HIV-1/2 nucleotide reverse transcriptase

替诺福韦艾拉酚胺 (GS-7340) 在所有细胞类型中的抗病毒活性范围为 5 至 7 nM，但 MT-4 和 MT-2 细胞的 CC50 范围为 4.7 至 42 μM。一组 HIV-1 和 HIV-2 分离株（包括 HIV-1 M 组亚型 A 至 G 以及 N 组和 O 组分离株）用于评估 TAF 的抗病毒活性。 TAF EC50 平均值为 3.5 nM，范围为 0.1 至 12 nM。相比之下，对于在 PBMC 中评估的 29 种主要 HIV-1 毒株，AZT（内部对照）的平均 EC50 为 11.8 nM。 TAF 对 HIV-2 分离株的平均 EC50 为 1.8 nM，而 AZT 的平均 EC50 为 6.4 nM [2]。

与富马酸替诺福韦二吡呋酯 (TDF) 相比，替诺福韦的酰胺化前药替诺福韦艾拉酚胺 (GS-7340) 半富马酸盐具有更好的口服生物利用度和血浆稳定性 [1]。

肠和肝S9稳定性[1]
将GS-7340与狗或人的肠道和肝脏S9级分在10μM下在37°C下以96孔板型孵育120分钟。在添加化合物后的指定时间点，用9体积的含有25%乙腈和50%甲醇的水溶液淬灭样品。将平板在3000g下离心30分钟，并通过LC–MS/MS分析10μL所得溶液。数据（分析物与内标峰面积之比）以半对数标度绘制，并使用指数拟合进行拟合。假设一阶动力学，确定半衰期和代谢速率。使用充分搅拌的肝脏清除模型，通过已报道的方法从半衰期计算预测的肝脏提取。

Caco-2渗透率[1]
如先前报道的，使用接种在12孔板中的人结肠癌细胞系caco-2的融合单层进行双向渗透性研究。研究了浓度（10、100或1000μM）或外排转运体抑制对GS-7340渗透的影响。在细胞单层与10μM环孢菌素a（CsA）在转运缓冲液中预孵育30分钟以使转运蛋白结合位点饱和后，评估包括P-糖蛋白（Pgp）在内的外排转运蛋白的抑制作用。预培养后，加入含有CsA和GS-7340的新鲜测定缓冲液，并开始测定。每种测定重复进行，并测定对照化合物（阿替洛尔、普萘洛尔和地高辛）的渗透性，以满足每批测定板的验收标准。

Animals [1]
Male beagle dogs between the ages of 6 and 18 months were used for the in life portion of this study. The animals were housed in accordance with the standards of the American Association for Accreditation of Laboratory Animal Care and were receiving a standard commercial diet. Animals were handled in strict accordance with the Guide for the Care and Use of Laboratory Animals, (22) and the protocol was reviewed by the Institutional Animal Care and Use Committee at SRI International. Animals were between 7 and 11 kg at dosing.
Drug Administration[1]
For intravenous administration, GS-7340 was formulated in 5% dextrose in water. For oral administration, GS-7340 was formulated in 50 mM citric acid (pH 5.0) at doses of 2 to 10 mg/kg. For the 20 mg/kg dose, GS-7340 was formulated in 50 mM citric acid (pH 5.5) with 0.1% Polysorbate 20. To test the effect of efflux transport inhibition, dogs were administered 2 mg/kg GS-7340 1 h following pretreatment with a 75 mg capsule of CsA.
Plasma and PBMC Sample Collection[1]
Blood (approximately 0.5 mL) was collected at specified time points over 24 h from the jugular vein (intraportal vein infusion and oral pharmacokinetic studies), the jugular and portal veins (oral administration to portal vein cannulated dogs), or the cephalic vein (jugular vein infusion). Plasma was isolated in Vacutainer tubes containing EDTA as an anticoagulant by centrifugation. At select time points (1, 4, 8, and 24 h postdose) in the 5 mg/kg oral pharmacokinetic study, 8 to 10 mL of blood was collected into Vacutainer cell preparation tubes (Becton Dickinson) for isolation of peripheral blood mononuclear cells (PBMC) and processed following manufacturer instructions. Small aliquots (10 μL) of isolated PBMC pellets diluted in 0.9% NaCl were maintained at room temperature and used to determine cell count. 500 μL of 70% methanol was added to the remaining PBMC pellets and, together with plasma samples, stored at −80 °C until shipment for further processing and analysis.

10 mg/kg, p.o.

Beagle dogs

Absorption, Distribution and Excretion
As compared to the parent molecule, [tenofovir], tenofovir alafenamide presents a lipophilic group that masks the negative charge of the parent moiety which improves its oral bioavailability. Tenofovir alafenamide is highly stable in plasma and, after administration of this prodrug, there is a low concentration of tenofovir in plasma. After oral administration, tenofovir alafenamide is rapidly absorbed by the gut. When a single dose is administered, a peak concentration of 16 ng/ml of the parent compound, corresponding to about 73% of the dose, is observed after 2 hours with an AUC of 270 ng\*h/mL. Once inside the body, tenofovir alafenamide enters hepatocytes by passive diffusion regulated by the organic anion transporters 1B1 and 1B3 for its activation. Administration of tenofovir alafenamide concomitantly with a high-fat meal results in an increase of about 65% in its internal exposure.
Tenofovir alafenamide has been registered to present a bile elimination that corresponds to 47% of the administered dose and a renal elimination the represents about 36%. From the recovered dose in urine, about 75% is represented as unchanged [tenofovir] followed by uric acid and a small dose of tenofovir alafenamide. On the other hand, in feces, 99% of the recovered dose corresponds to tenofovir.
In clinical trials, the reported volume of distribution of tenofovir alafenamide was higher than 100 L.
The reported clearance rate of tenofovir alafenamide is 117 L/h. In patients with severe renal impairment, this value can be decreased by 50%, reporting a rate of 61.7 L/h.

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Metabolism / Metabolites
To be activated, tenofovir alafenamide is required to be hydrolyzed to the parent compound [tenofovir] by the activity of cathepsin A or carboxylesterase 1. Tenofovir alafenamide presents significant plasma stability and hence, its activation is performed inside the target cells. After activation, tenofovir is further processed and after 1-2 days, it is detected in plasma almost completely transformed to uric acid.

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Biological Half-Life
The reported half-life for tenofovir alafenamide is of 0.51 hours.

Protein Binding
Tenofovir alafenamide is reported to bind to plasma proteins and _ex vivo_ studies have registered that approximately 80% of the administered dose of this drug is presented in a bound state.

[1]. Mechanism for effective lymphoid cell and tissue loading following oral administration of nucleotide prodrug GS-7340. Mol Pharm. 2013 Feb 4;10(2):459-66.

[2]. Antiviral activity, safety, and pharmacokinetics/pharmacodynamics of tenofovir alafenamide as 10-day monotherapy in HIV-1-positive adults. J Acquir Immune Defic Syndr. 2013 Aug 1;63(4):449-55.

Pharmacodynamics
Tenofovir alafenamide has been shown to be a potent inhibitor of hepatitis B viral replication. Tenofovir alafenamide presents a better renal tolerance when compared with the counterpart [tenofovir disoproxil]. This improved safety profile seems to be related to a lower plasma concentration of tenofovir. In clinical trials, tenofovir alafenamide was shown to present 5-fold more potent antiviral activity against HIV-1 when compared to tenofovir disoproxil.

C21H29N6O5P

476.47

476.193

C, 52.94; H, 6.13; N, 17.64; O, 16.79; P, 6.50

379270-37-8

Tenofovir alafenamide fumarate;379270-38-9;Tenofovir alafenamide hemifumarate;1392275-56-7

9574768

White to off-white solid

1.39±0.1 g/cm3

640.4±65.0 °C at 760 mmHg

104-107 ºC

341.1±34.3 °C

0.0±1.9 mmHg at 25°C

1.630

2.2

153.29

2

10

12

33

680

3

[P@](C([H])([H])OC([H])(C([H])([H])[H])C([H])([H])N1C([H])=NC2=C(N([H])[H])N=C([H])N=C12)(N([H])C([H])(C(=O)OC([H])(C([H])([H])[H])C([H])([H])[H])C([H])([H])[H])(=O)OC1C([H])=C([H])C([H])=C([H])C=1[H]

LDEKQSIMHVQZJK-AZFZMOAFSA-N

InChI=1S/C21H29N6O5P/c1-14(2)31-21(28)16(4)26-33(29,32-17-8-6-5-7-9-17)13-30-15(3)10-27-12-25-18-19(22)23-11-24-20(18)27/h5-9,11-12,14-16H,10,13H2,1-4H3,(H,26,29)(H2,22,23,24)/t15-,16+,33?/m1/s1

(S)-isopropyl 2-(((S)-((((R)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)(phenoxy)phosphoryl)amino)propanoate

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: ≥ 2.08 mg/mL (4.37 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 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (4.37 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 20.8 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: ≥ 2.08 mg/mL (4.37 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 20.8 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 4 中的溶解度: 40 mg/mL (83.95 mM) in PBS (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。