Tenofovir alafenamide (TAF) intracellular activation and tenofovir distribution to HIV-1 infected cells

Abstract

Tenofovir alafenamide undergoes intracellular activation primarily through the lysosomal protease cathepsin A, which cleaves TAF to form intermediates that ultimately convert to the active metabolite tenofovir diphosphate (TFV-DP). Liver carboxylesterase 1 provides an alternative hepatic activation pathway. This intracellular activation mechanism enables TAF to achieve 4- to 25-fold higher TFV-DP concentrations in peripheral blood mononuclear cells compared to tenofovir disoproxil fumarate (TDF), while reducing systemic tenofovir exposure by 86-97%. TAF demonstrates preferential distribution to lymphatic tissues and accumulates in epithelial cells and fibroblasts of the female reproductive tract at 10- to 100-fold higher concentrations than in CD4+ T cells, with these cells releasing antiretroviral activity over multiple days to protect CD4+ T cells.

The enhanced intracellular TFV-DP loading translates to superior antiviral activity, with TAF demonstrating >600-fold greater potency than parent tenofovir in CD4+ T cells and a 3.5-fold higher resistance barrier than TDF. In viral breakthrough assays at physiologically relevant concentrations, TAF inhibited 40 of 42 K65R-containing isolates while TDF inhibited only 32 of 42, and TAF prevented breakthrough in 65 of 68 isolates with thymidine analog-associated mutations compared to 53 of 68 for TDF. The covalent hepatitis C protease inhibitors telaprevir and boceprevir potently inhibit cathepsin A-mediated TAF activation (IC50 0.16-0.27 μM), reducing anti-HIV activity 3- to 21-fold, while medroxyprogesterone acetate differentially suppresses TAF activity in genital versus blood CD4+ T cells. These findings support TAF’s clinical efficacy at lower doses than TDF with improved safety profiles and activity against resistant HIV-1 strains.

Results

Characteristics of Included Studies

The review included 10 studies published between 2013 and 2023 examining TAF intracellular activation and tenofovir distribution to HIV-1 infected cells. Eight studies had full text available, while two were assessed using abstract-only information.

Study Characteristics

Studies employed diverse experimental approaches including enzyme assays, drug uptake studies, resistance selection experiments, viral breakthrough assays, and clinical pharmacokinetic measurements. Experimental durations ranged from 24-hour incubations to multi-week resistance selection experiments lasting up to 74 days and a 10-day clinical monotherapy trial.

TAF Activation Mechanisms

Enzymatic Pathways

TAF undergoes intracellular activation primarily through the lysosomal protease cathepsin A (CatA). The activation occurs in the lysosomal compartment, where CatA cleaves the carboxyester bond in TAF to form a metastable metabolite. This metabolite then converts to TFV-Ala, which spontaneously converts to TFV due to the acidic pH in lysosomes. Subsequently, TFV undergoes two sequential phosphorylation reactions to form the active metabolite TFV-DP, involving adenylate kinase for the first phosphorylation step and a kinase with nucleotide diphosphate kinase activity for the second step.

Alternative Activation Pathways

Liver carboxylesterase 1 (Ces1) represents an alternative pathway for TAF activation. Overexpression of Ces1 in HEK293T cells increased intracellular TAF hydrolysis 5-fold, and Ces1 is specifically important for the hepatic metabolism of TAF. TAF is converted to TFV by either CatA or Ces1 depending on the cell type, followed by intracellular phosphorylation to TFV-DP. This unique activation mechanism, distinct from TDF, contributes to TAF’s enhanced antiviral activity.

Intracellular Drug Concentrations and Distribution

Comparative TFV-DP Levels

TAF achieves substantially higher intracellular concentrations of the active metabolite TFV-DP compared to its predecessor TDF. The fold increases in TFV-DP concentrations varied across studies and doses:

TFV-DP Fold Increase Comparison

In blood and female reproductive tract (FRT) CD4+ T cells, intracellular TFV-DP concentrations were approximately 1-2 × 10^4 fmol/million cells after 24 hours of incubation with TFV or TAF.

Tissue-Specific Distribution

TAF demonstrates preferential distribution to lymphatic tissues and HIV-susceptible cells. In the female reproductive tract, epithelial cells and fibroblasts accumulate TFV-DP at substantially higher levels than CD4+ T cells, with epithelial cells showing 100-fold higher concentrations and fibroblasts showing 10-fold higher concentrations. Endometrial, endocervical, and ectocervical polarized epithelial cells pre-loaded with TFV or TAF released antiretroviral activity into their basolateral secretions 1, 2, and 3 days post-loading, providing sustained protection to CD4+ T cells. When TAF was added apically to epithelial cells, it was released basolaterally through Multidrug Resistant Protein transporters, taken up by fibroblasts, and released into secretions to partially protect CD4+ T cells.

Donor Variability

Levels of CatA and intracellular TAF metabolites differed minimally in CD4+ T cells and monocyte-derived macrophages among 13 demographically diverse donors tested. TAF’s relative range of antiviral activity across all tested donors was comparable to that of other HIV-1 reverse transcriptase inhibitors, with mean ±SD (range) EC50 values of 11.0 ±3.4 (6.6–19.9) nM in CD4+ T cells and 9.7 ±4.6 (2.5–15.7) nM in monocyte-derived macrophages.

Comparison with Tenofovir Disoproxil Fumarate

Potency and Activation Efficiency

TAF demonstrated markedly superior potency compared to parent TFV and the alternative prodrug TDF across multiple dimensions:

TAF Potency Comparison

This enhanced potency stems from TAF’s more efficient intracellular conversion to TFV-DP, allowing for lower dosing requirements. In the phase 1b monotherapy study, TAF at 25 mg demonstrated greater antiviral effect at less than 1/10th the dose of TDF.

Mechanistic Differences

TAF and TDF employ fundamentally different activation pathways. TAF is converted intracellularly by CatA or Ces1, whereas TDF is quickly metabolized to TFV by gut and serum esterases. This distinction confers TAF with superior plasma stability. While TAF maintained activity in the presence of human serum, TDF activity was significantly reduced, demonstrating TAF’s improved plasma stability over TDF.

Pharmacokinetic Profiles

The intracellular versus systemic exposure profiles strongly favor TAF for safety and efficacy:

Pharmacokinetic Comparison

This pharmacokinetic profile translates to clinical benefits, with TAF showing improved bone and kidney safety compared to TDF while achieving higher intracellular TFV-DP concentrations in PBMCs. Phase III data demonstrated that TAF was noninferior to TDF in virological suppression with more favorable effects on renal and bone parameters.

Activity Against Resistant HIV-1 Strains

Resistance Mutation Profiles

TAF activity was evaluated against multiple resistance mutations, with K65R and thymidine analog-associated mutations (TAMs: M41L, D67N, K70R, L210W, T215Y, K219Q) receiving particular attention.

The presence of the M184V mutation increased TAF sensitivity compared to viruses without M184V. In TAM-containing HIV-1 site-directed mutants, the presence of M184V significantly increased TAF sensitivity compared to mutants without M184V. This hypersusceptibility associated with M184V contributes to TAF’s higher resistance threshold.

Resistance Barrier Comparison

TAF demonstrated a substantially higher resistance barrier than TDF across multiple experimental systems. In viral breakthrough assays mimicking physiologically relevant concentrations:

The resistance threshold for TAF was estimated to be 3.5-fold higher than for TDF, with TAF capable of inhibiting viruses with up to four TAMs, and up to six TAMs when M184V is present. This enhanced resistance barrier stems directly from the higher intracellular TFV-DP levels achieved by TAF, supporting higher resistance cutoffs for TAF compared to TDF in genotypic and phenotypic resistance algorithms.

Factors Affecting TAF Activation and Distribution

Drug Interactions

The covalent hepatitis C virus protease inhibitors telaprevir and boceprevir potently inhibited CatA-mediated TAF activation, with IC50 values of 0.27 μM and 0.16 μM respectively. At pharmacologically relevant concentrations, these drugs reduced anti-HIV activity of TAF in primary CD4+ T lymphocytes by 21-fold and 3-fold respectively. Clinical recommendations indicate avoiding coadministration of telaprevir or boceprevir with TAF in HIV/HCV-coinfected individuals. In contrast, there was no inhibition of CatA or significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV protease inhibitors, or various prescribed inhibitors of host serine proteases.

TAF demonstrated additive to synergistic interactions when combined with antiretroviral drugs from different classes including NRTIs, NNRTIs, integrase strand transfer inhibitors, and protease inhibitors.

Hormonal Contraceptive Effects

Medroxyprogesterone acetate (MPA) exhibited differential effects on TAF and TFV activity depending on cell type. In blood CD4+ T cells, MPA suppressed the anti-HIV effect of TFV by reducing intracellular TFV-DP, but had no effect on TAF inhibition of infection or TFV-DP concentrations. Conversely, in genital CD4+ T cells, MPA suppressed TAF inhibition of HIV infection and lowered TFV-DP concentrations without affecting TFV protection. These findings suggest MPA may decrease antiretroviral protection in individuals using these drugs intermittently for prevention. Other progestins including levonorgestrel, norethisterone, and progesterone did not affect TFV or TAF activity.

Cellular and Environmental Factors

Alterations in the vaginal microbiome, particularly the presence of anaerobic bacteria, can decrease antiretroviral efficacy. Inflammation from vaginal gels or sexually-transmitted infections undermines TFV effectiveness by recruiting target cells to the mucosal surface. These environmental factors may modulate TAF distribution and efficacy in the female reproductive tract.

Host factors including cathepsin A expression levels play a critical role in TAF activation. Ces1 expression is particularly important for hepatic TAF metabolism. The consistent intracellular metabolism and antiretroviral potency of TAF across multiple donors of variable gender, age, and ethnicity supports its clinical applicability.

Clinical Implications

Dosing and Efficacy

The phase 1b monotherapy study established a dose-response relationship for viral load decrease up to 25 mg TAF. At steady state, 8, 25, and 40 mg TAF yielded mean TFV plasma exposures 97%, 86%, and 79% lower respectively compared to 300 mg TDF. For the 25 and 40 mg doses, mean intracellular PBMC TFV-DP area under the curve was approximately 7-fold and 25-fold higher relative to 300 mg TDF. Significant reductions in plasma HIV-1 RNA from baseline to day 11 were observed for all TAF dose groups compared with placebo (P < 0.01), with median decreases of 1.08–1.73 log10 copies per milliliter.

TAF demonstrates potent antiviral activity at lower doses than TDF, which may translate into greater antiviral efficacy, a higher barrier to resistance, and an improved safety profile. The higher intracellular TFV-DP exposures may offer clinical benefits including improved resistance profiles and treatment durability.

Safety Considerations

The reduced systemic TFV exposure with TAF compared to TDF has important safety implications. Lower plasma TFV concentrations may reduce known TFV-associated nephrotoxicity by decreasing concentrations in renal proximal tubule cells, which express the organic anion transporters OAT1 and OAT3. Clinical dosing with TAF showed improved bone and kidney safety compared to TDF.

Monitoring and Resistance Testing

TAF concentrations in cervical vaginal lavages may not accurately reflect tissue concentrations or protection, suggesting that cervical vaginal lavages should not be used to monitor adherence or efficacy. The mucosal environment, including factors like local inflammation, contraceptive use, microbiome composition, and sexually-transmitted infections, should be considered when evaluating TAF efficacy.

Higher resistance cutoffs should be applied for TAF compared to TDF in genotypic and phenotypic resistance algorithms. TAF may be more effective in treating HIV-1 infections with resistance mutations, particularly those with TAMs and M184V. Clinical studies demonstrated TAF’s efficacy in maintaining viral suppression in patients with NRTI resistance.

Knowledge Gaps and Future Directions

Clinical validation of TAF’s higher resistance threshold remains limited due to ethical concerns regarding resistance induction studies. Further research is needed on TAF distribution and activation in genital tissues, particularly under conditions of inflammation and sexually-transmitted infections. Long-term durability and safety profiles require additional study in larger and longer-term clinical trials. Understanding the mechanisms of antiretroviral distribution and efficacy in different clinical scenarios, especially in the context of inflammatory conditions, represents an important area for future investigation.

References

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