HIV-1/2 nucleotide reverse transcriptase

在MTT实验中，替诺福韦对HK-2细胞活力表现出细胞毒性作用，48小时和72小时的IC50值分别为2.77 μM。替诺福韦导致 HK-2 细胞的 ATP 水平下降。在 HK-2 细胞中，替诺福韦（3.0 至 28.8 μM）可增强蛋白质羰基化和氧化应激。此外，替诺福韦会导致HK-2细胞发生凋亡，这一过程是由线粒体损伤引起的[1]。当与 0.25% HEC 混合时，替诺福韦和 M48U1 会抑制激活的 PBMC 中 R5 向性 HIV-1BaL 和 X4 向性 HIV-1IIIb 的复制。此外，各种实验室毒株和患者来源的 HIV-1 分离株均受到抑制。 R5-tropic HIV-1BaL 的感染可通过 M48U1 和替诺福韦在 0.25% HEC 中的协同抗逆转录病毒作用来抑制，并且该制剂对 PBMC 无害[2]。

当给予 BLT 小鼠（20、50、140 或 300 mg/kg）时，富马酸替诺福韦二吡呋酯在 BLT 人源化小鼠中针对阴道 HIV 攻击表现出剂量依赖性功效。在 BLT 小鼠中，富马酸替诺福韦二吡呋酯（50、140 或 300 毫克/千克）可显着降低 HIV 传播[3]。在患有慢性 WHV 感染的土拨鼠中，富马酸替诺福韦二吡呋酯（0.5、1.5 或 5.0 mg/kg/天，口服）可导致血清病毒血症出现剂量依赖性下降。富马酸替诺福韦二吡呋酯在慢性 HBV 感染土拨鼠模型中的给药既安全又有效[4]。

替诺福韦（TFV）是一种经批准用于治疗人类免疫缺陷病毒（HIV）和乙型肝炎的抗病毒药物。TFV作为前药富马酸替诺福维尔二吡呋酯（TDF）口服给药，然后脱酯化为活性药物TFV。TFV诱导肾毒性，其特征为肾功能衰竭和范可尼综合征。由于实验模型有限，这种毒性的机制尚不清楚。本研究使用人肾近端小管上皮细胞系（HK-2）研究了细胞毒性的细胞机制。HK-2细胞生长48小时，然后暴露于0-28.8μM TFV或载体磷酸盐缓冲盐水（PBS）中24至72小时。MTT（MTT，3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物）和台盼蓝表明，与赋形剂相比，TFV在24-72小时时降低了细胞存活率。TFV增加了蛋白质羰基化和4-羟基壬烯醇（4-HNE）加合物形成的氧化应激生物标志物。在暴露于14.5和28.8μM TFV后，肿瘤坏死因子α（TNFα）释放到培养基中。与载体相比，TFV在72小时时诱导了Caspase 3和9的切割。这些研究表明，HK-2细胞是TFV细胞毒性的敏感模型，并表明用TFV处理的HK-2细胞中发生了线粒体应激和凋亡。[1]

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杀微生物剂被认为是预防人类免疫缺陷病毒（HIV-1）传播和疾病的一种有前景的策略。在本报告中，我们首先分析了在羟乙基纤维素（HEC）水凝胶中配制的miniCD4 M48U1肽在感染R5和X4嗜性HIV-1毒株的活化外周血单核细胞（PBMCs）中的抗病毒活性。结果表明，M48U1可以预防几种HIV-1毒株的感染，包括实验室毒株，以及从患者活化的PBMC中分离出的HIV-1 B和C亚型毒株。M48U1还抑制了两种HIV-1传播/创始人感染分子克隆（pREJO.c/2864和pTHRO.c/2626）的感染。此外，M48U1与替诺福韦联合给药，这两种抗逆转录病毒药物协同抑制HIV-1感染。在下一系列实验中，我们在HEC水凝胶中单独或与替诺福韦联合测试了M48U1，该水凝胶具有模仿人类宫颈阴道组织的器官样结构。我们证明了在没有明显组织毒性的情况下具有很强的抗病毒作用。总之，这些结果表明，与M48U1加替诺福韦联合治疗是一种有效的抗病毒策略，可作为一种新的局部杀微生物剂来预防HIV-1传播[2]。

The efficacy of HIV pre-exposure prophylaxis (PrEP) relies on adherence and may also depend on the route of HIV acquisition. Clinical studies of systemic tenofovir disoproxil fumarate (TDF) PrEP revealed reduced efficacy in women compared to men with similar degrees of adherence. To select the most effective PrEP strategies, preclinical studies are critically needed to establish correlations between drug concentrations (pharmacokinetics [PK]) and protective efficacy (pharmacodynamics [PD]). We utilized an in vivo preclinical model to perform a PK-PD analysis of systemic TDF PrEP for vaginal HIV acquisition. TDF PrEP prevented vaginal HIV acquisition in a dose-dependent manner. PK-PD modeling of tenofovir (TFV) in plasma, female reproductive tract tissue, cervicovaginal lavage fluid and its intracellular metabolite (TFV diphosphate) revealed that TDF PrEP efficacy was best described by plasma TFV levels. When administered at 50 mg/kg, TDF achieved plasma TFV concentrations (370 ng/ml) that closely mimicked those observed in humans and demonstrated the same risk reduction (70%) previously attained in women with high adherence. This PK-PD model mimics the human condition and can be applied to other PrEP approaches and routes of HIV acquisition, accelerating clinical implementation of the most efficacious PrEP strategies.[3]

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Tenofovir disoproxil fumarate (TDF) is a nucleotide analogue approved for treatment of human immunodeficiency virus (HIV) infection. TDF also has been shown in vitro to inhibit replication of wild-type hepatitis B virus (HBV) and lamivudine-resistant HBV mutants and to inhibit lamivudine-resistant HBV in patients and HBV in patients coinfected with the HIV. Data on the in vivo efficacy of TDF against wild-type virus in non-HIV-coinfected or lamivudine-naïve chronic HBV-infected patients are lacking in the published literature. The antiviral effect of oral administration of TDF against chronic woodchuck hepatitis virus (WHV) infection, an established and predictive animal model for antiviral therapy, was evaluated in a placebo-controlled, dose-ranging study (doses, 0.5 to 15.0 mg/kg of body weight/day). Four weeks of once-daily treatment with TDF doses of 0.5, 1.5, or 5.0 mg/kg/day reduced serum WHV viremia significantly (0.2 to 1.5 log reduction from pretreatment level). No effects on the levels of anti-WHV core and anti-WHV surface antibodies in serum or on the concentrations of WHV RNA or WHV antigens in the liver of treated woodchucks were observed. Individual TDF-treated woodchucks demonstrated transient declines in WHV surface antigen serum antigenemia and, characteristically, these woodchucks also had transient declines in serum WHV viremia, intrahepatic WHV replication, and hepatic expression of WHV antigens. No evidence of toxicity was observed in any of the TDF-treated woodchucks. Following drug withdrawal there was prompt recrudescence of WHV viremia to pretreatment levels. It was concluded that oral administration of TDF for 4 weeks was safe and effective in the woodchuck model of chronic HBV infection.[4]

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Dissolved in saline; 30 mg/kg; s.c. injection

Macaques

Absorption, Distribution and Excretion
Tenofovir as the active moiety presents a very low bioavailability when orally administered. Hence, the administration of this active agent is required to be under its two prodrug forms, [tenofovir disoproxil] and [tenofovir alafenamide]. This reduced absorption is suggested to be related to the presence of two negative charges among its structure. This negative charge limits its cellular penetration, and its passive diffusion across cellular membranes and intestinal mucosa hindering its availability for oral administration. Intravenous tenofovir has been shown to produce a maximum plasma concentration of 2500 ng/ml with an AUC of 4800 ng.h/ml.
Tenofovir is eliminated in the urine by tubular secretion and glomerular filtration. The elimination of this compound is driven by the activity of the human organic anion transporters 1 and 3 and its secretion is mainly ruled by the activity of the multidrug resistance-associated protein 4.
Accumulation of tenofovir in plasma is related to the presence of nephrotoxic effects. It is reported that tenofovir presents a volume of distribution of 0.813 L/kg.
The clearance of tenofovir is highly dependent on the patient renal stage and hence the clearance rate in patients with renal impairment is reported to be of 134 ml/min while in patients with normal function the clearance rate can be of 210 ml/min.

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Metabolism / Metabolites
Tenofovir activation is performed by a bi-phosphorylation which in order forms the biologically active compound, tenofovir biphosphate. This metabolic activation has been shown to be performed in hepG2 cells and human hepatocytes.

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Biological Half-Life
The reported half-life of tenofovir is of 32 hours.

Hepatotoxicity
Like all nucleoside analogues used as therapy of hepatitis B, tenofovir can cause transient increases in serum aminotransferases during or after therapy. These abnormalities appear to be due to an exacerbation or flare of the underlying hepatitis B. Three types of flares due to nucleoside analogue therapy have been described: transient flares during initiation of therapy (treatment flares), flares occurring in association with development of antiviral resistance (breakthrough flares) and flares occurring in the few months after stopping therapy (withdrawal flares). Treatment flares generally arise during the first few months of starting therapy, are usually mild, asymptomatic and self-limited and do not require dose modification or interruption of therapy. Breakthrough flares generally follow the development of antiviral resistance and subsequent rise in HBV DNA levels during nucleoside analogue therapy. Breakthrough flares can be symptomatic and severe. Because tenofovir is associated with a very low rate of antiviral resistance (
Tenofovir appears to have little or no direct hepatotoxicity. In patients without HBV and HIV infection, given tenofovir as a part of preexposure prevention, minor serum ALT and AST elevations are more frequent than with placebo, but are rarely above 5 times ULN (
Likelihood score: C (has been associated with flares of hepatitis when it is withdrawn and rarely with a sudden antiviral effect early during therapy and finally linked to episodes of lactic acidosis due to its effects on drug levels of other nucleosides that can cause lactic acidosis).

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Protein Binding
Tenofovir is minimally bound to plasma proteins and only about 7.2% of the administered dose is found in the bound state.

[1]. Establishment of HK-2 Cells as a Relevant Model to Study Tenofovir-Induced Cytotoxicity. Int J Mol Sci. 2017 Mar 1;18(3).

[2]. M48U1 and Tenofovir combination synergistically inhibits HIV infection in activated PBMCs and human cervicovaginal histocultures. Sci Rep. 2017 Feb 1;7:41018.

[3]. Predicting HIV Pre-exposure Prophylaxis Efficacy for Women using a Preclinical Pharmacokinetic-Pharmacodynamic In Vivo Model. Sci Rep. 2017 Feb 1;7:41098.

[4]. Menne S, Cote PJ, Korba BE, Antiviral effect of oral administration of tenofovir disoproxil fumarate in woodchucks with chronic woodchuck hepatitis virus infection. Antimicrob Agents Chemother. 2005 Jul;49(7):2720-8.

Pharmacodynamics
Tenofovir has been shown to be highly effective in patients that have never had an antiretroviral therapy and it seemed to have lower toxicity than other antivirals such as [stavudine]. In phase 3 clinical trials, tenofovir presented a similar efficacy than [efavirenz] in treatment-naive HIV patients. In hepatitis B infected patients, after one year of tenofovir treatment, the viral DNA levels were undetectable.

C9H14N5O4PEXACTMASS

287.2123

287.078

C, 37.64; H, 4.91; N, 24.38; O, 22.28; P, 10.78

147127-20-6

Tenofovir Disoproxil fumarate;202138-50-9;Tenofovir hydrate;206184-49-8;Tenofovir diphosphate;166403-66-3;Tenofovir maleate;1236287-04-9

464205

Typically exists as White to off-white solids at room temperature

1.8±0.1 g/cm3

616.1±65.0 °C at 760 mmHg

276-280°C

326.4±34.3 °C

0.0±1.9 mmHg at 25°C

1.740

-1.71

146.19

3

8

5

19

354

1

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

SGOIRFVFHAKUTI-ZCFIWIBFSA-N

InChI=1S/C9H14N5O4P/c1-6(18-5-19(15,16)17)2-14-4-13-7-8(10)11-3-12-9(7)14/h3-4,6H,2,5H2,1H3,(H2,10,11,12)(H2,15,16,17)/t6-/m1/s1

(R)-(((1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)phosphonic acid

GS 1278; GS1278; GS-1278; PMPA TDF GS1275; GS-1275; Tenofovir gel; GS 1275; (R)-9-(2-Phosphonomethoxypropyl)adenine; (R)-PMPA; Truvada; tenofovir (anhydrous); PMPA gel; Tenofovir TFV; gel PMPA

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)

DMSO : ~7.69 mg/mL (~26.77 mM)
H2O : ~2 mg/mL (~6.96 mM)

配方 1 中的溶解度: ≥ 0.77 mg/mL (2.68 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 7.7 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 0.77 mg/mL (2.68 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 7.7 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: ≥ 0.77 mg/mL (2.68 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 7.7 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: 1.96 mg/mL (6.82 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 (<60°C).

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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。