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.

---

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.

Rats 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.

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Pharmacokinetic studies. [1]
For 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.

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Studies in HCV-infected chimpanzees. [1]
The 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.

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Pharmacokinetics. [2]
Pharmacokinetic 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.

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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
Grazoprevir reaches peak plasma concentration 0.5-3 hours after administration. Grazoprevir has an absolute bioavailability of 27%. When taken with food the peak concentration of Grazoprevir increases 2.8 fold but this increase in exposure has not been deemed clinically relevant.
Grazoprevir is mainly eliminated in the feces (90%) with very little eliminated in the urine (<1%).
Grazoprevir has an estimated apparent volume of distribution of 1250 liters. It is thought to distribute primarily to the liver with its uptake facilitated by organic anion transporting polypeptide 1B1/3.
The clearance of Grazoprevir has not been determined.

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Metabolism / Metabolites
Grazoprevir is partially eliminated by oxidative metabolism meditated by CYP3A. No circulating metabolites of have been detected in human plasma.

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Biological Half-Life
The geometric mean apparent terminal half-life for Grazoprevir is 31 hours in HCV-infected subjects.

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The pharmacokinetic properties of the potassium salt of compound 15/Grazoprevir were evaluated in multiple species (Table 3). In rat, 15 showed a plasma clearance of 28 mL/min/kg and a plasma half-life of 1.4 h. When dosed orally at 5 mg/kg, the plasma exposure of 15 was good with an AUC of 0.7 μM h. The liver exposure of the compound is quite good (23 μM at 4 h), and 15 remains in liver 24 h after a single 5 mg/kg oral dose. At 24 h, the liver concentration of 15 is 0.2 μM, which is >25-fold higher than the IC50 in the replicon assay with 50% NHS.
When dosed to dogs, compound 15/Grazoprevir 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 15 after the 1 mg/kg oral dose is 1.4 μM at the 24 h time point. Similar to its behavior in rats, 15 demonstrates effective partitioning into liver tissue and maintains high liver concentration, relative to potency, 24 h after oral dosing in dogs. [2]

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Grazoprevir has not been studied in nursing mothers being treated for hepatitis C infection. Because it is greater than 98.9% bound to maternal plasma proteins, amounts in breastmilk are likely to be very low. Some sources recommend against breastfeeding when grazoprevir is used with ribavirin.
Hepatitis C is not transmitted through breastmilk and breastmilk has been shown to inactivate hepatitis C virus (HCV). However, the Centers for Disease Control recommends that mothers with HCV infection should consider abstaining from breastfeeding if their nipples are cracked or bleeding. It is not clear if this warning would apply to mothers who are being treated for hepatitis C.
Infants born to mothers with HCV infection should be tested for HCV infection; because maternal antibody is present for the first 18 months of life and before the infant mounts an immunologic response, nucleic acid testing is recommended.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not 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 proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens.[1]

---

A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (MK-5172), which is active against genotype 1-3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species.[2]

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Grazoprevir is an azamacrocyclic compound that is a hepatitis C protease inhibitor used in combination with elbasvir (under the brand name Zepatier) for treatment of chronic HCV genotypes 1 or 4 infection in adults. It has a role as an antiviral drug, a hepatoprotective agent and a hepatitis C protease inhibitor. It is an azamacrocycle, a carbamate ester, a lactam, an aromatic ether, a member of cyclopropanes, a N-sulfonylcarboxamide and a quinoxaline derivative.
Grazoprevir is a direct acting antiviral medication used as part of combination therapy to treat chronic Hepatitis C, an infectious liver disease caused by infection with Hepatitis C Virus (HCV). HCV is a single-stranded RNA virus that is categorized into nine distinct genotypes, with genotype 1 being the most common in the United States, and affecting 72% of all chronic HCV patients. Treatment options for chronic Hepatitis C have advanced significantly since 2011, with the development of Direct Acting Antivirals (DAAs) such as Grazoprevir. Grazoprevir is an inhibitor of NS3/4A, a serine protease enzyme, encoded by HCV genotypes 1 and 4 [synthesis]. These enzymes are essential for viral replication and serve to cleave the virally encoded polyprotein into mature proteins like NS3, NS4A, NS4B, NS5A and NS5B. The barrier for develoment of resistance to NS3/4A inhibitors is lower than that of NS5B inhibitors, another class of DAAs. Subtitutions at amino acid positions 155, 156, or 168 are known to confer resistance. The substitutions of the enzyme's catalytic triad consisting of H58, D82, and S139 are also likely to alter the affinity of the drug for NS3/4A or the activity of the enzyme itself. Despite this disadvantage Grazoprevir is still effective against HCV particularly when paired with [DB11574]. In a joint recommendation published in 2016, the American Association for the Study of Liver Diseases (AASLD) and the Infectious Diseases Society of America (IDSA) recommend Grazoprevir as first line therapy in combination with [DB11574] for genotypes 1a, 1b, and 4 of Hepatitis C. Grazoprevir and [DB11574] are used with or without [DB00811] with the intent to cure, or achieve a sustained virologic response (SVR), after 12 weeks of daily therapy. SVR and eradication of HCV infection is 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 available as a fixed dose combination product with [DB11574] (tradename Zepatier) used for the treatment of chronic Hepatitis C. Approved in January 2016 by the FDA, Zepatier is indicated for the treatment of HCV genotypes 1 and 4 with or without [DB00811] depending on the the presence of resistance associated amino acid substitutions in the NS5A protein and previous treatment failure with [DB00811], [DB00008], [DB00022], or other NS3/4A inhibitors like [DB08873], [DB06290], or [DB05521]. When combined together, Grazoprevir and [DB11574] as the combination product Zepatier have been shown to achieve a SVR between 94% and 97% for genotype 1 and 97% and 100% for genotype 4 after 12 weeks of treatment. It can be used in patients with compensated cirrhosis, human immunodeficiency virus co-infection, or severe kidney disease.
Grazoprevir anhydrous is a Hepatitis C Virus NS3/4A Protease Inhibitor. The mechanism of action of grazoprevir anhydrous is as a HCV NS3/4A Protease Inhibitor, and Breast Cancer Resistance Protein Inhibitor, and Cytochrome P450 3A Inhibitor.

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Drug Indication
Grazoprevir is indicated in combination with [DB11574] (as the fixed dose combination product Zepatier) with or without [DB00811] for treatment of chronic HCV genotypes 1a, 1b, or 4 infection in adults.
FDA Label
Treatment of chronic hepatitis C

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Mechanism of Action
Grazoprevir is a second generation NS3/4a protease inhibitor used to inhibit viral HCV replication. NS3/4a protease is an integral part of viral replication and mediates the cleavage the virally encoded polyprotein to mature proteins (NS3, NS4A, NS4B, NS5A and NS5B). Grazoprevir inhibits the NS3/4protease enzymes of HCV genotype 1a, 1B, and 4 with IC50 values of 7pM, 4pM, and 62pM, respectively.

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HCV NS3/4a protease inhibitors are proven therapeutic agents against chronic hepatitis C virus infection, with boceprevir and telaprevir having recently received regulatory approval as add-on therapy to pegylated interferon/ribavirin for patients harboring genotype 1 infections. Overcoming antiviral resistance, broad genotype coverage, and a convenient dosing regimen are important attributes for future agents to be used in combinations without interferon. In this communication, we report the preclinical profile of Grazoprevir/MK-5172, a novel P2-P4 quinoxaline macrocyclic NS3/4a protease inhibitor currently in clinical development. The compound demonstrates subnanomolar activity against a broad enzyme panel encompassing major hepatitis C virus (HCV) genotypes as well as variants resistant to earlier protease inhibitors. In replicon selections, MK-5172 exerted high selective pressure, which yielded few resistant colonies. In both rat and dog, MK-5172 demonstrates good plasma and liver exposures, with 24-h liver levels suggestive of once-daily dosing. When administered to HCV-infected chimpanzees harboring chronic gt1a or gt1b infections, MK-5172 suppressed viral load between 4 to 5 logs at a dose of 1 mg/kg of body weight twice daily (b.i.d.) for 7 days. Based on its preclinical profile, MK-5172 is anticipated to be broadly active against multiple HCV genotypes and clinically important resistance variants and highly suited for incorporation into newer all-oral regimens. [1]

---

Phenotypic assays showed that Grazoprevir/MK-5172 maintained potency across a genetically diverse panel of genotype 1a and 1b sequences derived from plasma of HCV-infected patients. In preclinical animal species, MK-5172 demonstrated a favorable pharmacokinetic profile with good plasma concentrations while maintaining the high liver concentrations as previously described with vaniprevir. Importantly, moderate oral doses achieved 24-h liver concentrations in preclinical species that were well above the in vitro EC50s. Resistance selections demonstrated that MK-5172 elicited few colonies even at low concentrations of the inhibitor. MK-5172 proved highly efficacious in vivo at moderate doses against chronic-HCV-infected chimpanzees, including greater viral load suppression than vaniprevir when dosed alternatively to the same animal at an otherwise identical dose and frequency. Collectively, these properties identified MK-5172 as an inhibitor of greater potency than current developmental HCV protease inhibitors with the potential to improve HCV treatment options. Indeed, early phase I studies in both healthy volunteers and HCV-infected patients showed that MK-5172 has a favorable preclinical profile that translates into a clinically efficacious drug, is broadly active across multiple HCV genotypes, and possesses favorable pharmacokinetics suggestive of once-daily (QD) dosing [1]

---

A new class of HCV NS3/4a protease inhibitors containing a P2 to P4 macrocyclic constraint was designed using a molecular modeling-derived strategy. Building on the profile of previous clinical compounds and exploring the P2 and linker regions of the series allowed for optimization of broad genotype and mutant enzyme potency, cellular activity, and rat liver exposure following oral dosing. These studies led to the identification of clinical candidate 15 (Grazoprevir), which is active against genotype 1-3 NS3/4a and clinically relevant mutant enzymes and has good plasma exposure and excellent liver exposure in multiple species.[2]
In summary, initial screening for gt 3a activity along with molecular modeling has led to the discovery of a series of P2 quinoline macrocycles with excellent broad activity vs NS3/4a genoytpes and clinically observed gt 1b mutant enzymes. This series was optimized for enzyme activity and liver exposure in preclinical species. Compound 15 emerged from this series via the introduction of a mildly basic quinoxaline P2 heterocycle to deal with disproportionation issues with the more basic quinoline P2 heterocycle. We believe the combination of good PK, and broad enzyme potency gives compound 15 the potential to be an important second generation NS3/4a protease inhibitor that could be a cornerstone of an all-oral treatment for hepatitis C. Further studies of 15 (Grazoprevir), including clinical investigations of the pharmacokinetic and efficacy profile, are ongoing.[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.)