The cation channel transient receptor potential ankyrin 1 (TRPA1) plays an important role in sensing potentially hazardous substances. However, TRPA1 species differences are substantial and limit translational research. TRPA1 agonists tested previously in humans also have other targets. Therefore, the sensation generated by isolated TRPA1 activation in humans is unknown. The availability of 2-chloro-N-(4-(4-methoxyphenyl)thiazol-2-yl)-N-(3-methoxypropyl)-acetamide (JT010), a potent and specific TRPA1 agonist, allowed us to explore this issue. To corroborate the specificity of JT010, it was investigated whether the TRPA1 antagonist (1E,3E)-1-(4-fluorophenyl)-2methyl-1-penten-3-one oxime (A-967079) abolishes JT010-elicited pain. Sixteen healthy volunteers of both sexes rated pain due to intraepidermal injections of different concentrations and combinations of the substances. The study design was a double-blind crossover study. All subjects received all types of injections, including a placebo without substances. Injections of the TRPA1 agonist dosedependently caused pain with a half-maximal effective concentration of 0.31 M. Coinjection of A-967079 dose-dependently reduced and at a high concentration abolished JT010-induced pain. Quantification of JT010 by HPLC showed that a substantial part is adsorbed when in contact with polypropylene surfaces, but that this was overcome by handling in glass vials and injection using glass syringes. Isolated TRPA1 activation in humans causes pain. Thus, intradermal JT010 injection can serve as a tool to validate new TRPA1 antagonists concerning target engagement. More importantly, TRPA1-specific tools allow quantification of the TRPA1-dependent component in physiology and pathophysiology.This study showed that activation of the ion channel transient receptor potential ankyrin 1 (TRPA1) alone indeed suffices to elicit pain in humans, independent of other receptors previously found to be involved in pain generation. The newly established TRPA1-specific pain model allows different applications. First, it can be tested whether diseases are associated with compromised or exaggerated TRPA1-dependent painful sensations in the skin. Second, it can be investigated whether a new, possibly systemically applied drug directed against TRPA1 engages its target in humans. Further, the general possibility of quantitative inhibition of TRPA1 allows identification of the TRPA1-dependent disease component, given that the substance reaches its target. This contributes to a better understanding of pathophysiology, can lay the basis for new therapeutic approaches, and can bridge the gap between preclinical research and clinical trials.
Cell-permeable orthosteric ligands can assist folding of G proteincoupled receptors in the endoplasmic reticulum (ER); this pharmacochaperoning translates into increased cell surface levels of receptors. Here we used a folding-defective mutant of human A 1 -adenosine receptor as a sensor to explore whether endogenously produced adenosine can exert a chaperoning effect. This A 1 -receptor-Y 288A was retained in the ER of stably transfected human embryonic kidney 293 cells but rapidly reached the plasma membrane in cells incubated with an A 1 antagonist. This was phenocopied by raising intracellular adenosine levels with a combination of inhibitors of adenosine kinase, adenosine deaminase, and the equilibrative nucleoside transporter: mature receptors with complex glycosylation accumulated at the cell surface and bound to an A 1 -selective antagonist with an affinity indistinguishable from the wild-type A 1 receptor. The effect of the inhibitor combination was specific, because it did not result in enhanced surface levels of two folding-defective human V 2 -vasopressin receptor mutants, which were susceptible to pharmacochaperoning by their cognate antagonist. Raising cellular adenosine levels by subjecting cells to hypoxia (5% O 2 ) reproduced chaperoning by the inhibitor combination and enhanced surface expression of A 1 -receptor-Y 288A within 1 hour. These findings were recapitulated for the wild-type A 1 receptor. Taken together, our observations document that endogenously formed adenosine can chaperone its cognate A 1 receptor. This results in a positive feedback loop that has implications for the retaliatory metabolite concept of adenosine action: if chaperoning by intracellular adenosine results in elevated cell surface levels of A 1 receptors, these cells will be more susceptible to extracellular adenosine and thus more likely to cope with metabolic distress.
Flunixin is a nonsteroidal anti‐inflammatory drug (NSAID) that has anti‐inflammatory, anti‐pyretic, and analgesic effects. Recently, a novel transdermal formulation was developed (Finadyne® Transdermal, MSD Animal Health) and is now the first NSAID registered to be administered as a pour‐on product in cattle. According to the manufacturer's instructions, the pour‐on product should be applied only to dry skin and exposure to rain should be avoided for at least 6 hr after application. The objective of the study was to evaluate the effect of simulated exposure to light or heavy rain on flunixin absorption and bioavailability within the first 4 hr after administration. Therefore, an isocratic HPLC method was developed to quantify flunixin concentrations in bovine serum by UV detection. Light rain decreased flunixin absorption only when rain started immediately after flunixin administration, while light rain starting more than 30 min after administration of flunixin had no effect on absorption. Absorption and bioavailability of flunixin was impacted under simulated heavy rain conditions, when exposure to rain occurred within one hour after the application of the pour‐on formulation, but not later.
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