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  53rd ICAAC Interscience Conference on
Antimicrobial Agents and Chemotherapy
September 10-13, 2013, Denver CO
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TAF, a Tenofovir Prodrug, Not Preferentially Loaded or Toxic in Osteoblasts
  53rd ICAAC, September 10-13, 2013, Denver
Mark Mascolini
Tenofovir alafenamide (TAF), an investigational prodrug of tenofovir, did not accumulate in primary osteoblasts (bone-forming cells) more than in peripheral blood mononuclear cells (PBMCs) and had no cytotoxic effects in osteoblasts at concentrations that would be used in humans [1]. The findings are in line with minimal bone mineral density (BMD) changes seen in phase 2 trials of TAF with elvitegravir, cobicistat, and emtricitabine [2,3].
Certain antiretrovirals, particularly tenofovir disoproxil fumarate (TDF) and protease inhibitors (PIs), are linked to decreasing BMD. TAF, an oral prodrug of tenofovir, delivers more tenofovir diphosphate (TFV-DP, the active form of the drug) to PBMCs than does TDF and has greater antiviral activity than TDF in clinical studies. Because TAF also yields about 90% lower tenofovir levels in plasma than TDF, there is hope that this new agent will be less toxic in humans.
Gilead Sciences investigators conducted the studies described here with several objectives in mind: (1) to establish clinically relevant TAF concentrations in PBMCs in vitro that result in TFV-DP levels comparable to those observed in vivo, (2) to compare TFV-DP levels in primary human osteoblasts to TFV-DP levels in PBMCs with equivalent TAF exposures, and (3) to evaluate the effect of clinically relevant TAF concentrations on TFV-DP formation on primary human osteoblast growth.
More than 95% of TAF gets eliminated from plasma 2 hours after dosing. To mimic that process, the researchers pulsed TAF into PBMCs and primary human osteoblasts for 2 hours, followed by a washout. They measured TFV-DP in cells collected at multiple points after dosing. The investigators conducted PBMC loading experiments with multiple TAF concentrations to find the concentration that results in intracellular TFV-DP levels similar to those seen in vivo (677 nM). They then evaluated similar TAF concentrations in primary osteoblasts. Next the Gilead team developed a primary osteoblast cell growth assay and evaluated TFV-DP levels after single and multiple TAF pulses. They assessed cell viability after treating primary osteoblasts with TAF for 3 days.
A 2-hour TAF pulse in PBMCs at concentrations from 124 to 370 nM yielded TFV-DP levels comparable to those seen in vivo with 25 mg of TAF, which results in a TAF maximum concentration (Cmax) of 484 nM. In primary osteoblasts, a single 2-hour pulse of the same TAF concentrations yielded TFV-DP levels comparable to those reached in PBMCs. Three days of daily 2-hour TAF pulses at 200 nM yielded similar TFV-DP levels.
The Gilead team saw no change in cell viability of primary osteoblasts exposed to clinically relevant TAF concentrations. The 50% cytotoxic concentration (CC50), a standard measure of cytotoxicity, was greater than 500 uM with the pulse method, which is more than 1033 times higher than TAF plasma Cmax (484 nM). For comparison, average CC50s for nelfinavir and lopinavir are 23.5 and 33.5 uM, or 3.4 and 1.8 times higher than their average plasma Cmax values (or about 34 and 18 times higher after adjustment for protein binding).
The investigators concluded that "primary osteoblasts were not preferentially loaded by TAF relative to PBMCs." As a result, intracellular levels of TFV-DP (the active form of tenofovir) are comparable in PBMCs (about 0.677 uM) and osteoblasts (0.395 uM). Furthermore, TAF concentrations similar to those given to humans were not toxic to osteoblasts. These findings could explain the minimal changes in bone mineral density seen in clinical trials of TAF so far [2,3].
1. Liu Y, Kitrinos K, Babusis D, et al. Lack of tenofovir alafenamide (TAF) effect on primary osteoblasts in vitro at clinically relevant drug concentrations. 53rd ICAAC. September 10-13, 2013. Denver. Abstract H-664.
2. Zolopa A, Ortiz R, Sax P, et al. Comparative study of tenofovir alafenamide vs tenofovir disoproxil fumarate, each with elvitegravir, cobicistat, and emtricitabine, for HIV treatment. 20th Conference on Retroviruses and Opportunistic Infections. March 3-6, 2013. Atlanta. Abstract 99LB. http://www.natap.org/2013/CROI/croi_23.htm
3. Sax P, Brar I, Elion R, et al. 48 week study of tenofovir alafenamide (TAF) vs. tenofovir disoproxil fumarate (TDF), each in a single tablet regimen with elvitegravir, cobicistat, and emtricitabine for initial HIV treatment. 53rd ICAAC. September 10-13, 2013. Denver. Abstract H-1464d.