Inhibition of Bitter Taste from Oral Tenofovir Alafenamide

Abstract

Children have difficulty swallowing capsules. Yet, when presented with liquid formulations, children often reject oral medications due to their intense bitterness. Presently, effective strategies to identify methods, reagents, and tools to block bitterness remain elusive. For a specific bitter-tasting drug, identification of the responsible bitter receptors and discovery of antagonists for those receptors can provide a method to block perceived bitterness. We have identified a compound (6-methylflavone) that can block responses to an intensely bitter-tasting anti-human immunodeficiency virus (HIV) drug, tenofovir alafenamide (TAF), using a primary human taste bud epithelial cell culture as a screening platform. Specifically, TAS2R39 and TAS2R1 are the main type 2 taste receptors responding to TAF observed via heterologously expressing specific TAS2R receptors into HEK293 cells. In this assay, 6-methylflavone blocked the responses of TAS2R39 to TAF. In human sensory testing, 8 of 16 subjects showed reduction in perceived bitterness of TAF after pretreating (or "prerinsing") with 6-methylflavone and mixing 6-methylflavone with TAF. Bitterness was completely and reliably blocked in two of these subjects. These data demonstrate that a combined approach of human taste cell culture-based screening, receptor-specific assays, and human psychophysical testing can successfully discover molecules for blocking perceived bitterness of pharmaceuticals, such as the HIV therapeutic TAF. Our hope is to use bitter taste blockers to increase medical compliance with these vital medicines. SIGNIFICANCE STATEMENT: Identification of a small molecule that inhibits bitter taste from tenofovir alafenamide may increase the compliance in treating children with human immunodeficiency virus infections.

Fig. 1

Primary human taste cell–based screening led to identification of 6-methylflavone as a suppressor of TAF-induced taste cell activity. (A) Critical path of bioassays in the MTS design to discover bitter blockers. Cutoff metrics for “hit picking” after the MTS step, validation steps, and counter screen assays are shown. (B) Concentration-response curve for 6-methylflavone in blocking TAF stimulation. TAF was the bitter stimulus in the assay. The IC₅₀ was approximately 5 µM for 6-methylflavone, and the efficacy of inhibition for 6-methylflavone was 90% or greater at 30 and/or 100 µM. Each compound was tested in triplicate in the MTS step, and every concentration was tested in quadruplicate in each step thereafter. A.U.: arbitrary units; LUC: luciferase; MTS: medium-throughput screening.

Fig. 2

TAF activates a limited number of bitter taste receptors. HEK293 cells transfected with human TAS2Rs, along with Gα16-gust44, were assayed for their responses to 0.1 mM (A, left) and 1 mM TAF (A, right). Quantitative analysis of responses is presented. Data are percentage change (means ± S.D.) in fluorescence [peak relative fluorescence units (RFU) − baseline RFU, denoted ΔF] from baseline fluorescence (denoted F) averaged from triplicates (n = 3). The experiment was replicated one more time. (B) Representative calcium mobilization traces of TAS2R1-, TAS2R8-, TAS2R14-, and TAS2R39-expressing cells to 1 mM TAF (black traces). Mock-transfected cells (Gα16-gust44 alone) were used as control (gray trace). (C) TAF activates TAS2R1, TAS2R8, TAS2R14, and TAS2R39 concentration-dependently. Each concentration was tested in triplicate (n = 3). Analysis of variance with Dunnett’s multiple comparisons test was used for statistical analysis. ***P < 0.001.

Fig. 3

6-methylflavone and its analogs block the responses of TAS2R39 to TAF and denatonium. HEK293 cells were transfected with TAS2R39 and Gα16-gust44 and assayed for responses to 6-methylflavone, its analogs, and other putative blockers identified in taste cell–based screening (Fig. 1). (A) Bar graph showing the responses of TAS2R39 to TAF (1 mM) in the presence of 6-methylflavone, its analogs, and other putative blockers. 6-methylflavone (0.1 mM) completely blocked the response of TAS2R39 to TAF. Two other analogs (compounds C5 and C7) at 0.1 mM largely blocked the response of TAS2R39 to TAF; three others (compounds C4, C6, and C10) at 0.1 mM partially blocked the response of TAS2R39 to TAF. Data are averaged from triplicates (n = 3). The experiment was replicated one more time. Analysis of variance with Dunnett’s multiple comparisons test was used for statistical analysis. ***P < 0.001. **P < 0.01, *P < 0.05. (B) Representative calcium mobilization traces of TAS2R39-expressing cells to TAF, TAF + 6-methylflavone (6-MF), TAF + C5, and TAF + C7. Compounds were added at 30 seconds after the start of the reading. (C) Concentration-response curve showing that 6-methylflavone blocked the responses of TAS2R39 to 1 mM TAF concentration-dependently (IC₅₀ = 0.02196 mM) (n = 3). (D) 6-methylflavone can block the response of TAS2R39 to the activator 3 mM denatonium (Den) (IC₅₀ = 0.01297 mM) (n = 3). The experiment was replicated three more times.

Fig. 5

Perceived bitterness of 6-methylflavone, TAF, and TAF with 6-methylflavone as prerinse and admixture in all subjects. (A) The graph shows the test-retest reliability of the subjects’ ratings of perceived bitterness for three conditions: 6-methylflavone, TAF, and TAF with 6-methylflavone as prerinse and admixture. The x-axis shows ratings for Session 1, whereas the y-axis shows ratings for Session 2. Pearson correlation coefficient = 0.76, P < 0.001, indicating consistency and reliability in the subjects’ bitterness ratings across the sessions. (B) The graph shows each subjects’ ratings of perceived bitterness of 6-methylflavone alone (green bar), TAF alone (blue bar), and TAF with 6-methylflavone as prerinse and admixture (orange bar) for all 16 subjects averaged over two replications. TAF was dosed at 0.5 mg/ml, and 6-methylflavone was dosed at 1 mM. The solution volume placed in the mouth was 10 ml. The subjects are sorted from highest to lowest rating of perceived bitterness of TAF. These data highlight the individual differences in bitter taste perception of TAF and bitter taste suppression of TAF with 6-methylflavone. (C) The graph shows subjects’ ratings of perceived bitterness for three conditions: 6-methylflavone, TAF, and TAF with 6-methylflavone as prerinse and admixture. Each condition was tested twice. TAF was dosed at 0.5 mg/ml, and 6-methylflavone was dosed at 1 mM. The solution volume placed in the mouth was 10 ml. The bitterness intensity of TAF is modestly reduced with 6-methylflavone, albeit without statistical significance; repeated measures analysis of variance, F (1, 60) = 0.71; P > 0.05. LMS: Labeled Magnitude Scale.

Fig. 4

Perceived bitterness of TAF with and without 1 mM 6-methylflavone (6-MF) as prerinse and admixture in two subjects. The top two graphs are ratings by one subject. The bottom two graphs are ratings by another individual subject. The graphs on the left are Session 1, and the graphs on the right are Session 2. In each graph the bar on the left (blue) is perceived bitterness of TAF, whereas the bar on the right (orange) is perceived bitterness of TAF with a 6-methylflavone prerinse and admixture with TAF. TAF was dosed at 0.5 mg/ml. 6-methylflavone was dosed at 1 mM. The solution volume put into the mouth was 10 ml. The bitterness of TAF treated with 6-methylflavone (orange) was nearly completely blocked in these two subjects. These two subjects were repeatedly tested with 6-methylflavone, four repetitions for one subject and five repetitions for the other, to establish the reliability of the observations. The result was very reliable for these two subjects: Pearson r = 0.87; P < 0.005. LMS: Labeled Magnitude Scale.