HIV-1/2 nucleotide reverse transcriptase

Tenofovir alafenamide hemifumarate (GS-7340 hemifumarate) exhibited comparable antiviral activity across all cell types, with CC50s ranging from 4.7 to 42 μM, respectively, for MT-4 and MT-2 cells. A panel of HIV-1 and HIV-2 isolates, comprising HIV-1 M group A to G subtypes and group N and O isolates, were used to assess TAF's antiviral activity. The average EC50 of 3.5 nM was found for TAF among the 29 main HIV-1 isolates tested in PBMC, while the average EC50 of 11.8 nM was found for AZT, the internal control. The average EC50 for AZT was 6.4 nM and for TAF was 1.8 nM for HIV-2 isolates [2].

The amidate prodrug of tenofovir, tenofovir alafenamide hemifumarate (GS-7340 hemifumarate), is comparable to tenofovir disoproxil fumarate. (TDF) has enhanced plasma stability and a favorable oral bioavailability profile [1].

Enzymatic assays. The rate of hydrolysis of amidate prodrugs of tenofovir with CatA was measured in a reaction buffer containing 25 mM morpholineethanesulfonic acid (MES) (pH 6.5), 100 mM NaCl, 1 mM dithiothreitol (DTT), 0.1% NP-40, and 1 μg/ml enzyme at 37°C. Inhibition of CatA-mediated TAF hydrolysis by protease inhibitors was performed by preincubation of the enzyme with serially diluted inhibitors for 10 min at 37°C. Ces1 and Ces2 (5 μg/ml) were assayed in 50 mM HEPES buffer (pH 7.2). The reactions were initiated by adding substrates to the reaction mixtures to the final concentration of 30 μM. At various time points, 100-μl aliquots were mixed with 200 μl of ice-cold methanol to stop the reactions. The samples were incubated at −20°C for 30 min and spun at 13,000 × g for 30 min at 4°C to remove denatured proteins. The supernatants were evaporated under vacuum, resuspended in 100 μl buffer A (25 mM potassium phosphate [pH 6.0], 5 mM tetrabutylammonium bromide) and injected onto a C18 reverse-phase column (5 μm, 2.1 by 100 mm, octadecyl silica 2 [ODS-2]) equilibrated with buffer A. The substrates and reaction products were eluted using a linear gradient of acetonitrile (0% to 65%, 10 min, 0.25 ml/min) in buffer A.[1]

---

Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 μM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4(+) T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF[1].

Tenofovir alafenamide (TAF) is an investigational oral prodrug of the HIV-1 nucleotide reverse transcriptase inhibitor tenofovir (TFV). Tenofovir disoproxil fumarate (TDF) is another TFV prodrug, widely used for the treatment of HIV-1 infection. TAF is converted mostly intracellularly to TFV and, in comparison to TDF, achieves higher tenofovir diphosphate (TFV-DP) levels in peripheral blood mononuclear cells. As a result, TAF has demonstrated potent anti-HIV-1 activity at lower doses than TDF in monotherapy studies. Here, the in vitro virology profile of TAF was evaluated and compared to that of TDF. TAF displayed potent antiviral activity against all HIV-1 groups/subtypes, as well as HIV-2. TAF exhibited minimal changes in the drug concentration needed to inhibit 50% of viral spread (EC50) upon removal of the prodrug, similar to TDF, demonstrating intracellular antiviral persistence. While TAF and TDF exhibited comparable potencies in the absence of serum pretreatment, TAF maintained activity in the presence of human serum, whereas TDF activity was significantly reduced. This result demonstrates TAF's improved plasma stability over TDF, which is driven by the different metabolic pathways of the two prodrugs and is key to TAF's improved in vivo antiviral activity. The activity of TAF is specific for HIV, as TAF lacked activity against a large panel of human viruses, with the exception of herpes simplex virus 2, where weak TAF antiviral activity was observed, as previously observed with TFV. Finally, in vitro combination studies with antiretroviral drugs from different classes showed additive to synergistic interactions with TAF, consistent with ongoing clinical studies with TAF in fixed-dose combinations with multiple other antiretroviral drugs for the treatment of HIV[2].

[1]. Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors. Antimicrob Agents Chemother. 2015 Oct 26;60(1):316-22.

[2]. In Vitro Virology Profile of Tenofovir Alafenamide, a Novel Oral Prodrug of Tenofovir with Improved Antiviral Activity Compared to That of Tenofovir Disoproxil Fumarate. Antimicrob Agents Chemother. 2015 Oct;59(10):5909-16.

Tenofovir alafenamide fumarate is a fumarate salt prepared from tenofovir alafenamide by reaction of one molecule of fumaric acid for every two molecules of tenofovir alafenamide. A prodrug for tenofovir, it is used in combination therapy for the treatment of HIV-1 infection. It has a role as an antiviral drug, a HIV-1 reverse transcriptase inhibitor and a prodrug. It contains a tenofovir alafenamide(1+).
Tenofovir alafenamide is an antiviral prescription medicine approved by the U.S. Food and Drug Administration (FDA) for the treatment of chronic hepatitis B virus infection (HBV) in adults and children 6 years of age and older who weigh at least 55 lb (25 kg) and who meet certain requirements, as determined by a health care provider.
HBV can be an opportunistic infection (OI) of HIV.
See also: Emtricitabine; Tenofovir Alafenamide Fumarate (component of) ... View More ...

---

Drug Indication
Vemlidy is indicated for the treatment of chronic hepatitis B (CHB) in adults and paediatric patients 6 years of age and older weighing at least 25 kg (see section 5. 1).

C46H62N12O14P2

1069.0195

1068.39836

C, 51.68; H, 5.85; N, 15.72; O, 20.95; P, 5.79

1392275-56-7

Tenofovir alafenamide;379270-37-8； 79270-38-9 (fumarate); 1392275-56-7 (hemifumarate)

71492247

White to off-white solid powder

7.601

6

24

26

74

799

6

O=C(O)/C=C/C(O)=O.NC1=NC=NC2=C1N=CN2C[C@@H](C)OC[P@](OC3=CC=CC=C3)(N[C@@H](C)C(OC(C)C)=O)=O.[0.5]

SVUJNSGGPUCLQZ-FQQAACOVSA-N

InChI=1S/2C21H29N6O5P.C4H4O4/c2*1-14(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)275-3(6)1-2-4(7)8/h2*5-9,11-12,14-16H,10,13H2,1-4H3,(H,26,29)(H2,22,23,24)1-2H,(H,5,6)(H,7,8)/b2-1+/t2*15-,16+,33+/m11./s1

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

GS734 GS-734 GS 734 GS 7340 GS-7340 GS7340 TAF Tenofovir alafenamide hemifumarate Tenofovir alafenamide fumarate (2

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~93.55 mM)
H2O : ~20 mg/mL (~37.42 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.68 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 25.0 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (4.68 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 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

View More

Solubility in Formulation 3: 2.5 mg/mL (4.68 mM) 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: 4.55 mg/mL (8.51 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

---

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