HCV NS5A (EC50 = 9 pM-50 pM)

体外活性：BMS-790052 是迄今为止报道的最有效的 HCV 复制抑制剂之一。对于 HCV 基因型 1a 和 1b 复制子，BMS-790052 的平均 EC50 值分别为 50 和 9 pM。 BMS-790052 显示出至少 105 的治疗指数 (CC50/EC50)，并且对一组 10 种 RNA 和 DNA 病毒无活性，EC50 高于 10 μM。这证实了 BMS-790052 对 HCV 的特异性。在含有 HCV 基因型 1b 复制子的 Huh7 细胞中，BMS-790052 可阻断短暂和稳定的 HCV 基因组复制，EC50 值范围为 1-15 pM。 BMS-790052（100 pM 或 1 nM）已被证明可以改变 NS5A 的亚细胞定位和生化分级。 BMS-790052 抑制含有 HCV 基因型 4 NS5A 基因的杂交复制子，EC50 为 7-13 pM。 NS5A 的残基 30 是杂种复制子中 BMS-790052 介导的抗性的重要位点。激酶测定：Daclatasvir 是一种有效的 HCV NS5A 蛋白抑制剂，针对基因型 1a 和 1b 复制子的平均 EC50 值分别为 50 和 9 pM。细胞测定：通过使用具有复制能力的 1a 或 1b 复制子构建杂交体来评估达卡他韦对基因型的抗病毒活性，其中整个 NS5A 编码区或来自不同基因型的 NS5A 的前 100 个氨基酸替换亲本复制子的相应序列。据报道，达卡他韦对所有 HCV 基因型均高度有效，半数有效浓度 (EC50) 范围为 9 至 146 pM

人源化肝脏嵌合小鼠的肝脏嵌合率估计超过 40%，静脉注射 100 µL HCV 阳性人血清样本。接种后，每 1-4 周从颈外静脉采集一次血液。 HCV RNA 水平通过 COBAS TaqMan HCV 测试在 100 倍稀释的血清中测量，测量范围较低为 3.2 log IU/mL 血清。 HCV RNA 血清水平达到平台水平后，给小鼠口服以下药物之一，每天一次，持续 4 周：40 mg/kg Asunaprevir 加 30 mg/kg Daclatasvir，15 mg/kg Ledipasvir 加 50 mg/kg。 kg GS-558093 和 50 mg/kg GS-558093 加 400 mg/kg Telaprevir。

肽 (Ac-Asp-Glu-Asp [EDANS]-Glu-Glu-Abu-[COO] Ala-Ser-Lys [DABCYL]-NH2) 含有荧光供体 {EDANS, 5-[(2-氨乙基)氨基]萘-1-磺酸}靠近肽的一端，受体{DABCYL，4-[(4-二甲基氨基)苯基]偶氮)苯甲酸}靠近另一端。供体和受体之间的分子间共振能量转移淬灭了肽的荧光，但当 NS3 蛋白酶切割肽时，产物从共振能量转移淬灭中释放出来。随着更多底物被 NS3 蛋白酶裂解，供体的荧光随着时间的推移而增强。检测试剂由 20 μM FRET 肽、150 mM NaCl 和 5× 荧光素酶细胞培养裂解液（用 dH₂O 稀释至 1×）组成。在 96 孔板中，添加 HCV-Huh-7 细胞并贴壁过夜（每孔 1×10⁴ 细胞）。第二天将 BMS-790052 添加到孔中后，将板孵育 72 小时。之后，用 PBS 清洗板，并通过向每个孔中添加 30 μL 前面提到的 FRET 肽测定试剂来准备 FRET 测试。通过使用 Cytoflor 4000 仪器在动力学模式下读取板来获取信号，该仪器被编程为在 340 nm（激发）/490 nm（发射）下以自动模式运行 20 个循环或更少。 FRET 完成后，每孔加入 40 μL 荧光素酶底物，并评估荧光素酶的量。

约 12 小时前，将 BMS-790052 添加到 96 孔板中，该板已在 200 µL 培养基中接种了 HCV 复制子细胞。孵育 72 小时后，检查细胞板的细胞毒性和复制活性。使用 CellTiter-Blue 测量细胞毒性后，除去培养基和染料，将板倒置，并用纸巾吸干剩余的液体。海肾荧光素酶用于测量 HCV 基因型 1a 细胞系的复制活性。向每个孔中添加 1× 海肾荧光素酶裂解缓冲液 (30 µL) 后，将板孵育 15 分钟，并轻轻摇动。然后使用 Top Count 光度计检测信号，该光度计配置为在添加 40 µL 海肾荧光素酶底物后进行光发射定量。仅使用 DMSO 的孔计算每个细胞系的 100% 活性；将含有化合物的孔的平均值除以含有DMSO的孔的平均值，以确定每个抑制剂浓度的活性百分比。

At the termination of experiments, all mice were euthanized by CO2 inhalation. To evaluate in vivo efficacy of antiviral agents on HCV, NOD/SCID mice bearing HCV-replicating Huh7 xenografts were used21. Briefly, HCV RNA-transfected cells mixed with Matrigel were injected into the large lobes of the livers of anesthetized immunodeficient NOD/SCID male mice (5 weeks of age, 18–20 g body weight). Four weeks after implantation, compounds dissolved in saline were orally administered to mice using a feeding needle. Serum HCV titer was monitored by RT-qPCR.[Sci Rep. 2018 Aug 20;8(1):12469]

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30 mg/kg; oral

Mice

Absorption, Distribution and Excretion
Studies demonstrated that peak plasma concentrations typically occurred within 2 hours after administration of multiple oral doses ranging from 1 - 100 mg once daily. Steady state is reached after approximately 4 days of once-daily daclatasvir administration. The absolute bioavailability of the tablet formulation is 67%.
Approximately 88% of total dose of daclatasvir is eliminated into bile and feces in which 53% remains as unchanged form, while 6.6% of the total dose is eliminated primarily unchanged in the urine.
The approximate volume of distribution of daclatasvir is 47 L in patients who was orally administered 60 mg tablet followed by 100 µg [13C,15N]-daclatasvir intravenously.
In subjects who received daclatasvir 60 mg tablet orally followed by 100 µg radiolabeled daclatasvir intravenously, the total clearance was 4.2 L/h.

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Metabolism / Metabolites
Daclastavir is a substrate of CYP3A enzymes where its metabolism is predominantly mediated by CYP3A4 isoform. Oxidative pathways included δ-oxidation of the pyrrolidine moiety, resulting in ring opening to an aminoaldehyde intermediate followed by an intramolecular reaction between the aldehyde and the proximal imidazole nitrogen atom. High proportion of the drug in the plasma (greater than 97%) is in the unchanged form.

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Biological Half-Life
Following multiple dose administration of daclatasvir in HCV-infected subjects, with doses ranging from 1 mg to 100 mg once daily, the terminal elimination half-life of daclatasvir ranged from approximately 12 to 15 hours.

Hepatotoxicity
In large randomized controlled trials, daclatasvir was not associated with serum enzyme elevations during therapy. A difficulty in assessing side effects of daclatasvir and other anti-HCV agents, however, was that they are never used as monotherapy, but were also combined with agents active against other HCV targets, such as the viral protease (NS3) or polymerase (NS5B). Daclatasvir was also commonly used in combination with the more traditional agents used for hepatitis C, such as peginterferon and ribavirin, both of which have prominent adverse effects. In combination with asunaprevir (an HCV protease inhibitor), daclatasvir was associated with serum ALT elevations in 3% to 11% of patients and with several instances of acute hypersensitivity and hepatitis, some of which were severe. The cause of the hypersensitivity reaction, however, appeared to be asunaprevir. In combination with sofosbuvir, daclatasvir was not associated with serum enzyme elevations or with clinically apparent liver injury.
Daclatasvir has, however, been implicated in rare instances of acute decompensation of HCV related cirrhosis. The role of daclatasvir and the other HCV antivirals in this syndrome, however, was unclear. The liver injury usually arose within 2 to 6 weeks of starting therapy, but occasionally later and even after discontinuation of therapy. The injury was marked by worsening jaundice and signs of hepatic failure. In some instances, lactic acidosis was present early. In most but not all instances, the serum enzymes increased minimally if at all, despite the worsening hepatic failure. Several instances resulted in death or need for emergency liver transplantation. For this reason, it was recommended that patients with cirrhosis undergoing antiviral therapy with potent direct acting agents should be monitored carefully, particularly during the first few weeks of treatment.
Finally, reactivation of hepatitis B has occurred in rare patients being treated for chronic hepatitis C some of whom had received daclatasvir. The relationship of HBV reactivation to the antiviral treatment of HCV infection is not clear, but it may be due to clearance of HCV replication which allows HBV DNA levels to increase.
Likelihood score: C (probable rare cause of clinically apparent liver injury in patients with cirrhosis or preexisting hepatitis B virus coinfection).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Daclatasvir has been removed from the US market. It has not been studied in nursing mothers being treated for hepatitis C infection. Because it is 99% bound to maternal plasma proteins, amounts in breastmilk are likely to be very low. If daclatasvir used alone or in combination with sofosbuvir is required by the mother, it is not a reason to discontinue breastfeeding. Some sources recommend against breastfeeding when daclatasvir 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
Daclatasvir is highly protein bound (99%).

[1]. Nature . 2010 May 6;465(7294):96-100.

[2]. Virology . 2011 May 25;414(1):10-8.

[3]. Antimicrob Agents Chemother . 2012 Mar;56(3):1588-90.

[4]. Antimicrob Agents Chemother . 2005 Apr;49(4):1346-53.

Pharmacodynamics
Daclatasvir is a direct-acting antiviral agent that targets the NS5A and causes a decrease in serum HCV RNA levels. It disrupts HCV replication by specifically inhibiting the critical functions of an NS5A protein in the replication complex. It is shown to cause downregulation of the hyperphosphorylation of NS5A. It does not appear to prolong the QT interval even when given at 3 times the maximum recommended dose.

C40H50N8O6

738.890

738.385

C, 65.02; H, 6.82; N, 15.17; O, 12.99

1009119-64-5

Daclatasvir dihydrochloride;1009119-65-6;Daclatasvir-d6;1801709-41-0;Daclatasvir-d16

25154714

Light yellow to yellow solid powder

1.3±0.1 g/cm3

1071.2±65.0 °C at 760 mmHg

601.7±34.3 °C

0.0±0.3 mmHg at 25°C

1.595

5.44

174.64

4

8

13

54

1190

4

O=C([C@@H](NC(OC)=O)C(C)C)N1CCC[C@H]1C2=NC=C(N2)C3=CC=C(C=C3)C4=CC=C(C5=CN=C([C@@H]6CCCN6C([C@@H](NC(OC)=O)C(C)C)=O)N5)C=C4

FKRSSPOQAMALKA-CUPIEXAXSA-N

InChI=1S/C40H50N8O6/c1-23(2)33(45-39(51)53-5)37(49)47-19-7-9-31(47)35-41-21-29(43-35)27-15-11-25(12-16-27)26-13-17-28(18-14-26)30-22-42-36(44-30)32-10-8-20-48(32)38(50)34(24(3)4)46-40(52)54-6/h11-18,21-24,31-34H,7-10,19-20H2,1-6H3,(H,41,43)(H,42,44)(H,45,51)(H,46,52)/t31-,32-,33-,34-/m0/s1

methyl N-[(2S)-1-[(2S)-2-[5-[4-[4-[2-[(2S)-1-[(2S)-2-(methoxycarbonylamino)-3-methylbutanoyl]pyrrolidin-2-yl]-1H-imidazol-5-yl]phenyl]phenyl]-1H-imidazol-2-yl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]carbamate dihydrochloride

BMS-790052; Daclatasvir; BMS790052; BMS 790052; EBP883; EBP 883; EBP-883; BMS 790052; Daclatasvir [USAN];Daklinza (trade name)

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 (3.38 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 (3.38 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (3.38 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;
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。