Abstract
The palatability of a pharmaceutical preparation is a significant obstacle in developing a patient-friendly dosage form. Bitter taste is an important factor for patients in (i) selecting a certain drug from generic products available in the market and (ii) adhering to a therapeutic regimen. The various methods developed for identification of bitter tasting and bitter-taste modulating compounds present a number of limitations, ranging from limited sensitivity to lack of close correlations with sensory data. In this study, we demonstrate a fluorescence-based assay, analyzing the bitter receptor TAS2R-linked intracellular pH (pHi) of human gastric parietal (HGT-1) cells as a suitable tool for the identification of bitter tasting and bitter-taste modulating pharmaceutical compounds and preparations, which resembles bitter taste perception. Among the fluorometric protocols established to analyze pHi changes, one of the most commonly employed assays is based on the use of the pH-sensitive dye SNARF-1 AM. This methodology presents some limitations; over time, the assay shows a relatively low signal amplitude and sensitivity. Here, the SNARF-1 AM methodology was optimized. The identified bicarbonate extrusion mechanisms were partially inhibited, and measurements were carried out in a medium with lower intrinsic fluorescence, with no need for controlling external CO2 levels. We applied the assay for the screening of flavonoids as potential bitter-masking compounds for guaifenesin, a bitter-tasting antitussive drug. Our findings revealed that eriodictyol, hesperitin and phyllodulcin were the most potent suitable candidates for bitter-masking activity, verified in a human sensory trial.
Highlights
Introduction published maps and institutional affilMany drugs taste bitter and are often aversive to children and adults
We present an in vitro assaying method for the determination of bitter tasting and bitter taste-masking pharmaceuticals
The signal-to-noise ratio from SNARF-measurements performed under Krebs-HEPES buffer (KRHP) and under DMEM were compared
Summary
The standard proton secretion-assay for HGT-1 cells uses DMEM as an extracellular medium during measurements. Krebs-HEPES buffer (KRHP) could be used instead, and whether it would present a better signal-to-noise ratio For this purpose, the signal-to-noise ratio from SNARF-measurements performed under KRHP and under DMEM were compared. We observed that, under atmospheric CO2 levels, vehicle-treated HGT-1 cells under KRHP presented significantly less change in intracellular pH after 35 min as compared with vehicle-treated cells under DMEM (Figure 2b). We compared both media in a 30-min long measurement, where recordings under KRHP presented a more consistent profile over long time, in addition to reduced variability (Figure 3)
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