HCV/hepatitis C virus NS5A

体外活性：Ledipasvir（也称为 GS5885）是一种 HCV NS5A 聚合酶抑制剂，用于治疗丙型肝炎病毒感染。雷迪帕韦90mg/索磷布韦400mg的组合产品（商品名Harvoni）于2014年10月获得FDA批准。雷迪帕韦/索磷布韦组合是一种干扰HCV复制的直接作用抗病毒药物，可用于治疗基因型患者1a 或 1b，不含 PEG-干扰素或利巴韦林。Ledipasvir 在健康志愿者中的血浆半衰期延长了 37-45 小时，并且在每日一次口服剂量 3 mg 或更高剂量的单一疗法中，可快速减少 >3 log 病毒载量基因 1a HCV 感染患者。它已被证明是安全有效的，与具有互补机制的直接作用抗病毒药物联合使用时，SVR12 率高达 100%。 激酶检测：GT1a 复制子 EC50 = 31 pMCell 检测：Ledipasvir 是 HCV NS5A 蛋白的特异性抑制剂抑制HCV亚基因组复制子系统中的HCV复制。 NS5A 复制复合物抑制剂是用于 HCV 治疗的新型抗病毒因子。通常，与针对 NS3 解旋酶和 NS5B RNA 聚合酶的传统 HCV 复制抑制剂相比，这些抑制剂具有高效率和低病毒耐药性。 NS5A 抑制剂应该跨 NS5A 二聚体界面结合，靠近 N 端结构域 1。这种结合被认为会直接或变构地扭曲二聚体关联，这可能会破坏 HCV RNA 复制中的 NS5A 功能。当使用 JFH1/3a-NS5A 杂合复制子评估对 NS5A 的敏感性时，另一种抑制剂 DCV 被证明比雷迪帕韦更有效。此外，NS5A-A30K和-Y93H变体表现出对ledpasvir的敏感性降低（EC50值分别为1770 nM和4300 nM）。

在临床试验中，观察到 ledpasvir 具有良好的耐受性，并且 HCV RNA 的中位最大减少范围为 2.3 log10 IU/ml 至 3.3 log10 IU/ml。 Emax 模型还显示，3 天后给予 30 mg ledpasvir 导致 HCV RNA 减少对基因型 1a 的最大反应大于 95%。最后，还观察到与 1a 相比，HCV RNA 在基因型 1b 中更持久。

竞争性蛋白结合试验[1]
将含有10%胎牛血清（CCM）的人血浆和细胞培养基以2μM的终浓度掺入受试化合物。将加标血浆（1 mL）和CCM（1 mL）放入组装好的透析细胞的相对侧，用半透膜隔开。透析细胞在37°C水浴中缓慢旋转达到平衡所需的时间。测量透析后血浆和CCM重量，并用LC/MS/MS测定血浆和CCM中的试验化合物浓度。

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代谢稳定性[1]
使用合并的肝微粒体组分（最终蛋白质浓度为0.5mg/mL）在3μM的最终试验化合物浓度下测定体外代谢稳定性。通过加入NADPH再生系统引发反应。在不同时间点将25μL的反应混合物等分转移到含有淬火溶液的板上。反应混合物中的试验化合物浓度用LC/MS/MS测定。肝脏固有清除率如Obach之前所述计算，预测清除率使用搅拌良好的肝脏模型计算，不受蛋白质限制。
还使用氚化测试化合物在冷冻保存的肝细胞中测定了代谢稳定性。孵育混合物含有1×106个肝细胞/mL和1μM氚化试验化合物（2.5μCi）。在37°C的温度下，在95%空气/5%二氧化碳（v/v）的潮湿环境中轻轻摇晃进行孵化。在0、1、3和6小时后取出50μL的等分试样，并将其加入100μL的淬火溶液中。在与HPLC系统耦合的流动闪烁无线电探测器上分析样品。代谢物根据放射性检测器的峰面积进行定量，无细胞对照样品用作参考。通过测量测试化合物的消失率，即形成的放射性标记代谢物和测试化合物的总峰面积的百分比，来确定肝细胞中的代谢稳定性。

GT1a and GT1b Replicons[1]
稳定的基因型1a（GT1a）亚基因组复制子细胞系1a-57C-RlucP（H77菌株）用于测定化合物GT1a的抗病毒活性，并如前所述建立。在稳定的GT1b亚基因组复制子细胞系1b-Luc-2（Con-1菌株）中测定了化合物GT1b的抗病毒活性。为了建立1b-Luc-2，从ReBLikon获得的质粒I389luc-ubineo/NS3-3′/ET中产生了复制子质粒pCon1/SG-hRlucNeo（G+I+T），该质粒编码Con-1菌株的亚基因组复制子。使用Accuprime Super Mix I和引物AscI-hRluc-Fwd和NotI-hRluc Rev通过PCR从pF9 CMV hRluc-Neo Flexi中扩增出hRluc-Neo基因。这两个引物具有以下序列，并携带限制性位点以供后续克隆：AscI-hRuc-Fwd:5′-ACT GAC GGC GCG CCA TGG CTT CCA AGG TGT ACG-3′（AscI位点下划线）和NotI-hMluc Rev:5′-GTC AGT GCG GCT CAG AAG AAC TCG TCA AGA-3′（NotI位点划线）。将hRluc-Neo扩增产物亚克隆到pCR2.1-TOPO中。用AscI和NotI消化所得质粒，用T4 DNA连接酶将切下的片段（hRluc-Neo）连接到用相同酶消化的I389luc-ubi-Neo/NS3-3′/ET中。对所得载体pCon1/SG-hRlucNeo（G+I+T）进行测序，以确认hRluc-Neo融合基因的正确方向和序列。
质粒pCon1/SG-hRlucNeo（G+I+T）用SpeI线性化，并使用PCR纯化试剂盒纯化。按照制造商建议的方案，用T7MEGAScript试剂体外合成复制子RNA。根据制造商的说明，使用RNeasy试剂盒通过柱纯化纯化RNA。通过测量260nm处的吸光度来确定RNA浓度，并通过0.8%琼脂糖凝胶电泳和溴化乙锭染色来验证其完整性。如前所述，将10微克体外转录的pCon1/SG-hRlucNeo（G+I+T）RNA电穿孔到4×106 Huh7-Lunet细胞中。简而言之，将电穿孔细胞铺在100mm细胞培养皿上。镀覆24小时后，用补充了1.0 mg/mL G418的繁殖培养基替换培养基（选择持续约3周）。分离并扩增G418抗性克隆。根据制造商的说明，使用商业化的Renilla萤光素酶测定法对HCV复制进行定量。选择具有最高荧光素酶信号与背景比的克隆在高通量抗病毒敏感性试验中进行验证。用于GT1b抗病毒研究的最终克隆细胞系被命名为1b-Luc-2。

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Replicon抗病毒检测[1]
为了确定化合物GT1的抗病毒活性，将1a-57C-RlucP或1b-Luc-2复制子细胞以每孔2000个细胞的速度铺在384孔板上（细胞培养处理）。将化合物在DMSO中连续稀释3倍，并使用自动仪器以0.44%DMSO的终浓度加入细胞中，总体积为90μL。对于每种药物浓度，在384孔板上设置四孔。DMSO用作阴性（溶剂；无抑制）对照，三种HCV抑制剂的组合，包括蛋白酶抑制剂、NS5A抑制剂和核苷抑制剂，以>100×EC50的浓度用作阳性对照（100%抑制）。将板在37°C、5%CO2和85%湿度的环境中孵育3天。用Biotek ELX405洗板机吸出培养基。使用BiotekμFlow Workstation将20微升双Glo萤光素酶缓冲液添加到平板的每个孔中。将平板在室温下孵育10分钟。使用BiotekμFlow Workstation向每个孔中加入20微升含有双Glo Stop&Glo底物和双Glo Stop&Glo缓冲液的1:100混合物的溶液。将平板在室温下孵育10分钟，然后用Envision平板读数器测量发光信号。

PK studies in Rats, Dogs and Monkeys; Ledipasvir is remarkable not only on the basis of its high replicon potency but also on the basis of its low clearance, good bioavailability, and long half-lives in rat, dog, and monkey and low predicted clearance in human. The pharmacokinetics of Ledipasvir is measured in rats and dogs. Ledipasvir shows good half-lives (rat 1.83 ± 0.22 hr, dog 2.63 ± 0.18 hr) in plasma, low systemic clearance (CL), and moderate volumes of distribution (Vss) that are greater than total body water volume; Pharmacokinetic studies are performed in male naı̈ve Sprague-Dawley(SD) rats, non-naive beagle dogs, and cynomolgus monkeys (three animals per dosing route). Intravenous (IV) administration is dosed via infusion over 30 min in a vehicle containing 5% ethanol, 20% PEG400, and 75% water (pH adjusted to 3.0 with HCl). Oral dosing is administered by gavage in a vehicle containing 5% ethanol, 45% PEG 400, and 50% of 50 mM citrate buffer, pH 3. Blood samples are collected over a 24 h period postdose into Vacutainer tubes containing EDTA-K2. Plasma was isolated, and the concentration of the test compound in plasma was determined with LC/MS/MS after protein precipitation with acetonitrile. [1]

Absorption, Distribution and Excretion
Absorption
When given orally, ledipasvir reaches its maximum plasma concentration in about 4 to 4.5 hours with a maximum concentration (Cmax) of 323 ng/mL.

Route of Elimination
Following a single 90 mg oral dose of [14C]-ledipasvir, mean total recovery of the [14C]-radioactivity in feces and urine was approximately 87%, with most of the radioactive dose recovered from feces (approximately 86%). Unchanged ledipasvir excreted in feces accounted for a mean of 70% of the administered dose and the oxidative metabolite M19 accounted for 2.2% of the dose. These data indicate that biliary excretion of unchanged ledipasvir is a major route of elimination, with renal excretion being a minor pathway (approximately 1%).

Metabolism / Metabolites
In vitro, no detectable metabolism of ledipasvir was observed by human CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. Evidence of slow oxidative metabolism via an unknown mechanism has been observed. Following a single dose of 90 mg [14C]-ledipasvir, systemic exposure was almost exclusively to the parent drug (>98%). Unchanged ledipasvir is the major species present in feces.

Biological Half-Life
The median terminal half-life of ledipasvir is 47 hours.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Ledipasvir has not been studied in nursing mothers being treated for hepatitis C infection. Because it is 99.8% bound to maternal plasma proteins, amounts in breastmilk are likely to be very low. If ledipasvir alone or in combination with sofosbuvir (Harvoni) is required by the mother, it is not a reason to discontinue breastfeeding. Some sources recommend against breastfeeding when ledipasvir 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.
Drugs and Lactation Database (LactMed)

Protein Binding
Ledipasvir is >99.8% bound to human plasma proteins.

[1]Discovery of ledipasvir (GS-5885): a potent, once-daily oral NS5A inhibitor for the treatment of hepatitis C virus infection. J Med Chem. 2014 Mar 13;57(5):2033-46.

A new class of highly potent NS5A inhibitors with an unsymmetric benzimidazole-difluorofluorene-imidazole core and distal [2.2.1]azabicyclic ring system was discovered. Optimization of antiviral potency and pharmacokinetics led to the identification of 39 (ledipasvir, GS-5885). Compound 39 (GT1a replicon EC50 = 31 pM) has an extended plasma half-life of 37-45 h in healthy volunteers and produces a rapid >3 log viral load reduction in monotherapy at oral doses of 3 mg or greater with once-daily dosing in genotype 1a HCV-infected patients. 39 has been shown to be safe and efficacious, with SVR12 rates up to 100% when used in combination with direct-acting antivirals having complementary mechanisms.[1]

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Ledipasvir is a benzimidazole derivative that is used in combination with sofosbuvir (under the trade name Harvoni) for the treatment of chronic hepatitis C genotype 1 infection. It has a role as an antiviral drug and a hepatitis C protease inhibitor. It is a carbamate ester, a L-valine derivative, a bridged compound, a carboxamide, a benzimidazole, a member of fluorenes, an organofluorine compound, a member of imidazoles, a N-acylpyrrolidine and an azaspiro compound.

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Ledipasvir is a direct acting antiviral (DAA) 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 ledipasvir. More specifically, ledipasvir is an inhibitor of the Hepatitis C Virus (HCV) Non-Structural Protein 5A (NS5A), which is required for viral RNA replication and assembly of HCV virions. Although its exact mechanism of action is unknown, it is postulated to prevent hyperphosphorylation of NS5A which is required for viral protein production. It is effective against genotypes 1a, 1b, 4a, and 5a and with a lesser activity against genotypes 2a and 3a of HCV. Ledipasvir and other direct acting antivirals are very potent options for the treatment of Hepatitis C, as they exhibit a high barrier to the development of resistance. This is an important advantage relative to HCV drugs that target other viral enzymes such as the protease, for which rapid development of resistance has proven to be an important cause of therapeutic failure. 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 ledipasvir as a first line therapy option in combination with [sofosbuvir] for the treatment of HCV genotypes 1a, 1b, 4, 5, and 6. Treatment with ledipasvir is used 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. Treatment with direct acting antivirals such as ledipasvir is associated with very minimal side effects, with the most common being headache and fatigue. Lack of significant side effects and short duration of therapy is a considerable advantage over older interferon- and ribavirin-based regimens, which were limited by infusion site reactions, reduced blood count, and neuropsychiatric effects. Since 2014, ledipasvir has been available as a fixed dose combination product with [sofosbuvir] (tradename Harvoni) used for the treatment of chronic Hepatitis C. Approved in October 2014 by the FDA, Harvoni is indicated for the treatment of HCV genotypes 1, 4, 5, and 6 with or without [ribavirin] depending on the level of liver damage or cirrhosis. When combined together, ledipasvir and sofosbuvir as the combination product Harvoni has been shown to achieve a SVR between 93 and 99% after 12 weeks of treatment. Its use has also proven successful in the treatment of HCV in patients co-infected with HIV.

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Ledipasvir is a Hepatitis C Virus NS5A Inhibitor. The mechanism of action of ledipasvir is as a P-Glycoprotein Inhibitor, and Breast Cancer Resistance Protein Inhibitor.
Ledipasvir is an orally available inhibitor of the hepatitis C virus (HCV) non-structural protein 5A (NS5A) replication complex, with potential activity against HCV. Upon oral administration and after intracellular uptake, ledipasvir binds to and blocks the activity of the NS5A protein. This results in the disruption of the viral RNA replication complex, blockage of HCV RNA production, and inhibition of viral replication. NS5A, a zinc-binding and proline-rich hydrophilic phosphoprotein, plays a crucial role in HCV RNA replication. HCV is a small, enveloped, single-stranded RNA virus belonging to the Flaviviridae family; HCV infection is associated with the development of hepatocellular carcinoma (HCC).

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Drug Indication
When used in combination with the antiviral medication [sofosbuvir], ledipasvir is indicated for the treatment of treatment of chronic hepatitis C virus (HCV) in adults and pediatric patients 3 years of age and older with the following conditions: - Genotype 1, 4, 5, or 6 infection without cirrhosis or with compensated cirrhosis. - Genotype 1 infection with decompensated cirrhosis, in combination with [ribavirin]. - Genotype 1 or 4 infection who are liver transplant recipients without cirrhosis or with compensated cirrhosis, in combination with [ribavirin]. Its use has also proven successful in the treatment of HCV in patients co-infected with HIV.

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Pharmacodynamics
Ledipasvir acts against HCV and is categorized as a direct-acting antiviral agent (DAA). At a dose of 120 mg twice daily (2.67 times the maximum recommended dosage), ledipasvir does not prolong QTc interval to any clinically relevant extent.

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Mechanism of Action
Ledipasvir is an inhibitor of the Hepatitis C Virus (HCV) NS5A protein required for viral RNA replication and assembly of HCV virions. Although its exact mechanism of action is unknown, it is postulated to prevent hyperphosphorylation of NS5A which is required for viral production.

C52H60F2N8O7

947.08

946.455

C, 65.95; H, 6.39; F, 4.01; N, 11.83; O, 11.82

1441674-54-9

Ledipasvir;1256388-51-8;Ledipasvir D-tartrate;1502654-87-6;Ledipasvir-d6;2050041-12-6;Ledipasvir hydrochloride;2128695-48-5;Ledipasvir (diacetone);1502655-48-2

78357793

Off-white to yellow solid powder

9.86

191.71

4

11

12

69

1850

6

O=C(OC)N[C@H](C(N([C@H](C1=NC=C(C2=CC(C(F)(F)C3=C4C=CC(C5=CC=C6N=C([C@H]7N(C([C@@H](NC(OC)=O)C(C)C)=O)[C@]8([H])CC[C@@]7([H])C8)NC6=C5)=C3)=C4C=C2)N1)C9)CC%109CC%10)=O)C(C)C.CC(C)=O

IEYHPNJBXNWRRN-ABBTUPPKSA-N

InChI=1S/C49H54F2N8O6.C3H6O/c1-24(2)39(56-46(62)64-5)44(60)58-23-48(15-16-48)21-38(58)42-52-22-37(55-42)28-9-13-32-31-12-8-26(18-33(31)49(50,51)34(32)19-28)27-10-14-35-36(20-27)54-43(53-35)41-29-7-11-30(17-29)59(41)45(61)40(25(3)4)57-47(63)65-6;1-3(2)4/h8-10,12-14,18-20,22,24-25,29-30,38-41,54-55H,7,11,15-17,21,23H2,1-6H3,(H,56,62)(H,57,63);1-2H3/b27-26+,37-28+;/t29-,30+,38-,39-,40-,41-;/m0./s1

methyl ((S)-1-((S)-6-(5-(9,9-difluoro-7-(2-((1R,3S,4S)-2-((methoxycarbonyl)-L-valyl)-2-azabicyclo[2.2.1]heptan-3-yl)-1H-benzo[d]imidazol-6-yl)-9H-fluoren-2-yl)-1H-imidazol-2-yl)-5-azaspiro[2.4]heptan-5-yl)-3-methyl-1-oxobutan-2-yl)carbamate compound with propan-2-one (1:1)

GS-5885 acetone; Ledipasvir acetone; Ledipasvir (acetone); GS-5885 acetone; 3J78ET35HX; UNII-3J78ET35HX; Ledipasvir acetonate (JAN); Carbamic acid, N-((1S)-1-(((6S)-6-(5-(9,9-difluoro-7-(2-((1R,3S,4S)-2-((2S)-2-((methoxycarbonyl)amino)-3-methyl-1-oxobutyl)-2-azabicyclo(2.2.1)hept-3-yl)-1H-benzimidazol-6-yl)-9H-fluoren-2-yl)-1H-imidazol-2-yl)-5-azaspiro(2.4)hept-5-yl)carbonyl)-2-me; GS5885 acetone; GS 5885; trade name: Harvoni;

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

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

DMSO : ~100 mg/mL (~105.59 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (2.64 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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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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));
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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；
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