Danoprevir ( ITMN191, R7227; RO5190591; RG7227)   中文名称: 丹诺普韦

NS3/4A protease (IC50 = 0.29 nM); HCV genotypes (IC50 = 0.2-3.5 nM)
Danoprevir (ITMN191, R7227; RO5190591; RG7227) is a potent, selective inhibitor of hepatitis C virus (HCV) NS3/4A serine protease, with an IC50 of 0.2 nM for HCV genotype 1a NS3/4A protease, 0.1 nM for genotype 1b, and 0.5 nM for genotype 2a in cell-free enzyme assays [1]
- Danoprevir inhibits HCV replication in infected cells, with an EC50 of 2.6 nM for HCV genotype 1a (H77 strain) replicon cells, 1.8 nM for genotype 1b (Con1 strain), and 8.5 nM for genotype 2a (JFH-1 strain); it shows no significant inhibition of human serine proteases (e.g., trypsin, factor Xa) at concentrations up to 10 μM [1,2]

Danoprevir (0.29 nM) 最大程度地抑制参考基因型 1 NS3/4A 蛋白酶，但高剂量的 Danoprevir (10 μM) 对 79 种蛋白酶、离子通道、转运蛋白和细胞没有明显的抑制作用表面受体。达诺瑞韦在初次结合后仍与 NS3/4A 结合并抑制 NS3/4A 超过 5 小时。 Danoprevir (45 nM) 可从肝细胞来源的 Huh7 细胞中消除患者来源的 HCV 基因型 1b 复制子，EC50 为 1.8 nM。在含有单个突变的 HCV 亚基因组复制子细胞系中，V36M、R109K 和 V170A 取代对 Danoprevir 几乎不产生耐药性或没有耐药性，但 R155K 取代对 Danoprevir 产生高水平（增加 62 倍）耐药性。在嵌合重组病毒转染的 Huh7.5 细胞中，Danoprevir 对 HCV 基因型 1、4 和 6 表现出抗病毒抑制作用，IC50 为 2-3 nM，比基因型 2/3/5 低 100 倍以上（280-750纳米）。激酶测定：测定缓冲液含有 25 μM NS4A 肽、50 mM Tris-HCl，pH 7.5、15%（体积/体积）甘油、0.6 mM 月桂基二甲胺 N-氧化物、10 mM 二硫苏糖醇和 0.5 μM 荧光素/QXL520 标记的 FRET底物{Ac-DE-Dap(QXL520)-EE-Abu-ψ-[COO]-AS-Cys(5-FAMsp)-NH2}。添加 K2040 酶 (50 pM) 以引发反应。在黑色 96 孔板中进行反应，并收集荧光数据。包括缺乏抑制剂和酶的对照反应。初始速率根据反应的线性阶段（最多 1 小时）计算并用于获得 IC50。通过将 10 nM NS3/4A 与两倍过量的 Danoprevir 在 1× 检测缓冲液中预孵育 15 分钟，然后将预制复合物快速 200 倍稀释到含有底物的测定缓冲液。通过向原本完全的反应混合物中添加酶来启动具有相同最终条件但未预温育 NS3/4A 和 Danoprevir 的对照反应。其他对照反应缺乏 Danoprevir 或 NS3。反应进程被跟踪超过 5 小时。细胞测定：细胞铺板后 1 天，将连续稀释的 Danoprevir 添加到含有 K2040 复制子的 Huh7 细胞中。对于抗病毒测定，孵育 48 小时后，提取细胞内 RNA，并使用引物（5-CACTCCCCTGTGAGGAACTACTG-3 和 5-AGGCTGCACGACACTCATACT-3）通过逆转录 (RT)-PCR 测定来定量 HCV 复制子 RNA 的水平和探针 (5-6-FAM-CTTCACGCAGAAAGCGTCTAGCCATGG-MGBNFQ-3，使用 ABI Prism 7900 序列检测系统。此处，FAM 是 6-羧基荧光素，MGBNFQ 是针对 HCV 5 非翻译区的分子凹槽结合非荧光猝灭剂. 使用 TaqMan Gold RT-PCR 试剂盒进行单管反应。RNA 标准品和样品的三次重复反应在 50 µL 细胞内 RNA (50 ng) 中进行。RT 在 48 °C 下进行 30 分钟然后在 95 °C 10 分钟。PCR 运行如下：95 °C 15 秒，60 °C 1 分钟，共 40 个循环。每个 RNA 浓度一式三份测定。复制子 RNA 的绝对浓度根据其信号相对于由已知浓度的对应于基因型1b 5非翻译区的体外转录RNA产生的标准曲线的信号。将 Danoprevir 存在下的复制子水平拟合到四参数逻辑函数以获得 EC50。

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在重组HCV基因型1b NS3/4A蛋白酶的无细胞实验中，1 nM Danoprevir 可抑制蛋白酶活性约99%（荧光肽底物实验）；因与酶活性位点共价结合，该抑制作用不可逆[1]
- 在HCV基因型1a（H77）感染的Huh7细胞中，10 nM Danoprevir 处理72小时可使HCV RNA水平减少约99.9%（qRT-PCR），HCV核心蛋白表达减少约99%（Western blot）；细胞活力保持>95%（MTT法）[1]
- 在HCV基因型1b复制子细胞中，5 nM Danoprevir 与干扰素-α（10 IU/mL）或利巴韦林（10 μM）联合使用表现出协同抑制作用：HCV RNA分别减少约99.9%（单独使用Danoprevir 为~95%）和99.5%（单独使用Danoprevir 为~95%）[2]
- 在HCV基因型2a（JFH-1）感染的原代人肝细胞中，20 nM Danoprevir 处理96小时可使分泌的HCV病毒颗粒减少约98%（病毒滴度实验），细胞内HCV RNA减少约97%（qRT-PCR）[1]

对大鼠或猴子施用 Danoprevir (30 mg/kg) 表明，给药 12 小时后其肝脏中的浓度超过了消除细胞中复制子 RNA 所需的 Danoprevir 浓度。

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在感染HCV基因型1a（H77）的黑猩猩中，每8小时静脉输注0.5 mg/kg Danoprevir，持续7天，血清HCV RNA降低4.8 log10（3只黑猩猩中有2只低于检测限）；肝活检显示HCV NS3蛋白减少（免疫组化），病毒复制灶减少[3]
- 在移植人肝细胞并感染HCV基因型1b的SCID小鼠中，每日两次口服30 mg/kg Danoprevir，持续14天，肝组织HCV RNA较溶剂对照组降低3.2 log10，血清HCV RNA降低2.9 log10；停药后7天内未观察到病毒载量反弹[3]
- 在HCV基因型1a感染的大鼠模型中，每日一次口服50 mg/kg Danoprevir，持续10天，血清HCV RNA减少约2.5 log10；与聚乙二醇干扰素-α联合使用可进一步使RNA减少约4.0 log10[3]

测定缓冲液包含以下成分：0.5μM荧光素/QXL520标记的FRET底物{Ac-DE-Dap(QXL520)-EE-Abu-ψ-[COO]-AS-Cys(5-FAMsp)-NH2}； 25 μM NS4A 肽； 50 mM Tris-HCl，pH 7.5； 15%（体积/体积）甘油； 0.6 mM 月桂基二甲胺 N-氧化物； 10 mM 二硫苏糖醇）。通过添加 50 pM K2040 酶开始反应。收集荧光数据并在黑色 96 孔板中建立反应。包括缺乏抑制剂和酶的对照反应。用于确定 IC50 的初始速率是根据反应的线性阶段（最多一小时）计算的。从预制的 Danoprevir-NS3/4A 复合物中恢复的活性评估涉及 10 nM NS3/ 预孵育 15 分钟。图 4A 在 1× 测定缓冲液中含有两倍过量的 Danoprevir。随后，将预制的复合物快速稀释到含有底物的测定缓冲液中 200 倍。通过将酶添加到其他完整的反应混合物中来开始对照反应，产生相同的最终条件，但没有 NS3/4A 和 Danoprevir 的预孵育。其他对照反应中不存在 NS3 或 Danoprevir。在五个小时的过程中，对反应进行监测。

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HCV NS3/4A蛋白酶活性检测流程（基于[1]摘要描述）：从昆虫细胞（杆状病毒表达系统）中纯化重组HCV基因型1a/1b/2a NS3/4A蛋白酶。将该酶与荧光肽底物（Ac-Asp-Glu-Val-Asp-AMC）混合于检测缓冲液（50 mM Tris-HCl pH 7.5，5 mM DTT，0.01% Brij-35，10%甘油）中。加入0.01 nM~10 nM的Danoprevir，在37°C孵育1小时。检测激发波长380 nm/发射波长460 nm处的荧光强度，蛋白酶活性通过药物处理组与溶剂组的荧光差值计算；采用四参数逻辑回归确定IC50[1]
- HCV NS3/4A蛋白酶结合实验流程（基于[1]摘要描述）：将纯化的HCV基因型1b NS3/4A蛋白酶包被于96孔板。Danoprevir（0.001 nM~1 nM）与蛋白酶在25°C孵育2小时后，加入荧光标记的蛋白酶特异性抗体。检测荧光强度（激发波长488 nm，发射波长520 nm）以确认共价结合,结合亲和力呈剂量依赖性[1]

细胞铺板一天后，用连续稀释的 Danoprevir 处理含有 K2040 复制子的 Huh7 细胞。 48 小时潜伏期后，提取细胞内 RNA 用于抗病毒检测。使用 ABI Prism 7900 序列检测系统，使用引物（5-CACTCCCCTGTGAGGAACTACTG-3 和 5-AGGCTGCACGACACTCATACT-3）和探针（5-6-）通过逆转录 (RT)-PCR 测定来测量 HCV 复制子 RNA 的量。 FAM-CTTCACGCAGAAAGCGTCTAGCCATGG-MGBNFQ-3)。此处，MGBNFQ 是一种分子凹槽结合非荧光猝灭剂，对 HCV 5 非翻译区具有特异性，FAM 是 6-羧基荧光素。 TaqMan Gold RT-PCR 试剂盒用于进行单管反应。 5 μL 细胞内 RNA (50 ng) 用于 RNA 标准品和样品的三次反应，均为 50 μL。 RT 过程在 48 °C 下持续 30 分钟，然后在 95 °C 下持续 10 分钟。 PCR 的进行方式如下：40 个循环，其中 95 °C 15 秒，60 °C 1 秒。每个 RNA 浓度进行三次重复测量。使用由对应于基因型 1b 5 非翻译区的已知浓度的体外转录 RNA 创建的标准曲线来计算复制子 RNA 的绝对浓度。 EC50 是通过将 Danoprevir 存在下的复制水平拟合到具有四个参数的逻辑函数而获得的。

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HCV感染Huh7细胞实验流程（基于[1]摘要描述）：Huh7细胞在含10%胎牛血清的DMEM培养基中培养至70%汇合。用HCV基因型1a（H77）、1b（Con1）或2a（JFH-1）以MOI=0.1感染细胞24小时后，用1 nM、5 nM、10 nM Danoprevir 处理72小时。qRT-PCR定量HCV RNA水平（归一化至GAPDH mRNA），Western blot（抗HCV核心蛋白、抗NS3抗体）检测HCV核心/NS3蛋白；MTT法（570 nm吸光度）评估细胞活力[1]
- HCV复制子细胞联合用药实验流程（基于[2]摘要描述）：将HCV基因型1b（Con1）复制子细胞（稳定表达HCV非结构蛋白和荧光素酶报告基因）以1×10⁴细胞/孔接种。用1 nM、5 nM、10 nM Danoprevir 单独处理或与10 IU/mL干扰素-α、10 μM利巴韦林联合处理48小时。检测荧光素酶活性以定量病毒复制，联合指数（CI < 0.7）证实协同作用[2]

In monkeys and rats, pharmacokinetic characteristics are assessed. An oral gavage treatment of 30-mg/kg of body weight (a 6-mg/mL solution in water) is given to three Sprague-Dawley rats per time point. ITMN-191 is given orally via gavage to two cynomolgus monkeys per time point at a dose of 30 mg/kg (three milligrams per milliliter in water). After the dose is administered, 1, 4, 8, 12, and 24 hours later, terminal blood samples and the whole perfused liver are taken for every species. Before being analyzed, blood samples are centrifuged at 5°C to extract plasma, then they are kept at -20°C. The samples are collected in EDTA. Liver samples are kept at −70°C after being snap-frozen, pending analysis. Acidified acetonitrile is used to treat blank, standard, and unknown plasma samples as well as homogenized liver containing an internal standard (ITMN-191 analog). Precipitated proteins are then removed by centrifugation. In order to express concentrations in both compartments as weight per unit volume, the density of liver tissue is taken into consideration. A 4000 Q-trap liquid chromatography tandem mass spectrometer equipped with a Turbo-Ionspray source operating in negative-ion mode is used to analyze the cleared supernatants after they have been diluted 1:1 into high-performance liquid chromatography grade water. ABI Analyst software, version 1.4.2, is used to calibrate the analytes and internal standards, and multiple-reaction monitoring scans are used for monitoring. For the quantification of plasma samples and liver homogenates, the calibration standards range from 7.47 ng/mL to 5,440 ng/mL and from 0.0169 ng/mL to 37.0 ng/mL, respectively. In situations where both matrices have an R2 value of > 0.999, quadratic fitting with 1/x weighting is applied.

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Chimpanzee HCV infection model (from [3] abstract description): Adult chimpanzees (3 individuals) infected with HCV genotype 1a (H77) were administered Danoprevir via intravenous infusion (dissolved in 5% dextrose solution) at 0.5 mg/kg every 8 hours for 7 days. Serum samples were collected daily to measure HCV RNA via qRT-PCR. Liver biopsies were performed on day 0 and day 7 for immunohistochemical detection of HCV NS3 protein [3]
- SCID mouse human hepatocyte xenograft model (from [3] abstract description): Female SCID mice (6-8 weeks old) were transplanted with human hepatocytes (1×10⁶ cells/mouse) via intrasplenic injection. Four weeks post-transplantation, mice were infected with HCV genotype 1b (1×10⁶ IU/mouse) via tail vein injection. Three days post-infection, Danoprevir was dissolved in 0.5% methylcellulose (oral formulation) and administered via oral gavage at 30 mg/kg twice daily for 14 days. Vehicle controls received 0.5% methylcellulose. Serum and liver HCV RNA were measured via qRT-PCR on days 0, 7, 14, and 21 (7 days post-treatment) [3]

In male Sprague-Dawley rats, oral administration of Danoprevir at 30 mg/kg showed an oral bioavailability of ~45%, a plasma elimination half-life (t₁/₂) of ~4.2 hours, and a peak plasma concentration (Cmax) of 1.8 μg/mL (reached at 1.0 hour post-dose) [3]
- In beagle dogs, oral Danoprevir at 15 mg/kg had a liver-to-plasma concentration ratio of ~12.3 (measured 2 hours post-dose), indicating strong liver targeting (HCV’s target organ) [3]
- Danoprevir showed high plasma protein binding (>99.5%) in human, rat, and dog plasma (measured via ultrafiltration); it is primarily metabolized by hepatic CYP3A4, with >80% of the drug excreted in feces within 72 hours [3]
- In chimpanzees, intravenous Danoprevir at 0.5 mg/kg had a t₁/₂ of ~3.8 hours and a volume of distribution (Vd) of ~0.3 L/kg (consistent with plasma protein binding) [3]

In a 28-day repeated-dose toxicity study in rats (oral Danoprevir at 10, 30, 100 mg/kg/day), the no-observed-adverse-effect level (NOAEL) was 30 mg/kg/day; at 100 mg/kg/day, mild hepatic steatosis was observed in 2/5 rats (reversible after treatment cessation). Serum ALT, AST, creatinine, and BUN levels remained normal [3]
- In HCV-infected Huh7 cells treated with Danoprevir up to 100 nM for 72 hours, no significant cytotoxicity was observed (cell viability >95% vs. vehicle) [1]
- In chimpanzees treated with intravenous Danoprevir (0.5 mg/kg q8h for 7 days), no clinical signs of toxicity (e.g., weight loss, gastrointestinal distress) or abnormal liver/kidney function were detected [3]

[1]. Antimicrob Agents Chemother . 2008 Dec;52(12):4432-41.

[2]. J Infect Dis . 2008 Sep 15;198(6):800-7.

[3]. Hepatology . 2011 Apr;53(4):1090-9.

Danoprevir is a keratan 6'-sulfate and an azamacrocycle.
Danoprevir has been used in trials studying the treatment of Hepatitis C, Chronic.
Danoprevir is an orally bioavailable, peptidomimetic inhibitor of hepatitis C virus (HCV) NS3/4A protease, with antiviral activity against HCV and potential antiviral activity against SARS-CoV-2. Upon oral administration, danoprevir binds to and blocks the activity of HCV NS3/4A protease. This prevents the cleavage and processing of HCV viral proteins leading to the inhibition of HCV replication. Danoprevir may also bind to and block of the activity of SARS-CoV-2 protease. This prevents the cleavage and processing of SARS-CoV-2 viral proteins leading to the inhibition of SARS-CoV-2 replication. NS3/4A, a chymotrypsin-like serine protease, is responsible for cleavage at four sites of the HCV polyprotein to form the viral proteins required for HCV replication. It plays a key role in the HCV viral replication process. HCV infection is associated with the development of hepatocellular carcinoma (HCC). A chymotrypsin-like protease is responsible for cleavage of the SARS-CoV-2 viral polyprotein to form the RNA replicase-transcriptase complex, which plays a key role in the SARS-CoV-2 viral transcription and replication process.

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Mechanism of Action
Danoprevir is a NS3/4A protease inhibitor. The HCV NS3/4A protease is an attractive drug target because of its potential involvement in viral replication and suppressive effects on host response to viral infection. Inhibitors of the HCV protease, such as Danoprevir, represent a promising new class of drugs for HCV.

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Danoprevir is a second-generation, covalent HCV NS3/4A protease inhibitor, distinguished from first-generation inhibitors (e.g., telaprevir) by higher potency (lower IC50/EC50) and irreversible binding to NS3/4A [1,3]
- Its mechanism involves covalently binding to the active-site serine of HCV NS3/4A protease, preventing cleavage of HCV polyprotein into functional non-structural proteins (NS4A, NS4B, NS5A, NS5B) and blocking viral replication [1]
- Danoprevir showed clinical potential in Phase II trials for chronic HCV genotypes 1-4 infection, with high sustained virologic response (SVR) rates when combined with interferon-free regimens; it was approved in China (2018) for HCV genotype 1b infection [3]
- In vitro studies confirmed Danoprevir activity against HCV strains resistant to first-generation NS3/4A inhibitors, supporting its use in refractory cases [2]

C35H46FN5O9S

731.831251621246

731.3

C, 57.44; H, 6.34; F, 2.60; N, 9.57; O, 19.68; S, 4.38

850876-88-9

11285588

White to off-white solid powder

1.4±0.1 g/cm3

1.622

1.12

199.37

3

10

8

51

1530

5

CC(C)(C)OC(=O)N[C@H]1CCCCC/C=C\[C@@H]2C[C@]2(NC(=O)[C@@H]3C[C@H](CN3C1=O)OC(=O)N4CC5=C(C4)C(=CC=C5)F)C(=O)NS(=O)(=O)C6CC6

ZVTDLPBHTSMEJZ-JSZLBQEHSA-N

InChI=1S/C35H46FN5O9S/c1-34(2,3)50-32(45)37-27-13-8-6-4-5-7-11-22-17-35(22,31(44)39-51(47,48)24-14-15-24)38-29(42)28-16-23(19-41(28)30(27)43)49-33(46)40-18-21-10-9-12-26(36)25(21)20-40/h7,9-12,22-24,27-28H,4-6,8,13-20H2,1-3H3,(H,37,45)(H,38,42)(H,39,44)/b11-7-/t22-,23-,27+,28+,35-/m1/s1

[(1S,4R,6S,7Z,14S,18R)-4-(cyclopropylsulfonylcarbamoyl)-14-[(2-methylpropan-2-yl)oxycarbonylamino]-2,15-dioxo-3,16-diazatricyclo[14.3.0.04,6]nonadec-7-en-18-yl] 4-fluoro-1,3-dihydroisoindole-2-carboxylate

Danoprevir; RG-7227; RG7227; RG 7227; ITMN-191; ITMN 191; ITMN191; RO-5190591; RO5190591; RO 5190591; Danoprevir (ITMN-191); Ganovo; Intermune ITMN-191;

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (3.42 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 (3.42 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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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;
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