HIV-1/2 nucleotide reverse transcriptase

In the MTT experiment, tenofovir exhibits cytotoxic effects on HK-2 cell viability, with IC50 values of 2.77 μM at 48 and 72 hours, respectively. Tenofovir causes HK-2 cells' ATP levels to drop. In HK-2 cells, tenofovir (3.0 to 28.8 μM) elevates protein carbonylation and oxidative stress. Moreover, tenofovir causes HK-2 cells to undergo apoptosis, and this process is brought on by mitochondrial damage[1]. The replication of R5-tropic HIV-1BaL and X4-tropic HIV-1IIIb in activated PBMCs is inhibited by tenofovir and M48U1, when compounded in 0.25% HEC. Additionally, various laboratory strains and patient-derived HIV-1 isolates are inhibited. Infection with R5-tropic HIV-1BaL is inhibited by the synergistic antiretroviral action of M48U1 and tenofovir coupled in 0.25% HEC, and this formulation is not harmful to PBMCs[2].

When given to BLT mice (20, 50, 140, or 300 mg/kg), tenofovir Disoproxil Fumarate exhibits dose-dependent efficacy in response to a vaginal HIV challenge in BLT humanized mice. In BLT mice, tenofovir Disoproxil Fumarate (50, 140, or 300 mg/kg) dramatically lowers HIV transmission[3]. In woodchucks with a chronic WHV infection, tenofovir Disoproxil Fumarate (0.5, 1.5, or 5.0 mg/kg/day, po) causes a dose-dependent decrease in serum viremia. The administration of tenofovir Disoproxil Fumarate in the woodchuck model of chronic HBV infection is both safe and effective[4].

Tenofovir (TFV) is an antiviral drug approved for treating Human Immunodeficiency Virus (HIV) and Hepatitis B. TFV is administered orally as the prodrug tenofovir disoproxil fumarate (TDF) which then is deesterified to the active drug TFV. TFV induces nephrotoxicity characterized by renal failure and Fanconi Syndrome. The mechanism of this toxicity remains unknown due to limited experimental models. This study investigated the cellular mechanism of cytotoxicity using a human renal proximal tubular epithelial cell line (HK-2). HK-2 cells were grown for 48 h followed by 24 to 72 h exposure to 0-28.8 μM TFV or vehicle, phosphate buffered saline (PBS). MTT (MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and Trypan blue indicated that TFV diminished cell viability at 24-72 h. TFV decreased ATP levels at 72 h when compared to vehicle, reflecting mitochondrial dysfunction. TFV increased the oxidative stress biomarkers of protein carbonylation and 4-hydroxynonenol (4-HNE) adduct formation. Tumor necrosis factor alpha (TNFα) was released into the media following exposure to 14.5 and 28.8 μM TFV. Caspase 3 and 9 cleavage was induced by TFV compared to vehicle at 72 h. These studies show that HK-2 cells are a sensitive model for TFV cytotoxicity and suggest that mitochondrial stress and apoptosis occur in HK-2 cells treated with TFV.[1]

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Microbicides are considered a promising strategy for preventing human immunodeficiency virus (HIV-1) transmission and disease. In this report, we first analyzed the antiviral activity of the miniCD4 M48U1 peptide formulated in hydroxyethylcellulose (HEC) hydrogel in activated peripheral blood mononuclear cells (PBMCs) infected with R5- and X4-tropic HIV-1 strains. The results demonstrate that M48U1 prevented infection by several HIV-1 strains including laboratory strains, and HIV-1 subtype B and C strains isolated from the activated PBMCs of patients. M48U1 also inhibited infection by two HIV-1 transmitted/founder infectious molecular clones (pREJO.c/2864 and pTHRO.c/2626). In addition, M48U1 was administered in association with tenofovir, and these two antiretroviral drugs synergistically inhibited HIV-1 infection. In the next series of experiments, we tested M48U1 alone or in combination with tenofovir in HEC hydrogel with an organ-like structure mimicking human cervicovaginal tissue. We demonstrated a strong antiviral effect in absence of significant tissue toxicity. Together, these results indicate that co-treatment with M48U1 plus tenofovir is an effective antiviral strategy that may be used as a new topical microbicide to prevent HIV-1 transmission[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

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)

Solubility in Formulation 1: ≥ 0.77 mg/mL (2.68 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 7.7 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: ≥ 0.77 mg/mL (2.68 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 7.7 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: ≥ 0.77 mg/mL (2.68 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 7.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 1.96 mg/mL (6.82 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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