Effect of alcohol exposure on the efficacy and safety of tenofovir alafenamide fumarate, a major medicine against human immunodeficiency virus

Abstract

Human immunodeficiency virus (HIV) continues to be a major health concern. AIDS-related deaths (acquired immunodeficiency syndrome) have decreased recently, but chronic liver disease is now a major cause of mortality among HIV patients. Widespread alcohol use is recognized to be a major contributing factor. Tenofovir alafenamide fumarate (TAF), one of the most used HIV drugs, requires hydrolysis followed by phosphorylation to produce tenofovir diphosphate, the ultimate anti-HIV metabolite. Carboxylesterase-1 (CES1), established to hydrolyze TAF, is known to catalyze transesterification in the presence of ethanol. The aim of the study was to test the hypothesis that metabolism-based interactions between TAF and ethanol negatively impact both efficacy and safety of TAF. To test this hypothesis, the metabolism of TAF was determined in human primary hepatocytes and with a large number of human liver samples (S9 fractions) in the presence or absence of ethanol. The metabolism was monitored by LC-MS/MS (liquid chromatography with tandem mass spectrometry) and the level of CES1 or CES2 was determined by Western blotting. Consistent with the hypothesis, TAF underwent transesterification in the presence of ethanol accompanied by decreased hydrolysis. The formation of tenofovir diphosphate (the therapeutically active metabolite) was significantly decreased. In addition, TAF but not its hydrolytic metabolite, was found to increase intracellular lipid retention, and the increase was enhanced by ethanol. These findings conclude that alcohol consumption, beyond commonly accepted poor adherence to HIV medications, directly impacts the efficacy and safety of TAF.

Keywords: Acquired immunodeficiency syndrome; Carboxylesterase-1 (CES1); Human immunodeficiency virus; Liver toxicity; Tenofovir alafenamide fumarate.

Fig. 1.

Structure, elution profile and m/z determination for TAF transesterification. (A) Structure showing displaced isopropyl alcohol with an ethyl alcohol (ethanol). An arrow indicates the displacement of isopropyl alcohol with an ethyl alcohol (boxed). (B and C) Elution profile of hydrolysis and transesterification by CES1. Lysates (10 μg) from CES1 transfected cells were incubated with TAF (20 μM) in the absence (B) or presence (C) of ethanol (20 mM) or the reaction buffer for 30 min and then mixed with acetonitrile at a final concentration of 66%. The incubation mixtures were subjected to centrifugation at 12,000g for 30 min. The supernatants were analyzed for elution profile by HPLC: incubation without (B) or with ethanol (C). (D) Determination of the formed ethyl TAF based on m/z. The incubation of transesterification was performed as described in the legend for Fig. 2 (below) with TAF being used at 1 μM and ethanol at 12.5 mM.

Fig. 2.. Transesterification of TAF as a function of ethanol.

(A) Transesterification by S9 fraction. Pooled liver S9 fractions (5 μg, n = 36) were incubated with TAF in the presence of ethanol at 0, 6.25, 12.5 or 25.0 mM for 60 min at 37°C. The reactions were stopped by adding two-volumes of acetonitrile termination buffer containing 0.15 μM d5-TFV (internal standard). The mixture was subjected to centrifugation at 12,000g and the supernatants were analyzed by LC-MS/MS for the formation of tenofovir or ethyl TAF. The same unit (nmole/μg protein/min) but different scales were used for the values of the Y-axis because the hydrolysis (formation of tenofovir) was more robust than transesterification (formation of ethyl TAF). *Statistical significant at p < 0.05, and **Statistical significant at p < 0.01 from the incubations without ethanol. (B) Extracellular concentrations of the hydrolytic metabolite tenofovir and the transesterification metabolite ethyl TAF. Pooled human primary hepatocytes (n = 4, 1 × 10⁶ cells/mL) were incubated in a total volume of 100 μL with a final TAF concentration of 1 μM and ethanol at 0, 6.25 and 12.5 mM. The incubation lasted for 60 min at 37°C. Media were collected and centrifuged at 12,000g for 5 min. The supernatant (30 μL) was mixed with two volumes of termination buffer containing the internal standard d5-TFV (0.15 μM). Tenofovir and ethyl TAF were quantified by LC-MS/MS. The amounts of tenofovir and ethyl TAF were expressed based on a total number of 10⁶ cells. *Statistical significant at p < 0.05, and **Statistical significant at p < 0.01 from incubations without ethanol. (C) Intracellular concentrations of the hydrolytic metabolite tenofovir and the transesterification metabolite ethyl TAF. The incubations were performed as described in B. Hepatocytes were collected, washed for two times by centrifugation and resuspended in 100 μL termination buffer containing the internal standard d5-TFV. The quantification of tenofovir and ethyl TAF was determined as described in B. *Statistical significant at p < 0.05, and **Statistical significant at p < 0.01 from incubations without ethanol.

Fig. 3.. Hydrolysis and transesterification of TAF among individual human S9 samples.

Individual human S9 samples (5 μg, n = 36) were incubated in a total volume of 100 μL with TAF (1 μM) in the absence (A) or presence (B) of ethanol at 12.5 mM for 60 min at 37°C. The reactions were stopped by adding two-volumes of acetonitrile termination buffer containing 0.15 μM d5-TFV (internal standard). The mixture was subjected to centrifugation at 12,000g and the supernatants were analyzed by LC-MS/MS. For Western blotting, individual or pooled (p) S9 samples (5 μg protein) were subjected to SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to nitrocellulose membrane and detected by the chemiluminescent detection system for CES1, CES2 or GAPDH. The intensity of immunostaining was captured and quantified by ChemiDoc Imaging system. Once again, the same unit (nmole/μg protein/min) but different scales were used for the values of the Y-axis because the hydrolysis (formation of tenofovir) was more robust than transesterification (formation of ethyl TAF).

Fig. 4.. Correlation analysis.

The correlation was performed with SPSS Statistics 20. (A) Correlation of hydrolysis (no ethanol) with CES1 (Left) or CES2 (Right) level. (B) Correlation of transesterification with CES1 (Left) or CES2 (Right) level. (C) Correlation of transesterification with hydrolysis (Left) or ethanol-reduced hydrolysis (Right). Correlation coefficients and corresponding p values are shown. The levels of CES1 or CES2 were normalized based on their respective ratios with the levels of GAPDH derived from Fig. 3A.

Fig. 5.. Ethanol-decreased formation of tenofovir diphosphate.

(A) Formation of tenofovir diphosphate in the presence of ethanol. Pooled human primary hepatocytes (n = 4, 1 × 10⁶ cells/mL) were incubated at 37°C for 60 min in a total volume of 100 μL with a final TAF concentration of 1 μM and ethanol at 0, 6.25 and 12.5 mM. Hepatocytes were collected, washed for two times by centrifugation and resuspended in 100 μL termination buffer containing the internal standard d5-TFV. The quantification of tenofovir diphosphate was determined by LC-MS/MS. *Statistical significant at p < 0.05, and **statistical significant at p < 0.01 from incubations without ethanol. (B) Representative mass spectrums. Tenofovir diphosphate (TFV-DP, m/z: 447.9) and the internal standard d5-tenofovir (TFV-d5, m/z, 293.5) are shown. Top shows the spectrum from incubation without ethanol and the Bottom shows the spectrum from incubation with ethanol.

Fig. 6.. Ethanol-TAF interactive enhancement of lipid retention.

(A) Lipid retention as a function of tenofovir or TAF in the presence or absence of ethanol. Huh7 cell line was seeding at 96-well plate at a density 10,000 cells/well. The cells were treated with tenofovir (TFV) or TAF at 5 or 10 μM at the absence or presence of ethanol (20 and 40 mM). The media were changed by every 6 h to minimize the impact of ethanol evaporation. The treatment lasted for a length of 48 h. The cells were then treated with 0.2 mM bovine serum albumin coupled-oleic acid for 24 h. The lipid retention was stained with Nile Red (1 μg/mL) and the nuclei were stained with Hoechst 3342 (10 μg/mL). The images were taken by confocal microscopy. For quantification, 8 images from each treatment were taken on 15 cells in each image. *Statistical significant at p < 0.01 from incubations from controls. (B) Representative images of lipid retention from different treatments.

Fig. 7.. Diagrammatical presentation of TAF-ethanol interaction.

TAF undergoes hydrolysis and transesterification in the presence of ethanol, and these two enzymatic reactions are catalyzed by CES1, constituting a competitive mechanism favoring transesterification. Additionally, the presence of ethanol decreases the phosphorylation of tenofovir, critical for the formation of the ultimate therapeutic anti-HIV metabolite tenofovir diphosphate.