HIV-1/2 nucleotide reverse transcriptase
Tenofovir Alafenamide (GS-7340) targets HIV reverse transcriptase (EC50 = 0.01 μM in HIV-1-infected MT-4 cells) [2]
Tenofovir Alafenamide (GS-7340) inhibits HIV reverse transcriptase via its active metabolite tenofovir diphosphate (TFV-DP), which competes with deoxyadenosine triphosphate (dATP) for incorporation into viral DNA (Ki = 0.008 μM for HIV-1 reverse transcriptase) [1]

The antiviral activity of tenofovir alafenamide (GS-7340) ranged from 5 to 7 nM in all cell types, although the CC50 for MT-4 and MT-2 cells varied from 4.7 to 42 μM. A panel of HIV-1 and HIV-2 isolates, comprising HIV-1 M group subtypes A to G and group N and O isolates, were used to assess the antiviral activity of TAF. The average TAF EC50 was 3.5 nM, with a range of 0.1 to 12 nM. In contrast, the average EC50 of AZT, the internal control, was 11.8 nM for 29 main HIV-1 strains evaluated in PBMC. TAF's average EC50 for HIV-2 isolates is 1.8 nM, while AZT's average EC50 is 6.4 nM [2].

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Tenofovir Alafenamide (GS-7340) exhibited potent antiviral activity against HIV-1 (subtype B) in primary human CD4+ T cells with an EC50 of 0.009 μM [2]
Tenofovir Alafenamide (GS-7340) showed low cytotoxicity in human peripheral blood mononuclear cells (PBMCs) with a CC50 > 10 μM, resulting in a selectivity index (SI) > 1111 [2]
Tenofovir Alafenamide (GS-7340) was efficiently taken up by lymphoid cells, with intracellular TFV-DP concentrations reaching 1200 pmol/10^6 cells after 24 h incubation with 1 μM drug [1]
Tenofovir Alafenamide (GS-7340) demonstrated antiviral activity against HIV-1 resistant to tenofovir disoproxil fumarate (TDF) in MT-4 cells, with an EC50 of 0.012 μM [2]
Tenofovir Alafenamide (GS-7340) was metabolized to TFV-DP in lymphoid cell lines (Jurkat and CEM) with a half-life of intracellular TFV-DP of 35 h [1]

When compared to Tenofovir disoproxil fumarate (TDF), Tenofovir alafenamide (GS-7340) hemifumarate, the amidate prodrug of Tenofovir, exhibits better oral bioavailability and plasma stability [1].

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Tenofovir Alafenamide (GS-7340) reduced HIV-1 viral load by a mean of 1.6 log10 copies/mL in HIV-1-positive adults after 10 days of oral monotherapy at 25 mg once daily [2]
Tenofovir Alafenamide (GS-7340) achieved higher TFV-DP concentrations in lymphoid tissues (spleen, lymph nodes) of mice than TDF, with a 3-fold higher concentration in splenic lymphocytes after oral administration of 10 mg/kg [1]
Tenofovir Alafenamide (GS-7340) showed sustained antiviral efficacy in mice implanted with HIV-infected human PBMCs, with viral load suppression lasting 7 days after the last dose [1]

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.

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HIV reverse transcriptase inhibition assay: Prepare a reaction mixture containing recombinant HIV-1 reverse transcriptase, poly(rC)-oligo(dG) as template-primer, and [3H]-dGTP. Incubate with serial dilutions of Tenofovir Alafenamide (GS-7340) or its metabolite TFV-DP at 37°C for 90 min. Terminate the reaction with EDTA, filter through DEAE-cellulose filters, and measure radioactivity to determine enzyme inhibition efficiency [1]
Nucleotide incorporation assay: Set up a primer extension reaction with HIV-1 reverse transcriptase, a DNA template-primer, and a mixture of dNTPs including [α-32P]-dATP. Add TFV-DP (metabolite of Tenofovir Alafenamide (GS-7340)) at various concentrations, run the reaction at 30°C for 60 min, separate products by denaturing polyacrylamide gel electrophoresis, and autoradiograph to analyze chain termination [1]

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.

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HIV-1 antiviral cell assay: Seed primary human CD4+ T cells in 96-well plates at 2×105 cells/well and infect with HIV-1 (MOI = 0.05). Add Tenofovir Alafenamide (GS-7340) at concentrations ranging from 0.001 to 10 μM and incubate for 7 days. Measure viral p24 antigen levels by ELISA to calculate EC50, and assess cell viability via MTT assay to determine CC50 [2]
Intracellular metabolite detection assay: Culture Jurkat cells at 1×106 cells/mL and treat with 1 μM Tenofovir Alafenamide (GS-7340) for 0–48 h. Harvest cells, extract intracellular nucleotides with perchloric acid, neutralize with potassium hydroxide, and separate TFV-DP by high-performance liquid chromatography (HPLC) with UV detection [1]
Lymphoid cell uptake assay: Incubate human PBMCs with [3H]-labeled Tenofovir Alafenamide (GS-7340) at 37°C for 2–24 h. Wash cells three times with cold phosphate-buffered saline (PBS), lyse with detergent, and measure radioactivity in the lysate to quantify cellular uptake [1]

10 mg/kg, p.o.
Beagle dogs 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, (22) 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.
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.
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.

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Mouse lymphoid tissue distribution assay: Female BALB/c mice (6–8 weeks old) are administered Tenofovir Alafenamide (GS-7340) via oral gavage at doses of 1, 10, or 30 mg/kg. The drug is formulated in 0.5% methylcellulose. Blood, spleen, and mesenteric lymph nodes are collected at 1, 4, 8, and 24 h post-administration. Tissue samples are homogenized, and TFV-DP concentrations are measured by LC-MS/MS [1]
HIV mouse model efficacy assay: Immunodeficient NSG mice are intravenously injected with HIV-1-infected human PBMCs. Two days post-infection, mice receive oral Tenofovir Alafenamide (GS-7340) at 10 mg/kg once daily for 14 days. Blood samples are collected every 3 days to measure viral load by RT-PCR, and spleen samples are harvested at study end to detect intracellular TFV-DP [1]

Absorption, Distribution and Excretion
Compared to the parent molecule [tenofovir], tenofovir alafenamide possesses a lipophilic group that masks the negative charge of the parent moiety, thereby improving its oral bioavailability. Tenofovir alafenamide is highly stable in plasma, and plasma concentrations of tenofovir are low after administration of this prodrug. Following oral administration, tenofovir alafenamide is rapidly absorbed from the intestine. Two hours after a single dose, a peak concentration of the parent compound of 16 ng/ml, approximately 73% of the administered dose, was observed, with an AUC of 270 ng/ml. Upon entering the body, tenofovir alafenamide enters hepatocytes via passive diffusion, a process regulated and activated by organic anion transporters 1B1 and 1B3. Concomitant administration of tenofovir alafenamide with a high-fat meal increases its in vivo exposure by approximately 65%.
Approximately 47% of the registered tenofovir alafenamide is excreted bile, and approximately 36% is excreted renally. Of the drug recovered in urine, approximately 75% is the parent drug (tenofovir), the remainder being uric acid and trace amounts of tenofovir alafenamide. On the other hand, 99% of the drug recovered in feces is tenofovir.
In clinical trials, the volume of distribution of tenofovir alafenamide was greater than 100 liters.
The clearance of tenofovir alafenamide is 117 L/h. In patients with severe renal impairment, this value can be reduced by 50%, with a reported rate of 61.7 L/h.
Metabolites/Metabolites
Tenofovir alafenamide requires hydrolysis by cathepsin A or carboxylesterase 1 to the parent compound [tenofovir] for activation. Tenofovir alafenamide has significant plasma stability, so its activation occurs within target cells. After activation, tenofovir is further metabolized and almost completely converted to uric acid after 1-2 days, and is detected in plasma.

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Biological half-life
The half-life of tenofovir alafenamide has been reported to be 0.51 hours.

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The oral bioavailability of tenofovir alafenamide (GS-7340) in mice is 39%[1].
Tenofovir alafenamide (GS-7340) is rapidly absorbed in humans, with a peak plasma concentration (Cmax) of 25.3 ng/mL and a median time to peak concentration (Tmax) of 0.5 hours after oral administration of 25 mg[2].
Tenofovir alafenamide (GS-7340) has an area under the plasma concentration-time curve (AUC0-24h) of 37.3 when administered once daily at 25 mg, with a plasma concentration of ng·h/mL [2]
Tenofovir alafenamide (GS-7340) is widely distributed in lymphoid tissues, with a concentration ratio of 12:1 in mouse spleen to plasma [1]
Tenofovir alafenamide (GS-7340) has a plasma elimination half-life (t1/2) of 0.51 hours in humans [2]
Tenofovir alafenamide (GS-7340) is metabolized in the liver and lymphocytes by carboxylesterase 1 (CES1) and cathepsin A, releasing tenofovir (TFV), which is further phosphorylated to active TFV-DP [1]
The renal excretion of tenofovir alafenamide (GS-7340) is 28% of the administered dose. In humans, 1.3% is excreted unchanged.[2]

Protein binding
It has been reported that tenofovir alafenamide can bind to plasma proteins, and in vitro studies have shown that about 80% of the administered dose of the drug exists in a bound state.

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Tenofovir alafenamide (GS-7340) did not show significant cytotoxicity against human liver cancer cells (HepG2) at concentrations up to 100 μM[1].
In HIV-1 positive adults, oral administration of 25 mg tenofovir alafenamide (GS-7340) once daily for 10 days was well tolerated with no grade 3-4 adverse events reported; the most common mild adverse event was headache (8% of subjects)[2]
Tenofovir alafenamide (GS-7340) did not cause significant changes in serum creatinine, alanine aminotransferase (ALT), or aspartate aminotransferase (AST) levels in human subjects[2]
Tenofovir alafenamide (GS-7340) had a plasma protein binding rate of 82%–86% in humans[2]
Tenofovir alafenamide (GS-7340) did not show any effect on cytochrome P450 in vitro. Inhibition of enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4) [2]

[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.

Pharmacodynamics
Tenofovir alafenamide has been shown to be a potent inhibitor of hepatitis B virus replication. It exhibits better renal tolerability compared to its class of drugs, tenofovir disoproxil fumarate. This improved safety profile appears to be related to lower plasma concentrations of tenofovir. In clinical trials, tenofovir alafenamide showed a 5-fold increase in antiviral activity against HIV-1 compared to tenofovir disoproxil fumarate. Tenofovir alafenamide (GS-7340) is a nucleotide prodrug of tenofovir designed to enhance its delivery to lymphocytes, a major target of HIV infection [1]. Tenofovir alafenamide (GS-7340) exerts its antiviral effect by conversion to TFV-DP, which inhibits HIV reverse transcriptase by terminating viral DNA synthesis [1].
In a phase 1b clinical trial, 10 days of once-daily 25 mg tenofovir alafenamide (GS-7340) monotherapy resulted in a reduction in viral load in 92% of HIV-1 positive subjects [2].
Compared to tenofovir disoproxil fumarate (TDF), GS-7340 showed better lymphatic penetration, thereby reducing systemic exposure and potentially reducing nephrotoxicity [1]

C21H29N6O5P

476.47

476.193

C, 52.94; H, 6.13; N, 17.64; O, 16.79; P, 6.50

379270-37-8

Tenofovir alafenamide fumarate;379270-38-9;Tenofovir alafenamide hemifumarate;1392275-56-7

9574768

White to off-white solid

1.39±0.1 g/cm3

640.4±65.0 °C at 760 mmHg

104-107 ºC

341.1±34.3 °C

0.0±1.9 mmHg at 25°C

1.630

2.2

2

10

12

33

680

3

[P@](C([H])([H])OC([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]

LDEKQSIMHVQZJK-AZFZMOAFSA-N

InChI=1S/C21H29N6O5P/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/h5-9,11-12,14-16H,10,13H2,1-4H3,(H,26,29)(H2,22,23,24)/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

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.37 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 (4.37 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 (4.37 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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Solubility in Formulation 4: 40 mg/mL (83.95 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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