Ombitasvir (ABT267; Viekira Pak)   中文名称: 奥米他韦

HCV (IC50 = 0.82 to 19.3 pM); HCV (IC50 = 366 pM)

体外活性：Ombitasvir（以前称为 ABT-267）是一种基于 N-苯基吡咯烷的丙型肝炎病毒（HCV）NS5A 抑制剂，具有出色的效力、代谢稳定性和药代动力学。奥比他韦被发现是一种泛基因型 HCV 抑制剂，针对 GT1a、-1b、-2a、-2b、-3a、-4a 和 -5a 的 EC50 范围为 1.7-19.3 pM，针对 GT6a 的 EC50 范围为 366 pM。在首次接受治疗的 HCV GT1 感染受试者中，在 3 天的单一治疗期间，它可将 HCV RNA 降低高达 3.10 log10 IU/mL。 Ombitasvir 已被美国食品药品监督管理局 (FDA) 批准与其他抗病毒药物（帕立瑞韦、利托那韦和达沙布韦）联合使用，商品名为 Viekira Pak。激酶测定：之前描述了在HCV复制子细胞培养测定中用于测量单个氨基酸变体对抑制剂活性的影响的方法。简言之，使用 Change-IT 多突变定点诱变试剂盒（Affymetrix，圣克拉拉）将 NS5A 中的抗性相关变体各自引入基因型 1a-H77 或 1b-Con1 或嵌合基因型 2 至 6 复制子之一中。 ，加利福尼亚州）。通过序列分析确认变体的存在后，将质粒线性化，并使用 TranscriptAid T7 高产转录试剂盒（Fermentas，Glen Burnie，MD）从质粒转录 HCV 亚基因组 RNA。在瞬时测定中，含有变体的复制子 RNA 通过电穿孔转染至 Huh-7 细胞系中。 EC50 的计算方法如前几节所述。细胞测定：构建了带有荧光素酶报告基因但不含 Neo 基因的基于基因型 1b-Con1 HCV 复制子的穿梭载体盒，用于评估源自感染 HCV 基因型 1 至 6 的个体的 NS5A 基因的表型。克隆了 NotI 限制位点NS4B 3'端NS5A上游90个核苷酸处插入1b-Con1亚基因组复制子载体，并在NS5A氨基酸413密码子后克隆ClaI位点。来自基因 1 型感染患者的 NS5A 区域插入到 NotI 和 ClaI 限制性位点之间。具有 NotI 和 BlpI 限制性位点（上一节中描述）的 1b-Con1 穿梭载体用于评估 ombitasvir 抑制包含非基因型 1 HCV 氨基酸 1 至 214 的 NS5A 区域的能力。临床样本中的 NS5A 基因被扩增并连接到穿梭载体中。在瞬时测定中，来自每个临床样本的含有 NS5A 基因的 HCV 亚基因组复制子 RNA 通过电穿孔转染至 Huh-7 衍生细胞系中。将细胞在ombitasvir存在下孵育4天。测量细胞中的荧光素酶活性，并如上所述计算EC50。

在一项为期3天的单一疗法研究中，对12名HCV基因型1感染患者进行了Ombitasvir的体内评估，每天给药5、25、50或200mg。研究中的所有患者都感染了HCV基因型1a，并且通过克隆测序确定在基线时没有预先存在的耐药变异。观察到HCV RNA减少高达3.1 log10IU/ml。在首次给药后48小时，在患者样本中检测到NS5A第28、30或93位的耐药性相关变异。克隆测序分析表明，在3天的单药治疗期间，ombitasvir在很大程度上抑制了野生型病毒，在高于5mg的剂量下，耐药变体M28V也受到了抑制。Ombitasvir在所有剂量下都具有良好的耐受性，没有发生严重或严重的不良事件。这些数据支持ombitasvir与靶向HCV NS3/4A蛋白酶（利托那韦的ABT-450）和HCV NS5B聚合酶（ABT-333，达沙布韦）的抑制剂联合治疗慢性HCV基因型1感染的临床开发。（研究M12-116在ClinicalTrials.gov上注册，注册号为NCT01181427）[1]。

先前描述的技术用于量化不同氨基酸变体如何影响 HCV 复制子细胞培养测定中的抑制剂活性。简而言之，使用 Change-IT 多重突变定点诱变试剂盒（Affymetrix，圣克拉拉，CA），将 NS5A 中的抗性相关变体分别引入基因型 1a-H77 或 1b-Con1 或嵌合体之一基因型 2 至 6 个复制子。序列分析验证变体的存在后，将质粒线性化，并使用 TranscriptAid T7 高产转录试剂盒（Fermentas，Glen Burnie，MD）提取 HCV 亚基因组 RNA。通过电穿孔，在临时实验中用含有变体的复制子 RNA 转染 Huh-7 细胞系。如前面部分所述，计算了 EC50。

创建了一个基于基因型 1b-Con1 HCV 复制子的穿梭载体盒，该载体缺乏 Neo 基因，但含有荧光素酶报告基因，用于评估感染 HCV 基因型 1 至 6 的患者的 NS5A 基因表型。 NS5A 上游 3' 端有 90 个核苷酸NS4B（NotI 限制性位点）被克隆到 1b-Con1 亚基因组复制子载体中，ClaI 位点被克隆到 NS5A 氨基酸 413 密码子之后。 NotI 和 ClaI 限制性位点与基因型 1 感染患者的 NS5A 区域交叉。使用具有 NotI 和 BlpI 限制性位点的 1b-Con1 穿梭载体（在上一节中讨论）评估了奥比他韦抑制非基因型 1 HCV NS5A 区域（包含氨基酸 1 至 214）的能力。使用临床样本将 NS5A 基因的扩增副本连接到穿梭载体中。一项瞬时测定涉及将每个临床样本的含有 NS5A 基因的 HCV 亚基因组复制子 RNA 电穿孔到源自 Huh-7 的细胞系中。奥比他韦在细胞培养的四天内一直存在。按照前面提到的程序，测定细胞中的荧光素酶活性。

Clinical study design. Study M12-116 (ClinicalTrials.gov registration no. NCT01181427) was the first study to evaluate the pharmacokinetics, safety, tolerability, antiviral activity, and resistance of ombitasvir in HCV-infected treatment-naive adults. All of the patients provided written informed consent. The study was performed in accordance with Good Clinical Practice guidelines and the principles of the Declaration of Helsinki, and the study protocol was approved by the relevant institutional review boards and regulatory agencies. Inclusion criteria included chronic HCV genotype 1 infection for at least 6 months prior to study enrollment, plasma HCV RNA level of >100,000 IU/ml at screening, and a liver biopsy within the past 3 years with histology consistent with HCV-related inflammation and fibrosis but no evidence of cirrhosis. Exclusion criteria included positive antibodies for hepatitis A or B virus or human immunodeficiency virus type 1 (HIV-1) or a history of clinically significant comorbidities. The primary endpoint was the maximum change from baseline in HCV RNA. The patients in the ombitasvir dose groups were enrolled sequentially, and within each group, patients were randomized (2:1) to either ombitasvir or placebo and treated under nonfasting conditions for 3 days while confined to the study site. The 200-mg dose group received a different formulation with higher bioavailability. Patients who received at least one dose of ombitasvir or placebo were provided the option to receive treatment with pegIFN/RBV for approximately 48 weeks once treatment with ombitasvir was completed. HCV RNA was measured using the Roche COBAS TaqMan HCV Test v2.0 real-time reverse transcriptase PCR assay (with a lower limit of quantification of 25 IU/ml and a lower limit of detection of 10 IU/ml). The virologic response was assessed as HCV RNA decrease from baseline in log10 IU/ml[1].

Absorption, Distribution and Excretion
Ombitasvir reaches peak plasma concentration 5 hours after administration. It has an absolute bioavailability of 48%. Taking ombitasvir with high or normal fat meals increases exposure by 1.76 or 1.82 fold respectively.
Ombitasvir is mainly excreted in the feces (90.2%) with very little excreted in the urine (1.91%). 87.8% and 0.03% of the dose excreted in the feces and urine respectively is present as the parent compound.
Ombitasvir has a volume of distribution at steady state of 173 liters.
Clearance of Ombitasvir has not been determined.

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Metabolism / Metabolites
Ombitasvir is mainly metabolized by amide hydrolysis followed by CYP2C8-mediated oxidative metabolism.

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Biological Half-Life
Ombitasvir has a half life of elimination of 21-25 hours

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Ombitasvir has not been studied in nursing mothers being treated for hepatitis C infection. Because it is 99.9% bound to maternal plasma proteins, amounts in breastmilk are likely to be very low. Some sources recommend against breastfeeding when ombitasvir is used with ribavirin.
Ritonavir used as a booster has been studied in several studies of breastfeeding mothers. It is excreted into milk in measurable concentrations and low levels can be found in the blood of some breastfed infants. No reports of adverse reactions in breastfed infants have been reported. For more information, refer to the LactMed record on ritonavir.
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
Ombitasvir is 99.9% bound to human plasma proteins.

[1]. In vitro and in vivo antiviral activity and resistance profile of ombitasvir, an inhibitor of hepatitis C virus NS5A. Antimicrob Agents Chemother. 2015 Feb;59(2):979-87.

[2]. Discovery of ABT-267, a pan-genotypic inhibitor of HCV NS5A. J Med Chem. 2014 Mar 13;57(5):2047-57.

Pharmacodynamics
Ombitasvir is classified as a direct acting antiviral and acts against HCV to inhibit viral replication.

C50H67N7O8

894.11

893.505

C, 67.17; H, 7.55; N, 10.97; O, 14.32

1258226-87-7

54767916

White to light yellow solid powder

1.2±0.1 g/cm3

1065.6±65.0 °C at 760 mmHg

598.2±34.3 °C

0.0±0.3 mmHg at 25°C

1.595

6.29

178.72

4

9

16

65

1540

6

CC(C)(C)C(C=C1)=CC=C1N2[C@H](C3=CC=C(NC([C@H]4N(C([C@@H](NC(OC)=O)C(C)C)=O)CCC4)=O)C=C3)CC[C@H]2C5=CC=C(NC([C@@H]6CCCN6C([C@@H](NC(OC)=O)C(C)C)=O)=O)C=C5

PIDFDZJZLOTZTM-KHVQSSSXSA-N

InChI=1S/C50H67N7O8/c1-30(2)42(53-48(62)64-8)46(60)55-28-10-12-40(55)44(58)51-35-20-14-32(15-21-35)38-26-27-39(57(38)37-24-18-34(19-25-37)50(5,6)7)33-16-22-36(23-17-33)52-45(59)41-13-11-29-56(41)47(61)43(31(3)4)54-49(63)65-9/h14-25,30-31,38-43H,10-13,26-29H2,1-9H3,(H,51,58)(H,52,59)(H,53,62)(H,54,63)/t38-,39-,40-,41-,42-,43-/m0/s1

methyl N-[(2S)-1-[(2S)-2-[[4-[(2S,5S)-1-(4-tert-butylphenyl)-5-[4-[[(2S)-1-[(2S)-2-(methoxycarbonylamino)-3-methylbutanoyl]pyrrolidine-2-carbonyl]amino]phenyl]pyrrolidin-2-yl]phenyl]carbamoyl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]carbamate

Viekira Pak (trade name); ABT-267; Ombitasvir(ABT-267); ABT267; CHEBI:85183; 2302768XJ8; ABT267; ABT 267

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 (2.80 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 (2.80 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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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。