Reducing the Bitter Taste of Pharmaceuticals Using Cell-Based Identification of Bitter-Masking Compounds.

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.

Effect of 158 herbal remedies on human TRPV1 and the two-pore domain potassium channels KCNK2, 3 and 9.

BACKGROUND AND AIM: Herbal medicines are used to treat a broad number of maladies. However, the pharmacological profile of most remedies is poorly understood. We investigated the effect of herbal remedies from kampo, traditional Chinese medicine (TCM) and other phytotherapies on human two-pore domain potassium channels (KCNK channels; TREK-1, TASK-1 and TASK-3) as well as the human TRPV1 channel. KCNK channels are responsible for the background potassium current of excitable cells, thus essential for the maintenance of the resting membrane potential. Hence, modulators of KCNK channels are of medical significance, e.g. for the treatment of sleep disorders and pain. The transient receptor potential channel TRPV1 is a pain detector for noxious heat. Agonists of this receptor are still used for the treatment of pain in ectopic applications. EXPERIMENTAL PROCEDURE: We evaluated the effect of 158 herbal remedies on these channels in a heterologous expression system (Xenopus laevis oocytes) using the two-electrode voltage-clamp technique with the aim of increasing the comprehension of their pharmacological profile. RESULTS AND CONCLUSION: Some remedies with modulating effects were identified such as Angelica pubescens (radix), which inhibit TASK-1 and TASK-3 channels. Furthermore, the modulatory effects of the most effective remedies on the two TASK family members TASK-1 and TASK-3 correlate positively, reflecting their close relation. For the TRPV1 channel Terminalia chebula and Alchemilla xanthochlora were identified as potentiators. This study identifies a variety of herbal remedies as modulators of human K2P and TRPV1 channels and gives new insights into the pharmacological profile of these herbal remedies.

The Effect of Pungent and Tingling Compounds from Piper nigrum L. on Background K+ Currents.

Black peppercorns (Piper nigrum L.) elicit a pungent and tingling oral impression. Their pungency is partially explained by the agonist activity of some of their active principles, especially piperine, on TRP channels. However, we recently showed that piperine, as well as other pungent compounds, also possess a marked effect on two-pore domain (KCNK, K2P) K+ channels. Members of this family play a key role in maintaining the resting membrane potential of excitable cells. Interestingly, tingling compounds have been shown to induce neuronal excitation by inhibiting KCNK channels. We addressed the question of whether it was plausible that KCNK channels could constitute a physiologically relevant target for the sensory active compounds present in black peppercorns. Because previous studies have demonstrated that mouse trigeminal neurons respond to several pungent compounds, to which humans are also sensitive, we used a primary culture of mouse trigeminal neurons to investigate whether the effect of piperine on these cell types could also be mediated by KCNK channels. We observed that even in the presence of classical TRP-antagonists, piperine was still able to activate a fraction of trigeminal neurons. Furthermore, our results showed that piperine is capable of inducing neuronal depolarization by a mechanism that does not require extracellular Na+ or Ca2+. This depolarization was mediated by the inhibition of a background K+ conductance, most likely corresponding to the KCNK channels of the TASK subfamily. We then performed a screening with 12 other pungent and/or tingling chemosensates isolated from black peppercorns. These compounds were evaluated on Xenopus laevis oocytes expressing the human orthologues of KCNK3, KNCK9 and KCNK18, which we previously showed to be inhibited by piperine. Remarkably, almost all of the isolated chemosensates inhibited the basal activity of hKCNK3, with 1-(octadeca-2E,4E,13/12Z-trienoyl)pyrrolidine acting as one of the most potent natural blockers for hKCNK3 found to date. Our results suggest that KCNK channels, especially KCNK3, are likely to play a complementary role to TRP channels in the complex orosensory impression elicited by black peppercorns, while they also help to expand the pharmacological knowledge of KCNK channels.

Nonivamide, a capsaicin analogue, exhibits anti-inflammatory properties in peripheral blood mononuclear cells and U-937 macrophages.

SCOPE: Inflammation-related diseases are a worldwide problem. The counteraction of inflammation with compounds activating the trigeminal nerve is one strategy to fight these diseases. Known trigeminally active compounds found in black or red pepper are the tingling t-pellitorine, the pungent capsaicin, and the less pungent nonivamide. The presented study compares the anti-inflammatory potential of nonivamide to the two known anti-inflammatory compounds, elucidating the mechanism of action and the role of transient receptor protein (TRP) channels. METHODS AND RESULTS: Primary peripheral blood mononuclear cells (PBMCs) and U-937 macrophages were stimulated with 1 µg/mL LPS from Escherichia coli (EC-LPS) to induce inflammation. Nonivamide attenuated the EC-LPS induced release of IL-6 and TNF-α in PBMCs and U-937 macrophages determined by magnetic bead kit analysis. This anti-inflammatory mechanism was independent from nuclear factor-kappa B pathway but mitogen-activated protein kinase (MAPK) pathway may be involved. In addition, cotreatment of U-937 with the trigeminally active compound and an antagonist of TRPV1 or TRPA1 abolished the anti-inflammatory activity. CONCLUSIONS: Nonivamide possessed similar anti-inflammatory potential as capsaicin and t-pellitorine. In U-937 macrophages, the tested compounds exploited an anti-inflammatory effect by inhibiting the EC-LPS induced activation of the MAPK pathway. In addition, the TRP channel activation plays a role in the anti-inflammatory capacity of capsaicin and nonivamide.

Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors.

Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore, the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzed as ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effective inhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis (cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identified for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing for a better understanding of their physiological effects and clinical applications.

Potentiating effect of glabridin from Glycyrrhiza glabra on GABAA receptors.

Extracts from Glycyrrhiza are traditionally used for the treatment of insomnia and anxiety. Glabridin is one of the main flavonoid compounds from Glycyrrhiza glabra and displays a broad range of biological properties. In the present work, we investigated the effect of glabridin on GABAA receptors. For this purpose, we employed the two-electrode voltage-clamp technique on Xenopus laevis oocytes expressing recombinant GABAA receptors. Through this approach, we observed that glabridin presents a strong potentiating effect on GABAA α1ß(1-3)γ2 receptors. The potentiation was slightly dependent on the ß subunit and was most pronounced at the α1ß2γ2 subunit combination, which forms the most abundant GABAA receptor in the CNS. Glabridin potentiated with an EC50 of 6.3±1.7 µM and decreased the EC50 of the receptor for GABA by approximately 12-fold. The potentiating effect of glabridin is flumazenil-insensitive and does not require the benzodiazepine binding site. Glabridin acts on the ß subunit of GABAA receptors by a mechanism involving the M286 residue, which is a key amino acid at the binding site for general anesthetics, such as propofol and etomidate. Our results demonstrate that GABAA receptors are strongly potentiated by one of the main flavonoid compounds from Glycyrrhiza glabra and suggest that glabridin could contribute to the reported hypnotic effect of Glycyrrhiza extracts.

Direct action and modulating effect of (+)- and (-)-nicotine on ion channels expressed in trigeminal sensory neurons.

Nicotine sensory perception is generally thought to be mediated by nicotinic acetylcholine (nACh) receptors. However, recent data strongly support the idea that other receptors (e.g., transient receptor potential A1 channel, TRPA1) and other pathways contribute to the detection mechanisms underlying the olfactory and trigeminal cell response to nicotine flavor. This is in accordance with the reported ability of humans to discriminate between (+)- and (-)- nicotine enantiomers. To get a more detailed understanding of the molecular and cellular basis underlying the sensory perception of nicotine, we studied the activity of (+)- and (-)-nicotine on cultured murine trigeminal sensory neurons and on a range of heterologously expressed receptors. The human TRPA1 channel is activated by (-)-nicotine. In this work, we show that (+)-nicotine is also an activator of this channel. Pharmacological experiments using nicotinic acetylcholine receptors and transient receptor potential blockers revealed that trigeminal neurons express one or more unidentified receptors that are sensitive to (+)- and/or (-)-nicotine. Results also indicate that the presence of extracellular calcium ions is required to elicit trigeminal neuron responses to (+)- and (-)-nicotine. Results also show that both (+)-nicotine and (-)-nicotine can block 5-hydroxytryptamine type 3 (5-HT3) receptor-mediated responses in recombinant expression systems and in cultured trigeminal neurons expressing 5-HT3 receptors. Our investigations broaden the spectra of receptors that are targets for nicotine enantiomers and give new insights into the physiological role of nicotine.

The pungent substances piperine, capsaicin, 6-gingerol and polygodial inhibit the human two-pore domain potassium channels TASK-1, TASK-3 and TRESK.

For a long time, the focus of trigeminal chemoperception has rested almost exclusively on TRP channels. However, two-pore domain (K2P) potassium channels have recently been identified as targets for substances associated with typical trigeminal sensations, such as numbing and tingling. In addition, they have been shown to be modulated by several TRP agonists. We investigated whether the pungent substances piperine, capsaicin, 6-gingerol and polygodial have an effect on human K2P channels. For this purpose, we evaluated the effects of these pungent substances on both wild-type and mutant K2P channels by means of two-electrode voltage-clamp experiments using Xenopus laevis oocytes. All four pungent substances were found to inhibit the basal activity of TASK-1 (K2P 3.1), TASK-3 (K2P 9.1), and TRESK (K2P 18.1) channels. This inhibitory effect was dose-dependent and, with the exception of polygodial on TASK-1, fully reversible. However, only piperine exhibited an IC50 similar to its reported EC50 on TRP channels. Finally, we observed for TASK-3 that mutating H98 to E markedly decreased the inhibition induced by piperine, capsaicin, and 6-gingerol, but not by polygodial. Our data contribute to the relatively sparse knowledge concerning the pharmacology of K2P channels and also raise the question of whether K2P channels could be involved in the pungency perception of piperine.

Transient receptor potential channels encode volatile chemicals sensed by rat trigeminal ganglion neurons.

Primary sensory afferents of the dorsal root and trigeminal ganglia constantly transmit sensory information depicting the individual's physical and chemical environment to higher brain regions. Beyond the typical trigeminal stimuli (e.g. irritants), environmental stimuli comprise a plethora of volatile chemicals with olfactory components (odorants). In spite of a complete loss of their sense of smell, anosmic patients may retain the ability to roughly discriminate between different volatile compounds. While the detailed mechanisms remain elusive, sensory structures belonging to the trigeminal system seem to be responsible for this phenomenon. In order to gain a better understanding of the mechanisms underlying the activation of the trigeminal system by volatile chemicals, we investigated odorant-induced membrane potential changes in cultured rat trigeminal neurons induced by the odorants vanillin, heliotropyl acetone, helional, and geraniol. We observed the dose-dependent depolarization of trigeminal neurons upon application of these substances occurring in a stimulus-specific manner and could show that distinct neuronal populations respond to different odorants. Using specific antagonists, we found evidence that TRPA1, TRPM8, and/or TRPV1 contribute to the activation. In order to further test this hypothesis, we used recombinantly expressed rat and human variants of these channels to investigate whether they are indeed activated by the odorants tested. We additionally found that the odorants dose-dependently inhibit two-pore potassium channels TASK1 and TASK3 heterologously expressed In Xenopus laevis oocytes. We suggest that the capability of various odorants to activate different TRP channels and to inhibit potassium channels causes neuronal depolarization and activation of distinct subpopulations of trigeminal sensory neurons, forming the basis for a specific representation of volatile chemicals in the trigeminal ganglia.

2-Aminoethoxydiphenyl borate activates the mechanically gated human KCNK channels KCNK 2 (TREK-1), KCNK 4 (TRAAK), and KCNK 10 (TREK-2).

Two-pore domain K(+) (KCNK, K2P) channels underlie the "leak" (background) potassium conductance in many types of excitable cells. They oppose membrane depolarization and cell excitability. These channels have been reported to be modulated by several physical and chemical stimuli. The compound 2-aminoethoxydiphenyl borate (2-APB) was originally described as an inhibitor of IP3-induced Ca(2+) release but has been shown to act as either a blocker or an activator for several ion channels. Here, we report the effects of this compound on members of the TREK (TWIK related K(+) channels) subfamily of human KCNK channels. We injected Xenopus laevis oocytes with cRNAs (complementary RNAs) encoding several KCNK channels and measured their response using the two-electrode voltage clamp technique. 2-APB was found to be an effective activator for all members of the TREK subfamily (hKCNK2, hKCNK4, and hKCNK10), with the highest efficacy in hKCNK10. We also found that 2-APB was able to activate these channels in cell-excised patches of HEK293 (human embryonic kidney 293) cell transfected with hKCNK4 or hKCNK10, demonstrating direct activation. TREK channels are widely expressed in the central nervous system and peripheral tissues, where they play roles in several key processes. However, little is known regarding their pharmacology; therefore, the identification of a common, stable and inexpensive agonist should aid further investigations of these channels. Additionally, 2-APB has been used to study native receptors in cell systems that endogenously express members of the TREK subfamily (e.g., rat dorsal root ganglia); our results thus warn against the use of 2-APB at high concentrations in these systems.