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)

体外活性：MK-5172 (Grazoprevir) 在针对常见耐药突变的主要基因型和变异体的生化检测中有效，Ki 为 0.01±<0.01 nM (gt1b)、0.01±0.01 nM (gt1a)、0.08±0.02 nM (gt2a)、0.15±0.06nM(gt2b)、0.90±0.2nM(gt3a)、0.07±0.01nM(gt1bR155K)、0.14±0.03nM(gt1bD168V)、0.30±0.04nM(gt1bD168Y)、5.3±0.9nM(gt1bD168Y) 1bA156T ) 和 12±2 nM (gt1bA156V)。在复制子测定中，MK-5172 对基因型 1a、1b 和 2a 表现出亚纳摩尔至低纳摩尔 EC50，对于 gt1bcon1、gt1a 和 gt2a 的 EC50 分别为 0.5±0.1 nM、2±1 nM 和 2±1 nM，分别。 MK-5172 对一组 HCV 复制突变体 NS5A (Y93H) (EC50=0.7±0.3 nM)、NS5B 核苷 (S282T) (EC50=0.3±0.1 nM) 和 NS5B (C316Y) (EC50=0.4±0.2) 有效。 ）。 MK-5172 保持了针对 gt 3a 酶以及多种突变酶的优异功效，在复制子系统中具有优异的功效 [gt1b IC50(50% NHS)=7.4 nM； gt1a IC50(40% NHS)=7 nM]，并且显示出优异的大鼠肝脏暴露。激酶测定：从大肠杆菌中表达并纯化重组 HCV NS3/4A 酶。酶序列源自基因型 1a (gt1a) H77、gt1b con1、gt2a JFH1、gt2b HCJ8 和 gt3a NZL1。在时间分辨荧光测定中测定含有 MK-5172 (Grazoprevir)、Vaniprevir 或参考化合物 Danoprevir 和 TMC435 的反应混合物中 HCV NS3/4A 蛋白酶活性的抑制。基于细胞的 HCV 复制子测定是在 10% 胎牛血清 (FBS) 或 40% 正常人血清 (NHS) 存在下，在基因型 1b (con1) 稳定细胞系 HB1 或 gt2a 细胞系 (JFH) 中进行。使用基于 TaqMan 的测定法确定针对一组基因型或突变复制子细胞系的 50% 有效浓度 (EC50)。使用 MTS 测定在 HCV 复制子细胞系中测定 50% 细胞毒性浓度 (CC50)。使用基于细胞的瞬时表型测定来确定针对临床基因型 1 NS3/4A 序列的效力。 NS3/4A 患者分离株是从感染 HCV 的人血浆中克隆出来的。 MDS Pharma Services 进行了广泛的反筛选，评估 MK-5172 在 10 μM 浓度下的抑制效力。细胞测定：将HB1细胞（每孔30,000个）按照药物浓度接种到6孔组织培养板中。第二天（第 0 天），用新鲜培养基和适当药物浓度的 MK-5172 补充培养基。在第 0、1 和 2 天从每个药物浓度的单个孔中收获细胞，洗涤并冷冻保存直至评估。第四个孔在第 3.5 天以类似方式收获，不同之处在于用新鲜培养基和适当药物浓度的 MK-5172 重新接种 30,000 个细胞。对于其他时间点，每半周传代并收获细胞，持续 2 周。第三周，对细胞进行类似处理，不同之处在于细胞接受含有0.5mg/ml G418且不含蛋白酶抑制剂的补充培养基。

MK-5172（Grazoprevir）对慢性 HCV 感染的黑猩猩表现出高体内功效。当给狗给药时，MK-5172 在静脉给药后显示出 5 mL/min/kg 的低清除率和 3 小时的半衰期，并且在口服 1 mg/kg 剂量后具有良好的血浆暴露（AUC=0.4 μM h）。狗肝活检研究表明，口服 1 mg/kg 剂量后 MK-5172 在 24 小时时间点的肝脏浓度为 1.4 μM。与在大鼠中的行为类似，MK-5172 在狗口服给药 24 小时后表现出有效分配到肝组织中并相对于效力保持较高的肝脏浓度。

重组 HCV NS3/4A 酶从大肠杆菌中表达和纯化。酶序列源自基因型 1a (gt1a) H77、gt1b con1、gt2a JFH1、gt2b HCJ8 和 gt3a NZL1。在时间分辨荧光测定中测定含有 MK-5172 (Grazoprevir)、Vaniprevir 或参考化合物 Danoprevir 和 TMC435 的反应混合物中 HCV NS3/4A 蛋白酶活性的抑制。基于细胞的 HCV 复制子测定在 10% 胎牛血清 (FBS) 或 40% 正常人血清 (NHS) 存在下，在基因型 1b (con1) 稳定细胞系 HB1 或 gt2a 细胞系 (JFH) 中进行。使用基于 TaqMan 的测定法确定针对一组基因型或突变复制子细胞系的 50% 有效浓度 (EC50)。使用 MTS 测定在 HCV 复制子细胞系中测定 50% 细胞毒性浓度 (CC50)。使用基于细胞的瞬时表型测定来确定针对临床基因型 1 NS3/4A 序列的效力。 NS3/4A 患者分离株是从感染 HCV 的人血浆中克隆出来的。 MDS Pharma Services 进行了广泛的反筛选，评估 MK-5172 在 10 μM 浓度下的抑制效力。

将 HB1 细胞（每孔 30,000 个）按照药物浓度接种到 6 孔组织培养板中。第二天（第 0 天），用新鲜培养基和适当药物浓度的 MK-5172 补充培养基。在第 0、1 和 2 天从每个药物浓度的单个孔中收获细胞，洗涤并冷冻保存直至评估。第四个孔在第 3.5 天以类似方式收获，不同之处在于用新鲜培养基和适当药物浓度的 MK-5172 重新接种 30,000 个细胞。对于其他时间点，每半周传代并收获细胞，持续 2 周。第三周，对细胞进行类似处理，不同之处在于细胞接受含有0.5mg/ml G418且不含蛋白酶抑制剂的补充培养基。

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.

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.

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.

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Protein Binding
Grazoprevir is more than 98.8% bound to plasma proteins. It binds both human serum albumin and α1-acid glycoprotein.

[1]. MK-5172, a selective inhibitor of hepatitis C virus NS3/4a protease with broad activity across genotypes and resistant variants. Antimicrob Agents Chemother. 2012 Aug;56(8):4161-7.

[2]. Discovery of MK-5172, a Macrocyclic Hepatitis C Virus NS3/4a Protease Inhibitor. ACS Med Chem Lett. 2012 Mar 2;3(4):332-6.

[3]. Bardoxolone and bardoxolone methyl, two Nrf2 activators in clinical trials, inhibit SARS-CoV-2 replication and its 3C-like protease. Signal Transduct Target Ther. 2021 May 29;6(1):212.

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.

C38H50N6O9S

766.9

766.335

C, 59.51; H, 6.57; N, 10.96; O, 18.78; S, 4.18

1350514-68-9

Grazoprevir potassium salt;1206524-86-8;Grazoprevir hydrate;1350462-55-3;Grazoprevir sodium salt;1425038-27-2

44603531

White to off-white solid powder

1.4±0.1 g/cm3

1.633

3.93

203.6

3

11

8

54

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

OBMNJSNZOWALQB-NCQNOWPTSA-N

InChI=1S/C38H50N6O9S/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)/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

MK5172; MK 5172; MK-5172; Trade name: Zepatier‎; 1350514-68-9; MK-5172; Grazoprevir anhydrous; MK5172; Grazoprevir [INN]; MK-5172 ANHYDROUS; 8YE81R1X1J;

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 : 50~100 mg/mL ( 65.20~130.39 mM )
Ethanol : 66.67~100 mg/mL(86.93 mM )

配方 1 中的溶解度: ≥ 2.5 mg/mL (3.26 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (3.26 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (3.26 mM)

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配方 4 中的溶解度: 12.5 mg/mL (16.30 mM) in 100% PEG-300 (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。