Relative Mitochondrial Toxicity of Tenofovir Alafenamide (TAF) vs. Tenofovir Disoproxil Fumarate (TDF)

Increased comorbidities such as cardiovascular disease (CVD), bone and kidney disease are emerging problems in HIV infection but the mechanisms are unclear. Understanding how antiretrovirals can minimize morbidity in treated HIV infection is a research priority. Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) are included in all HIV treatment regimens. Tenofovir (TFV) disoproxil fumarate (TDF) has been associated with an increased risk of nephrotoxicity and bone disease compared with other NRTIs. Tenofovir alafenamide (TAF) is an oral prodrug of TFV, but is much more stable in plasma as compared with TDF and markedly lower plasma levels of TFV are thought to lead to the favorable safety profile of TAF. Mitochondrial dysfunction has a key role in HIV pathogenesis and may be the common denominator that drives pathogenesis of several comorbidities. Despite the better safety profile of newer (such as TDF) compared to older NRTIs, there remain concerns for the potential for longer term toxicity of the current NRTIs especially since the exact cellular effects of NRTIs remain unclear. It is unknown whether a four-fold increase in intracellular drug levels seen in peripheral blood mononuclear cells (PBMCs) with TAF may increase toxicity in mitochondria. Better understanding of these effects could provide insights into mechanisms of HIV pathogenesis and selection of NRTIs that improve morbidity in chronic HIV infection.

Hypothesis: Despite higher intracellular levels, TAF has minimal/no overall mitochondrial toxicity compared to TDF in vivo. The investigators anticipate that the preliminary data and the proposed experimental approach will set the basis for future large scale studies to directly compare subtle potential mitochondrial toxicities of newer NRTIs in large HIV cohorts.

Primary Objective: This research application will explore the relative mitochondrial toxicity of newer NRTIs (TAF, TDF) as a possible mechanism for differential NTRI-related toxicities. These data will allow selection of NRTIs that may improve morbidity in chronic treated HIV infection, given the known possible role of mitochondrial dysfunction in pathogenesis of HIV-related comorbidities. Towards this aim, the investigators will use a robust experimental approach to study NRTI-related mitochondrial dysfunction using novel methods, human cell lines and PBMC. The specific aims of this project are: Aim 1: To evaluate the relative in vitro effects of TAF and TDF compared to an older NRTI (ddC) on 5 independent measures of mitochondrial function in the human cell line HepG2 and PBMC. Hypothesis: Despite higher intracellular levels, TAF has minimal/no overall mitochondrial toxicity compared to TDF in both the HepG2 line and PBMC. ddC will be used as a positive control for NRTI-induced mitochondrial dysfunction. Aim 2: To explore in vivo whether there is increased mitochondrial dysfunction with the use of TAF vs. TDF in chronic treated HIV infection. The overall goal of this proposal is to determine the in vitro and in vivo effects of TAF on mitochondria.

Treatment regimen: Healthy HIV-uninfected > 18 years old with no known comorbidities including metabolic disease will be recruited for PBMC isolation for the in vitro studies described in Aim 1 (n=30). For Aim 1, cells will be exposed to 10 μM (and other dose that will be determined after discussions with Gilead) NRTIs (TDF, TAF, ddC) for up to 14 days of treatment. To evaluate the relative mitochondrial toxicity of TDF vs. TAF in vivo (Aim 2), an open-label switch study will be conducted in virologically-suppressed, HIV-1 positive participants. The switch regimen will be Stribild® (elvitegravir 150 mg/cobicistat 150 mg/emtricitabine 200 mg/tenofovir disoproxil fumarate 300 mg; E/C/F/TDF) to Genvoya® (elvitegravir 150 mg/cobicistat 150 mg/emtricitabine 200 mg/tenofovir alafenamide 10 mg; E/C/F/TAF). For inclusion and exclusion criteria see respective sections.

Primary Endpoint: Overall, the above in vitro and in vivo measurements of mitochondrial function before and after TAF and TDF will test the hypotheses that newer NRTIs have minimal overall mitochondrial toxicity in humans. From all the measures of mitochondrial function, cellular oxygen consumption (COC; as determined by Seahorse technology) will be considered the "gold standard" given it is a measure of the most important mitochondrial function (oxidative phosphorylation) that relates to clinical outcome. Given the uncertainty of what measure of mitochondrial function can best predict NRTI-induced mitochondrial toxicity, comparison of relative fold induction of mitochondrial dysfunction in PBMC vs. HepG2 cells in response to a specific concentration of a known NRTI inducer of mitochondrial dysfunction (ddC) will be used to guide analysis. The baseline measurement will be used to control for confounders of NRTI-related mitochondrial dysfunction (each person will serve as his/her own control) and results will be expressed as relative-fold change compared to baseline. In addition, using similar approach, Aim 2 will explore whether switch from TDF to a TAF-based regimen will induce any changes in measures of mitochondrial function. This approach ensures that any changes in the mitochondrial function at 6 and/or 12 months will be secondary only to the NRTI switch.

Secondary Endpoints: It is accepted that two independent measures of mitochondrial function can more reliably predict mitochondrial dysfunction compared to each one of the measures separately. The investigators will define NRTI-induced mitochondrial toxicity as significant change in any of other major measures of mitochondrial toxicity [reduction in membrane potential (Δψm), increase in mitochondrial reactive oxygen species (mito-ROS), decrease in the amount of mitochondrial DNA (mtDNA), abnormal mitochondrial dynamics (increase fragmentation of mitochondria)). Finally, other exploratory secondary endpoints (based on available left-over resources) will be measurement of intracellular levels of TAF metabolites. Flow cytometry and live cell imaging will be determined as needed.

The investigators will define NRTI-induced mitochondrial toxicity as significant change in any of the measures of mitochondrial toxicity (COC, Δψm, mito-ROS, mtDNA, mitochondrial dynamics). The baseline measurement will be used to control for confounders of NRTI-related mitochondrial dysfunction (each person will serve as his/her own control) and results will be expressed as relative-fold change compared to baseline. In addition, using similar approach, Aim 2 will explore whether switch from TDF to a TAF-based regimen will induce any changes in measures of mitochondrial function. This approach ensures that any changes in the mitochondrial function at 6 and/or 12 months will be secondary only to the NRTI switch.

Since each donor will contribute samples to all experimental groups (baseline, follow up measurement), paired t-test, linear mixed effects model and nonparametric test (as needed) will be used to compare expression measures between groups adjusting for donor effects (age, sex, race.

The results from this research will yield useful information on NRTI-related overall mitochondrial dysfunction that can further be validated in large studies with data on clinical outcomes.