks of daily treatment. SVR and eradication of hepatitis C virus infection are associated with significant long-term health benefits, including reduced liver-related damage, improved quality of life, reduced incidence of hepatocellular carcinoma, and reduced all-cause mortality. Grazoprevir is a fixed-dose combination product with [DB11574] (brand name Zepatier) for the treatment of chronic hepatitis C. Zepatier was approved by the FDA in January 2016 for the treatment of HCV genotypes 1 and 4, with or without [DB00811], depending on the presence of resistance-related amino acid substitutions in the NS5A protein and failure to respond to previous treatment with [DB00811], [DB00008], [DB00022], or other NS3/4A inhibitors (such as [DB08873], [DB06290], or [DB05521]). Grazoprevir, when used in combination with [DB11574] to form the combination formulation Zepatier, showed a sustained virological response rate (SVR) of 94% to 97% in genotype 1 patients and 97% to 100% in genotype 4 patients after 12 weeks of treatment. This combination therapy can be used for patients with compensated cirrhosis, concurrent human immunodeficiency virus infection, or severe renal disease. Anhydrous granzoprovir is a hepatitis C virus NS3/4A protease inhibitor. The mechanism of action of anhydrous granzoprovir is as an HCV NS3/4A protease inhibitor, a breast cancer resistance protein inhibitor, and a cytochrome P450 3A inhibitor. Drug Indications Granzoprovir, in combination with [DB11574] (in the form of the fixed-dose combination formulation Zepatier), or alone or in combination with [DB00811], is used to treat chronic HCV genotype 1a, 1b, or 4 infection in adults. FDA Label Treatment of Chronic Hepatitis C Mechanism of Action Granzoprovir is a second-generation NS3/4a protease inhibitor used to inhibit HCV viral replication. NS3/4a proteases are components of viral replication, mediating the cleavage of viral-encoded polyproteins into mature proteins (NS3, NS4A, NS4B, NS5A, and NS5B). Grazoprevir inhibits the NS3/4 proteases of HCV genotypes 1a, 1B, and 4, with IC50 values of 7 pM, 4 pM, and 62 pM, respectively. HCV NS3/4a protease inhibitors have proven effective in treating chronic hepatitis C virus infection. Bosepvir and traprovir have recently been approved as adjunctive therapy to pegylated interferon/ribavirin combination therapy for patients with genotype 1 infection. Overcoming antiviral resistance, broad genotype coverage, and convenient dosing regimens are important characteristics for future drugs that can be used in combination without interferon. This article reports preclinical results of a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor, Grazoprevir/MK-5172, which is currently in clinical development. This compound exhibited sub-nanomolar activity against a wide range of enzymes, including major hepatitis C virus (HCV) genotypes and early protease inhibitor-resistant variants. In replicon selection, MK-5172 exerted high selective pressure, producing only minimally resistant colonies. In rats and dogs, MK-5172 demonstrated good plasma and liver exposure, with 24-hour liver concentrations suggesting once-daily dosing is sufficient. When administered to chimpanzees infected with chronic gt1a or gt1b HCV, MK-5172 at a dose of 1 mg/kg body weight twice daily (bid) for 7 days reduced viral load by 4 to 5 log units. Based on its preclinical results, MK-5172 is expected to have broad activity against multiple HCV genotypes and clinically significant resistant variants, making it ideally suited for inclusion in novel all-oral treatment regimens. [1] Phenotypic analysis showed that glazoprevir/MK-5172 maintained activity against genetically diverse samples of genotype 1a and 1b from plasma of HCV-infected patients. In preclinical animal models, MK-5172 exhibited favorable pharmacokinetic characteristics with good plasma concentrations while maintaining high liver concentrations similar to those previously reported with vaniprevir. Importantly, moderate oral doses resulted in 24-hour liver concentrations in preclinical animal models that were significantly higher than in vitro EC50 values. Resistance screening experiments showed that even at low concentrations, MK-5172 induced only a small number of colonies. MK-5172 demonstrated moderate-dose efficacy in vivo against chimpanzees infected with chronic hepatitis C virus (HCV), including better viral load inhibition compared to alternating administration of vaniprevir at the same dose and frequency in the same animals. These characteristics together suggest that MK-5172 is a more potent inhibitor than currently developed HCV protease inhibitors and has the potential to improve HCV treatment regimens. In fact, early Phase I studies in healthy volunteers and HCV-infected patients have shown that MK-5172 has good preclinical characteristics that can be translated into a clinically effective drug with broad activity against multiple HCV genotypes and good pharmacokinetic characteristics that suggest once-daily (QD) dosing. [1]

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A novel HCV NS3/4a protease inhibitor containing P2 to P4 macrocyclic constraints was designed using a molecular modeling strategy. Based on the characteristics of previous clinical compounds and exploring the P2 and linker regions of this series of compounds, we optimized its inhibitory efficacy against multiple genotypes and mutant enzymes, cellular activity, and rat liver exposure after oral administration. These studies ultimately identified clinical candidate 15 (glazoprevir), which is active against NS3/4a genotypes 1–3 and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple animal models. [2] In summary, through preliminary screening and molecular modeling of the 3a genotype activity, we identified a series of P2 quinoline macrocyclic compounds that exhibit excellent broad-spectrum activity against both the NS3/4a genotype and the clinically observed 1b genotype mutant enzyme. We optimized the enzyme activity and liver exposure of this series of compounds in preclinical animal models. Compound 15 was obtained by introducing a weakly basic quinoxaline P2 heterocycle into this series of compounds to address the issue of disproportionation reactions with the more basic quinoline P2 heterocycle. We believe that the favorable pharmacokinetic properties and broad-spectrum enzyme inhibitory activity of compound 15 make it a promising second-generation NS3/4a protease inhibitor and a potential cornerstone of all-oral treatment regimens for hepatitis C. Further studies of compound 15 (glazoprevir) are currently underway, including clinical studies of its pharmacokinetics and efficacy. [2]

C38H52N6O10S

784.93

784.347

C, 58.15; H, 6.68; N, 10.71; O, 20.38; S, 4.08

1350462-55-3

Grazoprevir;1350514-68-9;Grazoprevir potassium salt;1206524-86-8;Grazoprevir sodium salt;1425038-27-2

71576667

White to off-white solid powder.

5.576

4

12

8

55

1580

7

S(C1([H])C([H])([H])C1([H])[H])(N([H])C([C@]1(C([H])([H])[C@@]1([H])C([H])=C([H])[H])N([H])C([C@]1([H])C([H])([H])[C@]2([H])C([H])([H])N1C([C@]([H])(C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])N([H])C(=O)O[C@]1([H])C([H])([H])[C@@]1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1C(=NC3C([H])=C(C([H])=C([H])C=3N=1)OC([H])([H])[H])O2)=O)=O)=O)(=O)=O.O([H])[H]

RXSARIJMSJWJLZ-CIAYNJNFSA-N

InChI=1S/C38H50N6O9S.H2O/c1-6-22-19-38(22,35(47)43-54(49,50)25-13-14-25)42-32(45)29-18-24-20-44(29)34(46)31(37(2,3)4)41-36(48)53-30-16-21(30)10-8-7-9-11-27-33(52-24)40-28-17-23(51-5)12-15-26(28)39-27;/h6,12,15,17,21-22,24-25,29-31H,1,7-11,13-14,16,18-20H2,2-5H3,(H,41,48)(H,42,45)(H,43,47);1H2/t21-,22-,24-,29+,30-,31-,38-;/m1./s1

(33R,35S,91R,92R,5S)-5-(tert-butyl)-N-((1R,2S)-1-((cyclopropylsulfonyl)carbamoyl)-2-vinylcyclopropyl)-17-methoxy-4,7-dioxo-2,8-dioxa-6-aza-1(2,3)-quinoxalina-3(3,1)-pyrrolidina-9(1,2)-cyclopropanacyclotetradecaphane-35-carboxamide hydrate

MK5172 hydrate; MK 5172; MK-5172 hydrate; 1350462-55-3; MK-5172 (hydrate); MK-5172 hydrate; Grazoprevir [USAN]; Grazoprevir monohydrate; 4O2AB118LA; Grazoprevir (USAN); Trade name: Zepatier‎.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~63.70 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.19 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.19 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (3.19 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)