HIV-1/2 nucleotide reverse transcriptase

根据MTT实验，替诺福韦对HK-2细胞活力有有害影响，48小时和72小时的IC50值分别为2.77 μM和9.21 μM。替诺福韦可降低 HK-2 细胞中的 ATP 水平。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，替诺福韦能够导致 HK-2 细胞凋亡，这是由线粒体损伤引起的[1]。活化的 PBMC 中 R5 向性 HIV-1BaL 和 X4 向性 HIV-1IIIb 的复制被替诺福韦和 M48U1（在 0.25% HEC 中复合）抑制。此外，一些实验室毒株和患者来源的 HIV-1 分离株也受到抑制。除了对 PBMC 无毒之外，M48U1 和替诺福韦在 0.25% HEC 中的组合制剂还表现出针对 R5 倾向性 HIV-1BaL 感染的协同抗逆转录病毒功效 [2]。

在 BLT 人源化小鼠中，富马酸替诺福韦二吡呋酯（20、50、140 或 300 mg/kg）给药在阴道 HIV 攻击期间产生剂量依赖性活性。富马酸替诺福韦二吡呋酯（50、140 或 300 mg/kg）可显着减少 BLT 小鼠中的 HIV 传播[3]。在慢性感染 WHV 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）会导致血清病毒血症出现剂量依赖性下降。对于治疗土拨鼠持续性 HBV 感染，富马酸替诺福韦二吡呋酯既安全又有效 [4]。

将细胞接种到48孔组织培养板（39000个细胞/mL）中，使其生长48小时，然后用赋形剂或替诺福韦处理。治疗期后，使用MTT法评估细胞存活率。MTT法依赖于NAD（P）H依赖性氧化还原酶将四唑染料3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑（MTT）转化为甲氮[1]。

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.[5]

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Twenty adult chronic WHV carrier woodchucks are stratified equally by age, sex, body weight, and serum GGT activity into five treatment groups consisting of four animals each: (i) Tenofovir Disoproxil Fumarate at 15.0 mg/kg once per day, (ii) Tenofovir Disoproxil Fumarate at 5.0 mg/kg/day, (iii) Tenofovir Disoproxil Fumarate at 1.5 mg/kg/day, (iv) Tenofovir Disoproxil Fumarate at 0.5 mg/kg/day, and (v) a placebo control. The woodchucks are treated daily for 4 weeks and observed for an additional 12 weeks following cessation of drug treatment.

Absorption, Distribution and Excretion
After oral administration of tenofovir disoproxil to patients with HIV infection, tenofovir disoproxil is quickly absorbed and metabolized to tenofovir. Administration of tenofovir disoproxil 300 mg tablets after a high-fat meal increases the oral bioavailability of this drug, as demonstrated by an increase in tenofovir AUC0-∞ of about 40% as well as an increase in Cmax of about 14%. On the contrary, the administration of tenofovir disoproxil with a light meal did not exert a relevant effect on the pharmacokinetics of tenofovir when compared to administration under fasting conditions. The presence of ingested food slows the time to tenofovir Cmax by approximately 1 hour. Cmax and AUC of tenofovir are 0.33 ± 0.12 μg/mL and 3.32 ± 1.37 μg•hr/mL after several doses of tenofovir disoproxil 300 mg once daily in the fed state when meal content is not controlled.
Following IV administration of tenofovir, approximately 70–80% of the dose is recovered in the urine as unchanged tenofovir within 72 hours of dosing. Tenofovir is eliminated by a combination of glomerular filtration and active tubular secretion. There may be competition for elimination with other compounds that are also eliminated by the kidneys.
The volume of distribution at steady-state is 1.3 ± 0.6 L/kg and 1.2 ± 0.4 L/kg, following intravenous administration of tenofovir 1.0 mg/kg and 3.0 mg/kg. After oral administration of tenofovir disoproxil, tenofovir is distributed to the majority tissues with the highest concentrations measured in the kidney, liver and the intestinal contents (based on data from preclinical studies).
The clearance of tenofovir is highly dependent on renal function and may vary greatly. Total clearance has been estimated to be approximately 230 ml/h/kg (approximately 300 ml/min). On average, renal clearance has been estimated to be approximately 160 ml/h/kg (approximately 210 ml/min), which is in excess of the glomerular filtration rate. This shows that active tubular secretion is an essential part of the elimination of tenofovir. The FDA label provides specific guidelines for dosing according to renal function. It is important to consult product labeling before administering tenofovir to individuals with renal dysfunction, as the clearance of this drug may vary greatly among these patients.
Following IV administration of tenofovir, approximately 70-80% of the dose is recovered in the urine as unchanged tenofovir within 72 hours of dosing. Following single dose, oral administration of tenofovir, the terminal elimination half-life of tenofovir is approximately 17 hours. After multiple oral doses of tenofovir 300 mg once daily (under fed conditions), 32 + or - 10% of the administered dose is recovered in urine over 24 hours. Tenofovir is eliminated by a combination of glomerular filtration and active tubular secretion. There may be competition for elimination with other compounds that are also renally eliminated.
In vitro binding of tenofovir to human plasma or serum proteins is less than 0.7 and 7.2%, respectively, over the tenofovir concentration range 0.01 to 25 ug/mL. The volume of distribution at steady-state is 1.3 + or - 0.6 L/kg and 1.2 + or - 0.4 L/kg, following intravenous administration of tenofovir 1.0 mg/kg and 3.0 mg/kg.
Viread is a water soluble diester prodrug of the active ingredient tenofovir. The oral bioavailability of tenofovir from Viread in fasted subjects is approximately 25%. Following oral administration of a single dose of Viread 300 mg to HIV-1 infected subjects in the fasted state, maximum serum concentrations (Cmax) are achieved in 1.0 + or - 0.4 hr. Cmax and AUC values are 0.30 + or - 0.09 ug/mL and 2.29 + or - 0.69 ug hr/mL, respectively.
Administration of Viread 300 mg tablets following a high-fat meal (approximately 700 to 1000 kcal containing 40 to 50% fat) increases the oral bioavailability, with an increase in tenofovir AUC of approximately 40% and an increase in Cmax of approximately 14%. However, administration of Viread with a light meal did not have a significant effect on the pharmacokinetics of tenofovir when compared to fasted administration of the drug. Food delays the time to tenofovir Cmax by approximately 1 hour. Cmax and AUC of tenofovir are 0.33 + or - 0.12 ug/mL and 3.32 + or - 1.37 ug hr/mL following multiple doses of Viread 300 mg once daily in the fed state, when meal content was not controlled.
For more Absorption, Distribution and Excretion (Complete) data for TENOFOVIR DISOPROXIL FUMARATE (6 total), please visit the HSDB record page.

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Metabolism / Metabolites
Tenofovir disoproxil fumarate is the fumarate salt of the prodrug _tenofovir disoproxil_. Tenofovir disoproxil is absorbed and converted to its active form, _tenofovir_, a nucleoside monophosphate (nucleotide) analog. Tenofovir is then converted to the active metabolite, _tenofovir diphosphate_, a chain terminator, by constitutively expressed enzymes in the cell. Two phosphorylation steps are required to convert tenofovir disoproxil to the active drug form. The cytochrome P450 enzyme system is not involved with the metabolism of tenofovir disoproxil or tenofovir.
Tenofovir disoproxil fumarate is a prodrug and is not active until it undergoes diester hydrolysis in vivo to tenofovir and subsequently is metabolized to the active metabolite (tenofovir diphosphate).

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Biological Half-Life
When a single oral dose is given, the terminal elimination half-life is approximately 17 hours.
Following single dose, oral administration of Viread, the terminal elimination half-life of tenofovir is approximately 17 hours.

Protein Binding
_In vitro_ binding of tenofovir to human plasma or serum proteins is <0.7 and <7.2%, respectively, over the tenofovir concentration range 0.01 to 25 μg/mL.

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Interactions
Potential pharmacokinetic interaction with drugs that reduce renal function or that may compete with tenofovir for active renal tubular secretion (i.e., acyclovir, cidofovir, ganciclovir, valacyclovir, valganciclovir); increased plasma concentrations of tenofovir or the concomitantly administered drug may occur.
The manufacturer of tenofovir states that tenofovir should not be used with adefovir for the treatment of hepatitis B virus (HBV) infection.
Pharmacokinetic interaction with atazanavir sulfate (decrease plasma concentrations and AUC of atazanavir (minimum concentration decreased 40%) and increased plasma concentrations and AUC of tenofovir when atazanavir 400 mg and tenofovir disoproxil fumarate 300 mg given once daily). Pharmacokinetic interaction with ritonavir-boosted atazanavir sulfate (decrease plasma concentrations and AUC of atazanavir (minimum concentration decreased 23%) and increased plasma concentrations and AUC of tenofovir when atazanavir 300 mg, ritonavir 100 mg, and tenofovir disoproxil fumarate 300 mg given once daily). If used concomitantly, a dosage regimen of atazanavir 300 mg, ritonavir 100 mg, and tenofovir disoproxil fumarate 300 mg given once daily with food is recommended; atazanavir should not be used with tenofovir unless low-dose ritonavir is a component of the regimen. Monitor for tenofovir toxicity and discontinue the drug if tenofovir-associated adverse effects occur. If atazanavir is used concomitantly with tenofovir and a histamine H2-receptor antagonist, the recommended dosage for treatment-experienced patients is atazanavir 400 mg, ritonavir 100 mg, and tenofovir disoproxil fumarate 300 mg given once daily with food.
Pharmacokinetic interaction with the buffered didanosine preparation (pediatric oral solution admixed with antacid; Videx) or delayed-release capsules containing enteric-coated pellets of didanosine (Videx EC) resulting in increased plasma concentrations and AUC of didanosine; no change in tenofovir pharmacokinetics. Potential for early virologic failure, rapid selection of resistant mutations, immunologic nonresponse (e.g., decline in CD4+ T-cell count), and increased risk of didanosine-associated adverse effects (e.g., pancreatitis, neuropathy). Caution is advised if didanosine and tenofovir are used concomitantly and patients should be monitored closely for didanosine-associated adverse effects; didanosine should be discontinued if such effects occur. If didanosine delayed-release capsules are used with tenofovir disoproxil fumarate, the recommended dosage of didanosine is 250 mg once daily for those weighing 60 kg or more with creatinine clearances of 60 mL/minute or greater and 200 mg once daily for those weighing less than 60 kg with creatinine clearances of 60 mL/minute or greater. Didanosine delayed-release capsules and tenofovir may be taken at the same time with a light meal (no more than 400 kcal, no more than 20% fat) or in the fasted state.
For more Interactions (Complete) data for TENOFOVIR DISOPROXIL FUMARATE (10 total), please visit the HSDB record page.

[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.

Therapeutic Uses
Anti-HIV Agents, Reverse Transcriptase Inhibitors
Tenofovir disoproxil fumarate is used in conjunction with other antiretroviral agents for the treatment of human immunodeficiency virus type 1 (HIV-1) infections in adults. /Included in US product labeling/
Tenofovir is used for the management of chronic hepatitis B virus (HBV) infection in adults. This indication is based on histologic, virologic, biochemical, and serologic responses in adults with hepatitis B e antigen (HBeAg)-positive or -negative chronic HBV with compensated liver function.
Tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and efavirenz (EFV) are the three components of the once-daily, single tablet regimen (Atripla) for treatment of HIV-1 infection. Previous cell culture studies have demonstrated that the double combination of tenofovir (TFV), the parent drug of TDF, and FTC were additive to synergistic in their anti-HIV activity, which correlated with increased levels of intracellular phosphorylation of both compounds. In this study, /researchers/ demonstrated the combinations of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV synergistically inhibit HIV replication in cell culture and synergistically inhibit HIV-1 reverse transcriptase (RT) catalyzed DNA synthesis in biochemical assays. Several different methods were applied to define synergy including median-effect analysis, MacSynergyII and quantitative isobologram analysis. We demonstrated that the enhanced formation of dead-end complexes (DEC) by HIV-1 RT and TFV-terminated DNA in the presence of FTC-triphosphate (TP) could contribute to the synergy observed for the combination of TFV+FTC, possibly through reduced terminal NRTI excision. Furthermore, /researchers/ showed that EFV facilitated efficient formation of stable, DEC-like complexes by TFV- or FTC-monophosphate (MP)-terminated DNA and this can contribute to the synergistic inhibition of HIV-1 RT by TFV-diphosphate (DP)+EFV and FTC-TP+EFV combinations. This study demonstrated a clear correlation between the synergistic antiviral activities of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV combinations and synergistic HIV-1 RT inhibition at the enzymatic level. /Researchers/ propose the molecular mechanisms for the TFV+FTC+EFV synergy to be a combination of increased levels of the active metabolites TFV-DP and FTC-TP and enhanced DEC formation by a chain-terminated DNA and HIV-1 RT in the presence of the second and the third drug in the combination. This study furthers the understanding of the longstanding observations of synergistic anti-HIV-1 effects of many NRTI+NNRTI and certain NRTI+NRTI combinations in cell culture, and provides biochemical evidence that combinations of anti-HIV agents can increase the intracellular drug efficacy, without increasing the extracellular drug concentrations.

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Drug Warnings
/BOXED WARNING/ WARNING: LACTIC ACIDOSIS/SEVERE HEPATOMEGALY WITH STEATOSIS and POST TREATMENT EXACERBATION OF HEPATITIS. Lactic acidosis and severe hepatomegaly with steatosis, including fatal cases, have been reported with the use of nucleoside analogs, including Viread, in combination with other antiretrovirals. Severe acute exacerbations of hepatitis have been reported in HBV-infected patients who have discontinued anti-hepatitis B therapy, including Viread. Hepatic function should be monitored closely with both clinical and laboratory follow-up for at least several months in patients who discontinue anti-hepatitis B therapy, including Viread. If appropriate, resumption of anti-hepatitis B therapy may be warranted.
Lactic acidosis and severe hepatomegaly with steatosis (sometimes fatal) have been reported rarely in patients receiving nucleoside reverse transcriptase inhibitors alone or in conjunction with other antiretroviral agents. Most reported cases have involved women; obesity and long-term therapy with a nucleoside reverse transcriptase inhibitor also may be risk factors. Caution should be observed when nucleoside analogs are used in patients with known risk factors for liver disease; however, lactic acidosis and severe hepatomegaly with steatosis have been reported in patients with no known risk factors. Tenofovir therapy should be interrupted in any patient with clinical or laboratory findings suggestive of lactic acidosis or pronounced hepatotoxicity (signs of hepatotoxicity include hepatomegaly and steatosis even in the absence of marked increases in serum aminotransferase concentrations).
Redistribution or accumulation of body fat, including central obesity, dorsocervical fat enlargement (buffalo hump), peripheral wasting, facial wasting, breast enlargement, and general cushingoid appearance, has been reported with antiretroviral therapy.
The most common adverse effects in HIV-infected patients receiving tenofovir disoproxil fumarate are rash, diarrhea, headache, pain, depression, asthenia, and nausea. The most common adverse effect in HIV-infected patients receiving tenofovir disoproxil fumarate is nausea.
For more Drug Warnings (Complete) data for TENOFOVIR DISOPROXIL FUMARATE (14 total), please visit the HSDB record page.

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Pharmacodynamics
This drug prevents viral DNA chain elongation through inhibition of enzymes necessary for host cell infection viral replication in HIV-1 and Hepatitis B infections,. **In vitro effects** The antiviral activity of tenofovir against in laboratory and clinical isolates of HIV-1 was studied in lymphoblastoid cell lines, primary monocyte/macrophage cells, in addition to peripheral blood lymphocytes. The EC50 (50% effective concentration) values of tenofovir against HIV-1 virus ranged between 0.04 μM to 8.5 μM. **Combination of tenofovir disoproxil with other drugs** In drug combination studies of tenofovir with nucleoside reverse transcriptase inhibitors (abacavir, didanosine, lamivudine, stavudine, zalcitabine, zidovudine), non-nucleoside reverse transcriptase inhibitors (delavirdine, efavirenz, nevirapine), and protease inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, saquinavir), additive and synergistic effects were seen. Tenofovir demonstrated antiviral activities in cell cultures against HIV-1.

C19H30N5O10P

519.4478

519.173

C, 43.93; H, 5.82; N, 13.48; O, 30.80; P, 5.96

201341-05-1

Tenofovir Disoproxil fumarate;202138-50-9; 201341-05-1 (free) ; 147127-20-6 (Tenofovir); 206184-49-8 (hydrate); 379270-37-8 (alafenamide); 1571075-19-8 (aspartate)

5481350

White to off-white solid powder

1.5±0.1 g/cm3

642.7±65.0 °C at 760 mmHg

113-115

342.5±34.3 °C

0.0±1.9 mmHg at 25°C

1.578

2.04

195.25

1

14

17

35

698

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)(OC([H])([H])OC(=O)OC([H])(C([H])([H])[H])C([H])([H])[H])OC([H])([H])OC(=O)OC([H])(C([H])([H])[H])C([H])([H])[H]

JFVZFKDSXNQEJW-CQSZACIVSA-N

InChI=1S/C19H30N5O10P/c1-12(2)33-18(25)28-9-31-35(27,32-10-29-19(26)34-13(3)4)11-30-14(5)6-24-8-23-15-16(20)21-7-22-17(15)24/h7-8,12-14H,6,9-11H2,1-5H3,(H2,20,21,22)/t14-/m1/s1

[[(2R)-1-(6-aminopurin-9-yl)propan-2-yl]oxymethyl-(propan-2-yloxycarbonyloxymethoxy)phosphoryl]oxymethyl propan-2-yl carbonate

GS 4331; GS-4331; GS4331; Bis(POC)PMPA; PMPA prodrug; 9-((R)-2-((Bis(((isopropoxycarbonyl)oxy)methoxy)phosphinyl)methoxy)propyl)adenine; (r)-bis(poc)pmpa; bis-POC-PMPA; tenofovir bis(isopropyloxycarbonyloxymethyl) ester; Viread.

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 : ≥ 38 mg/mL (~73.16 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.00 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 (4.00 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 (4.00 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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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。