Tenofovir alafenamide hemifumarate

Cell lines

MT-2 cells

Preparation Method

MT-2 cells (2.4 million) were incubated with virus for 3h at 37˚C in 1mL screw-cap tubes. Virus was normalized to yield a signal-to-noise ratio (uninfected cells to infected cells) between 4 to 7, which was equivalent to a multiplicity of infection (MOI) ~0.003 for wild-type HIV-1 IIIB. Five-fold Tenofovir alafenamide hemifumarate gradient dilutions (0.01nM-1µM) were transferred in triplicate to the inside wells of 96-well plates (50µL per well). After incubation, infected MT-2 cells were seeded at 8500 per well in 100µL of media. After incubation for 5 days at 37˚C, 100µL of CellTiterGlo reagent was added to each well of the assay plates and luminescence was measured using an Envision plate reader.

Reaction Conditions

0.01nM-1µM; 5 days

Applications

Treatment of MT-2 cells with Tenofovir alafenamide hemifumarate showed potent antiviral activity in vitro with EC₅₀ values of 5nM.

Animal models

Naive Male Sprague-Dawley rats

Preparation Method

Naive male Sprague-Dawley rats were singly housed in polycarbonate caging with free access to food and water for the duration of the study, unless otherwise noted. At the time of dose initiation, animals were 11 to 14 weeks old and weighed between 372 and 549g. After an acclimation period of at least 7 days, rats were surgically implanted with a s.c. cannula while under isoflurane anesthesia. Cannulas were routed dorsally through the s.c. tissue extending from the lumbar region to an exteriorization site at the base of the neck. The cannulas were tethered through a swivel system outside the cage, and jackets were placed on the rats to hold the tethered system in place. Saline infusions were maintained at a rate of 0.04ml/h until study initiation, which occurred after a recovery period of at least 10 days. Prior to study initiation, animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. The study consisted of five treatment groups, with each group receiving Tenofovir alafenamide hemifumarate at a dose of 0, 30, 100, 300, or 1,000mg/kg/day. Within each treatment group, five animals were assigned to the main study group, and six animals were assigned to the recovery group. Each treatment group also contained two separate satellite groups: one group of six animals for Histopathology analysis and one group of eighteen animals for PBMC sample collection.

Dosage form

0-1000mg/kg/day; 28 days; subcutaneous infusion

Applications

Tenofovir alafenamide hemifumarate resulted in increased incidence and severity of fibrosis and mononuclear cell inflammation at the infusion site in male rats.

References:
[1] Callebaut C, Stepan G, Tian Y, Miller MD. 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;59(10):5909-5916.
[2] Zane D, Roller S, Shelton J, et al. A 28-Day Toxicity Study of Tenofovir Alafenamide Hemifumarate by Subcutaneous Infusion in Rats and Dogs. Microbiol Spectr. 2021;9(1):e0033921.