HIV-1/2 nucleotide reverse transcriptase
The active moiety, tenofovir, targets HIV-1 and HIV-2 reverse transcriptase, an enzyme critical for the replication of retroviruses. By inhibiting this enzyme, tenofovir prevents the virus from converting its RNA into DNA, thereby blocking viral replication.

Tenofovir alafenamide fumarate (GS-7340 fumarate) exhibited comparable antiviral activity across all cell types, with a CC50 ranging from 4.7 to 42 μM for MT-4 and MT-2 cells, respectively. The fumarate varied from 5 to 7 nM. 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].
In vitro, Tenofovir alafenamide fumarate exhibits potent antiviral activity against a panel of HIV-1 and HIV-2 isolates, including various subtypes. Its antiviral activities are similar across all cell types, ranging from 5 to 7 nM.

The amidate prodrug of tenofovir is called GS-7340 fumarate, or tenofovir alafenamide fumarate. It has better plasma stability and good oral bioavailability when compared to tenofovir disoproxil fumarate (TDF) [1].
In vivo, the prodrug is designed to deliver tenofovir more efficiently to target cells, particularly lymphocytes and macrophages, resulting in higher intracellular concentrations of the active drug with lower systemic exposure. It has good oral bioavailability and increased plasma stability compared to tenofovir disoproxil fumarate (TDF).

Intestinal and Hepatic S9 Stability[1]
GS-7340 was incubated at 10 μM with either dog or human intestinal and hepatic S9 fractions for 120 min at 37 °C in a 96-well plate format. At specified time points following compound addition, samples were quenched with 9 volumes of an aqueous solution containing 25% acetonitrile and 50% methanol. Plates were centrifuged at 3000g for 30 min, and 10 μL of the resulting solution was analyzed by LC–MS/MS. Data (analyte to internal standard peak area ratio) were plotted on a semi log scale and fitted using an exponential fit. Assuming first order kinetics, the half-life and rate of metabolism were determined. Predicted hepatic extraction was calculated from the half-life by reported methods using the well-stirred model for hepatic clearance.[1]

---

Plasma Stability[1]
GS-7340 was incubated at 2 μM in either dog or human plasma for 4 h at 37 °C. At specified time points, an aliquot from the incubation was quenched by addition of 9 volumes of 100% acetonitrile and internal standard. Following the last collection, samples were centrifuged at 3000g for 30 min and supernatants were transferred to a new plate containing an equal volume of water for analysis by liquid chromatography coupled to triple quadrupole mass spectrometry (LC–MS/MS).
The in vitro antiviral assay measures the inhibition of HIV-1 replication in cell culture. Cells are infected with HIV-1 in the presence of varying concentrations of the compound, and viral replication is quantified by measuring the production of viral proteins or viral RNA.

Caco-2 Permeability[1]
Bidirectional permeability studies were done using confluent monolayers of the human colon carcinoma cell line caco-2 seeded in 12-well plates as previously reported. The effects of either concentration (10, 100, or 1,000 μM) or efflux transporter inhibition on the permeation of GS-7340 were studied. Effect of the inhibition of efflux transporters including P-glycoprotein (Pgp) was assessed following a 30 min preincubation of cell monolayers with 10 μM cyclosporin A (CsA) in transport buffer to allow for saturation of transporter binding sites. Following preincubation, fresh assay buffer containing CsA and GS-7340 was added and the assay was started. Each determination was performed in duplicate, and the permeability of control compounds (atenolol, propranolol, and digoxin) was determined to meet acceptance criteria for each batch of assay plates.
In vitro cell-based assays are performed in various cell lines, including MT-4 and MT-2 cells, to determine the antiviral activity (IC50) and cytotoxicity (CC50) of the compound. The IC50 values typically range from 5 to 7 nM, while the CC50 varies from 4.7 to 42 µM.

Animals[1]
Male beagle dogs between the ages of 6 and 18 months were used for the in life portion of this study. The animals were housed in accordance with the standards of the American Association for Accreditation of Laboratory Animal Care and were receiving a standard commercial diet. Animals were handled in strict accordance with the Guide for the Care and Use of Laboratory Animals, and the protocol was reviewed by the Institutional Animal Care and Use Committee at SRI International. Animals were between 7 and 11 kg at dosing.[1]

---

Drug Administration[1]
For intravenous administration, GS-7340 was formulated in 5% dextrose in water. For oral administration, GS-7340 was formulated in 50 mM citric acid (pH 5.0) at doses of 2 to 10 mg/kg. For the 20 mg/kg dose, GS-7340 was formulated in 50 mM citric acid (pH 5.5) with 0.1% Polysorbate 20. To test the effect of efflux transport inhibition, dogs were administered 2 mg/kg GS-7340 1 h following pretreatment with a 75 mg capsule of CsA.[1]

---

Plasma and PBMC Sample Collection[1]
Blood (approximately 0.5 mL) was collected at specified time points over 24 h from the jugular vein (intraportal vein infusion and oral pharmacokinetic studies), the jugular and portal veins (oral administration to portal vein cannulated dogs), or the cephalic vein (jugular vein infusion). Plasma was isolated in Vacutainer tubes containing EDTA as an anticoagulant by centrifugation. At select time points (1, 4, 8, and 24 h postdose) in the 5 mg/kg oral pharmacokinetic study, 8 to 10 mL of blood was collected into Vacutainer cell preparation tubes (Becton Dickinson) for isolation of peripheral blood mononuclear cells (PBMC) and processed following manufacturer instructions. Small aliquots (10 μL) of isolated PBMC pellets diluted in 0.9% NaCl were maintained at room temperature and used to determine cell count. 500 μL of 70% methanol was added to the remaining PBMC pellets and, together with plasma samples, stored at −80 °C until shipment for further processing and analysis.
In vivo efficacy is evaluated in animal models of HIV or HBV infection and in clinical trials. The prodrug is administered orally, and its effect on viral load is measured. Its improved pharmacokinetic profile allows for lower doses and reduced side effects.

Tenofovir alafenamide is an orally bioavailable prodrug. It is rapidly absorbed and converted to tenofovir intracellularly. This results in higher intracellular concentrations of the active drug in target cells and lower plasma levels, reducing the risk of renal and bone toxicity compared to TDF.

The toxicity profile of Tenofovir alafenamide is improved compared to tenofovir disoproxil fumarate. It has a lower risk of renal impairment and bone mineral density loss, which are known side effects of TDF. It is generally well-tolerated in clinical use.

[1]. Mechanism for effective lymphoid cell and tissue loading following oral administration of nucleotide prodrug GS-7340. Mol Pharm. 2013 Feb 4;10(2):459-66.

[2]. Antiviral activity, safety, and pharmacokinetics/pharmacodynamics of tenofovir alafenamide as 10-day monotherapy in HIV-1-positive adults. J Acquir Immune Defic Syndr. 2013 Aug 1;63(4):449-55.

Tenofovir alafenamide is an antiviral prescription drug approved by the U.S. Food and Drug Administration (FDA) for the treatment of chronic hepatitis B virus (HBV) infection in adults and children aged 6 years and older, weighing at least 25 kg (55 lbs), and meeting certain criteria determined by a healthcare provider. HBV may be an opportunistic infection (OI) of HIV.
Drug Indications
Vemlidy is indicated for the treatment of chronic hepatitis B (CHB) in adults and children aged 6 years and older, weighing at least 25 kg (see Section 5.1).
Tenofovir alafenamide fumarate is an FDA-approved drug for the treatment of HIV-1 infection and chronic hepatitis B. It is marketed under the brand name Vemlidy for hepatitis B and is a component of several fixed-dose combination products for HIV, such as Genvoya and Descovy.

C25H33N6O9P

593.55

592.204

C, 50.68; H, 5.61; N, 14.18; O, 24.30; P, 5.23

379270-38-9

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

68516365

White to off-white solid powder

3.656

4

14

14

41

799

3

[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)(N([H])[C@]([H])(C(=O)OC([H])(C([H])([H])[H])C([H])([H])[H])C([H])([H])[H])(=O)OC1C([H])=C([H])C([H])=C([H])C=1[H].O([H])C(/C(/[H])=C(\[H])/C(=O)O[H])=O

MEJAFWXKUKMUIR-WIUYAKJJSA-N

InChI=1S/C21H29N6O5P.C4H4O4/c1-14(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)27;5-3(6)1-2-4(7)8/h5-9,11-12,14-16H,10,13H2,1-4H3,(H,26,29)(H2,22,23,24);1-2H,(H,5,6)(H,7,8)/b;2-1+/t15-,16+,33?;/m1./s1

(S)-isopropyl 2-(((S)-((((R)-1-(6-amino-9H-purin-9-yl)propan-2-yl)oxy)methyl)(phenoxy)phosphoryl)amino)propanoate fumarate

GS734; GS-734; GS 734; GS 7340; GS-7340; GS7340; TAF; Tenofovir alafenamide fumarate; GS-7340 fumarate; 379270-38-9; GS-7340 monofumarate; H2S5S51WW6; GS-7340 (fumarate); GS-7339 monofumarate; L-Alanine, N-((S)-(((1R)-2-(6-amino-9H-purin-9-yl)-1-methylethoxy)methyl)phenoxyphosphinyl)-, 1-methylethyl ester, (2E)-2-butenedioate (1:1); trade name: Genvoya.

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 : ≥ 36 mg/mL (~60.76 mM)
H2O : ≥ 25 mg/mL (~42.19 mM)

Solubility in Formulation 1: 6.67 mg/mL (11.26 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

---

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