HIV-1/2 nucleotide reverse transcriptase

According to the MTT experiment, tenofovir has a deleterious effect on HK-2 cell viability, with IC50 values of 2.77 μM and 9.21 μM at 48 and 72 hours, respectively. ATP levels in HK-2 cells are lowered by tenofovir. In HK-2 cells, tenofovir (3.0 to 28.8 μM) elevates protein carbonylation and oxidative stress. Furthermore, tenofovir has the ability to cause HK-2 cells to undergo apoptosis, which is brought on by damage to the mitochondria [1]. The replication of R5-tropic HIV-1BaL and X4-tropic HIV-1IIIb in activated PBMC was suppressed by tenofovir and M48U1, which were compounded in 0.25% HEC. Additionally, several laboratory strains and patient-derived HIV-1 isolates were inhibited. In addition to being non-toxic to PBMC, the combination formulation of M48U1 and tenofovir in 0.25% HEC demonstrated synergistic antiretroviral efficacy against R5-tropic HIV-1BaL infection [2].

In BLT humanized mice, tenofovir disoproxil fumarate (20, 50, 140, or 300 mg/kg) administration resulted in dose-dependent activity during vaginal HIV challenge. HIV transmission in BLT mice is dramatically decreased by tenofovir disoproxil fumarate (50, 140, or 300 mg/kg) [3]. In woodchucks that are chronically infected with WHV, tenofovir disoproxil fumarate (0.5, 1.5, or 5.0 mg/kg/day) causes a dose-dependent decrease in serum viremia. For treating a persistent HBV infection in woodchucks, tenofovir disoproxil fumarate is both safe and efficacious [4].

Cells are plated into 48-well tissue culture plates (39,000 cells/mL) and allowed to grow for 48 h followed by treatment with vehicle or Tenofovir. Following the treatment period, cell viability is assessed using the MTT assay. The MTT assay relies on the conversion of tetrazolium dye 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan by NAD(P)H-dependent oxidoreductases[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
Following oral administration of tenofovir disoproxil fumarate to HIV-infected patients, tenofovir disoproxil fumarate is rapidly absorbed and metabolized into tenofovir. Taking a 300 mg tenofovir disoproxil fumarate tablet after a high-fat meal improves the oral bioavailability of the drug, as evidenced by an approximately 40% increase in AUC0-∞ and an approximately 14% increase in Cmax. Conversely, taking tenofovir disoproxil fumarate with a small amount of food has no significant effect on the pharmacokinetics of tenofovir compared to taking it on an empty stomach. The presence of food prolongs the time for tenofovir to reach Cmax by approximately 1 hour. In a meal-fed state, without controlling food intake, after a once-daily dose of 300 mg tenofovir disoproxil fumarate, the Cmax and AUC of tenofovir are 0.33 ± 0.12 μg/mL and 3.32 ± 1.37 μg•hr/mL, respectively. Following intravenous administration of tenofovir, approximately 70-80% of the dose is excreted unchanged in the urine within 72 hours. Tenofovir is primarily cleared via glomerular filtration and active tubular secretion. Competition for clearance with other compounds cleared by the kidneys is possible. The steady-state volumes of distribution after intravenous administration of 1.0 mg/kg and 3.0 mg/kg tenofovir were 1.3 ± 0.6 L/kg and 1.2 ± 0.4 L/kg, respectively. After oral administration of tenofovir disoproxil fumarate, tenofovir is distributed to most tissues, with the highest concentrations in the kidneys, liver, and intestinal contents (based on preclinical data). The clearance of tenofovir is highly dependent on renal function and can vary considerably among individuals. Total clearance is estimated to be approximately 230 ml/h/kg (approximately 300 ml/min). On average, renal clearance is estimated to be approximately 160 ml/h/kg (approximately 210 ml/min), higher than glomerular filtration rate. This indicates that active tubular secretion is a crucial component of tenofovir clearance. The FDA drug label provides specific guidance on adjusting the dosage based on renal function. For patients with renal insufficiency, it is essential to consult the product information leaflet before administering tenofovir, as clearance can vary significantly among these patients. Following intravenous administration of tenofovir, approximately 70-80% of the dose is excreted unchanged in the urine within 72 hours. After a single oral dose of tenofovir, the terminal elimination half-life is approximately 17 hours. With a once-daily oral dose of 300 mg tenofovir (after food), approximately 32 ± 10% of the administered dose is excreted in the urine within 24 hours. Tenofovir is primarily cleared via glomerular filtration and active tubular secretion. Competition for clearance with other compounds excreted by the kidneys is possible.
Within the range of tenofovir concentrations from 0.01 to 25 μg/mL, the in vitro binding rates of tenofovir to human plasma or serum proteins were less than 0.7% and 7.2%, respectively. The steady-state volumes of distribution after intravenous administration of tenofovir at doses of 1.0 mg/kg and 3.0 mg/kg were 1.3 ± 0.6 L/kg and 1.2 ± 0.4 L/kg, respectively.
Viread is a water-soluble diester prodrug of the active ingredient tenofovir. The bioavailability of tenofovir is approximately 25% after oral administration of Viread in fasting subjects. In fasting HIV-1 infected individuals, after a single oral dose of 300 mg Viread, peak plasma concentration (Cmax) was reached within 1.0 ± 0.4 hours. The Cmax and AUC values were 0.30 ± 0.09 μg/mL and 2.29 ± 0.69 μg·hr/mL, respectively.
Taking 300 mg Viread tablets after a high-fat meal (approximately 700 to 1000 kcal, containing 40% to 50% fat) improves oral bioavailability, increasing tenofovir's AUC by approximately 40% and Cmax by approximately 14%. However, taking Viread after consuming a small amount of food has no significant effect on the pharmacokinetics of tenofovir compared to taking it on an empty stomach. Food delays the time to reach Cmax for tenofovir by approximately 1 hour. In a fed state, after a once-daily dose of 300 mg Viread, the Cmax and AUC of tenofovir were 0.33 ± 0.12 μg/mL and 3.32 ± 1.37 μg·h/mL, respectively (meal components not controlled).
For more complete data on the absorption, distribution, and excretion of tenofovir disoproxil fumarate (6 types), please visit the HSDB records page.

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Metabolism/Metabolite
Tenofovir disoproxil fumarate is the fumarate salt of the prodrug tenofovir disoproxil fumarate. After absorption, tenofovir disoproxil fumarate is converted to its active form, tenofovir, a nucleoside monophosphate (nucleotide) analog. Tenofovir is then converted to the active metabolite, tenofovir disoproxil fumarate (a chain terminator), by constitutively expressed enzymes within the cell. The conversion of tenofovir disoproxil fumarate to its active drug form requires two phosphorylation steps. The cytochrome P450 enzyme system is not involved in the metabolism of tenofovir disoproxil fumarate or tenofovir.
Tenofovir disoproxil fumarate is a prodrug that becomes active only after diester hydrolysis in vivo to tenofovir, which is then metabolized to the active metabolite (tenofovir disoproxil fumarate).

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Biological Half-Life
The terminal elimination half-life after a single oral administration is approximately 17 hours.
After a single oral dose of Viread, the terminal elimination half-life of tenofovir is approximately 17 hours.

Protein Binding
At concentrations ranging from 0.01 to 25 μg/mL, tenofovir's in vitro binding rates to human plasma and serum proteins are <0.7% and <7.2%, respectively. Interactions Pharmacokinetic interactions may occur with drugs that reduce renal function or may compete with tenofovir for active tubular secretion (e.g., acyclovir, cidofovir, ganciclovir, valacyclovir, valganciclovir); elevated plasma concentrations of tenofovir or concomitant drugs may occur. The manufacturer of tenofovir states that tenofovir should not be used to treat hepatitis B virus (HBV) infection. Pharmacokinetic interactions exist with atazanavir sulfate (when taken once daily at 400 mg atazanavir and 300 mg tenofovir disoproxil fumarate, plasma concentrations and AUC of atazanavir decreased (minimum concentration decreased by 40%), while plasma concentrations and AUC of tenofovir increased). Atazanavir sulfate, which enhances the efficacy of ritonavir, exhibits pharmacokinetic interactions (when taken once daily at 300 mg atazanavir, 100 mg ritonavir, and 300 mg tenofovir disoproxil fumarate, plasma concentrations and AUCs of atazanavir decrease (lowest concentration decreased by 23%), while plasma concentrations and AUCs of tenofovir increase). If used concomitantly, it is recommended to take 300 mg atazanavir, 100 mg ritonavir, and 300 mg tenofovir disoproxil fumarate once daily with food; atazanavir should not be used with tenofovir unless a low-dose ritonavir regimen is part of this regimen. Tenofovir toxicity should be monitored, and treatment should be discontinued if adverse reactions related to tenofovir occur. If atazanavir is used concomitantly with tenofovir and histamine H2 receptor antagonists, the recommended dose for previously treated patients is atazanavir 400 mg, ritonavir 100 mg, and tenofovir disoproxil fumarate 300 mg, once daily with food. Pharmacokinetic interactions exist with buffered didanosin formulations (pediatric oral solution mixed with an antacid; Videx) or extended-release capsules containing enteric-coated didanosin granules (Videx EC), resulting in increased plasma concentrations and AUC of didanosin; tenofovir pharmacokinetics remain unchanged. This may lead to early virological failure, rapid selection of resistance mutations, immune non-response (e.g., decreased CD4+ T cell count), and an increased risk of didanosin-related adverse reactions (e.g., pancreatitis, neuropathy). Caution should be exercised when didanosin and tenofovir are used concomitantly, and patients should be closely monitored for didanosin-related adverse reactions; if such adverse reactions occur, didanosin should be discontinued. If didanoxin extended-release capsules are used in combination with tenofovir disoproxil fumarate, the recommended dose is: 250 mg once daily for patients weighing ≥60 kg with a creatinine clearance ≥60 mL/min; and 200 mg once daily for patients weighing <60 kg with a creatinine clearance ≥60 mL/min. Didanoxin extended-release capsules and tenofovir can be taken concurrently with a small amount of food (not exceeding 400 kcal, fat content not exceeding 20%) or on an empty stomach. For more complete data on interactions of tenofovir disoproxil fumarate (one of 10), please visit the HSDB records 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
Tenofovir disoproxil fumarate, an anti-HIV drug and reverse transcriptase inhibitor, is used in combination with other antiretroviral drugs to treat human immunodeficiency virus type 1 (HIV-1) infection in adults. /US product label includes/ Tenofovir is used to treat chronic hepatitis B virus (HBV) infection in adults. This indication is based on histological, virological, biochemical, and serological responses in adult patients with chronic HBV who are HBeAg positive or negative and have compensated liver function. Tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and efavirenz (EFV) are three components of a once-daily single-tablet combination formulation (Atripla) for the treatment of HIV-1 infection. Previous cell culture studies have shown that the dual combination of tenofovir (TFV, the parent drug of TDF) and emtricitabine (FTC) has an additive or synergistic effect in anti-HIV activity, which is associated with increased intracellular phosphorylation levels of both compounds. In this study, researchers demonstrated that 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 analyses. The researchers employed various methods to define the synergistic effect, including median effect analysis, MacSynergy II, and quantitative equivalence plot analysis. We found that the enhanced formation of the dead-end complex (DEC) between HIV-1 RT and TFV-terminated DNA in the presence of FTC-triphosphate (TP) may be the reason for the observed synergistic effect of the TFV+FTC combination, possibly achieved by reducing the excision of terminal nucleoside reverse transcriptase inhibitors (NRTIs). Furthermore, the researchers found that EFV can promote the efficient formation of stable DEC-like complexes from TFV or FTC monophosphate (MP)-terminated DNA, which contributes to the synergistic inhibitory effect of the TFV diphosphate (DP)+EFV and FTC-TP+EFV combinations on HIV-1 reverse transcriptase (RT). This study confirms a clear correlation between the synergistic antiviral activity of TFV+FTC, TFV+EFV, FTC+EFV, and TFV+FTC+EFV combinations and their synergistic inhibitory effect on HIV-1 RT at the enzyme level. Researchers propose that the molecular mechanism of the synergistic effect of TFV+FTC+EFV is as follows: in the presence of the second and third drugs in the combination, the levels of the active metabolites TFV-DP and FTC-TP are increased, enhancing the formation of DEC in terminal DNA and HIV-1 RT. This study further deepens the long-term observation of the synergistic anti-HIV-1 effects of various NRTI+NNRTI combinations and certain NRTI+NRTI combinations in cell culture, and provides biochemical evidence that anti-HIV drug combinations can improve intracellular drug efficacy without increasing extracellular drug concentration.

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Drug Warning
/Black Box Warning/ Warning: Lactic acidosis/Severe hepatomegaly with steatosis and exacerbation of hepatitis after treatment.
Reports have indicated that lactic acidosis and severe hepatomegaly with steatosis, sometimes fatal, can occur when nucleoside analogues (including Verede) are used in combination with other antiretroviral drugs. There have also been reports of severe acute hepatitis exacerbations in patients with hepatitis B virus infection after discontinuing anti-hepatitis B treatment, including Verede. Patients who have discontinued anti-hepatitis B treatment (including Verede) should be closely monitored for liver function for at least several months, with clinical and laboratory follow-up. Reinstatement of anti-hepatitis B treatment may be considered if necessary. Rare reports of lactic acidosis and severe hepatomegaly with steatosis (sometimes even fatal) have emerged in patients receiving monotherapy or combination therapy with nucleoside reverse transcriptase inhibitors. Most cases are in women; obesity and long-term use of nucleoside reverse transcriptase inhibitors may also be risk factors. Caution should be exercised when using nucleoside analogues in patients with known risk factors for liver disease; however, there have been reports of lactic acidosis and severe hepatomegaly with steatosis even in patients without known risk factors. Tenofovir treatment should be discontinued in any patient with clinical or laboratory findings suggestive of lactic acidosis or significant hepatotoxicity (signs of hepatotoxicity include hepatomegaly and steatosis, even if serum transaminase levels are not significantly elevated). Antiretroviral therapy has been reported to cause redistribution or accumulation of body fat, including central obesity, back and neck fat hyperplasia (buffalo hump), emaciation of the extremities, facial emaciation, breast enlargement, and Cushing's syndrome-like appearance. The most common adverse reactions in HIV-infected patients receiving tenofovir disoproxil fumarate include rash, diarrhea, headache, pain, depression, fatigue, and nausea. Nausea is the most common adverse reaction in HIV-infected patients receiving tenofovir disoproxil fumarate. For more complete data on drug warnings for tenofovir disoproxil fumarate (14 in total), please visit the HSDB record page.

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Pharmacodynamics
This drug inhibits DNA strand elongation required for viral replication in HIV-1 and hepatitis B virus infections by inhibiting enzymes necessary for host cell infection. In vitro effects The antiviral activity of tenofovir against laboratory and clinically isolated HIV-1 virus was studied in lymphoblastic cell lines, primary monocytes/macrophages, and peripheral blood lymphocytes. The EC50 (50% effective concentration) values of tenofovir against HIV-1 virus ranged from 0.04 μM to 8.5 μM. Combination Therapy of Tenofovir Disoproxil Flavescent Alcohol with Other Drugs Additive and synergistic effects have been observed in studies of combination therapy of tenofovir with nucleoside reverse transcriptase inhibitors (abacavir, didanosine, lamivudine, stavudine, zalcitabine, zidovudine), non-nucleoside reverse transcriptase inhibitors (delavirin, efavirenz, nevirapine), and protease inhibitors (ampenavir, indinavir, nelfinavir, ritonavir, saquinavir). Tenofovir has demonstrated antiviral activity against HIV-1 in cell culture.

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

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.00 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.00 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.00 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)