Pharmacological challenge models are deployed to evaluate drug effects during clinical development. Intradermal injection of Substance P (SP) neuropeptide, a potential challenge agent for... Show morePharmacological challenge models are deployed to evaluate drug effects during clinical development. Intradermal injection of Substance P (SP) neuropeptide, a potential challenge agent for investigating local mediators, is associated with wheal and flare response mediated by the MRGPRX2 receptor. Although dose-dependent data on SP effects exist, full characterization and information on potential carryover effect after repeated challenge are lacking. This open-label, two-part, prospective enabling study of SP intradermal challenge in healthy participants aimed to understand and distinguish between wheal and flare responses following various SP doses. Part 1 included one challenge visit to determine optimum SP dose range for evaluation in part 2, which determined variability in 20 participants and used intradermal microdialysis (IDM) for SP-challenged skin sampling. At 5, 15, 50, and 150 pmol doses, respectively, posterior median area under the curve (AUC; AUC0-2h ) was 4090.4, 5881.2, 8846.8, and 9212.8 mm2 /min, for wheal response, and 12020.9, 38154.3, 65470.6, and 67404.4 mm2 /min for flare response (SP-challenge visit 2). When the challenge was repeated ~2 weeks later, no carryover effect was observed. IDM histamine levels were relatively low, resulting in low confidence in the data to define temporal characteristics for histamine release following SP challenge. No safety concerns were identified using SP. Wheal and flare responses following intradermal SP challenge were dose-dependent and different. The results indicate that this challenge model is fit-for-purpose in future first-in-human studies and further assessment of novel drugs targeting dermal inflammatory disease responses, such as chronic spontaneous urticaria, chronic inducible urticaria, and pseudo-allergic reactions. Show less
Schlegel, A.; Reeven, M. van; Croome, K.; Parente, A.; Dolcet, A.; Widmer, J.; ... ; Muiesan, P. 2021
Background: The R192Q mutation of the CACNA1A gene, encoding for the alpha 1 subunit of voltage-gated P/Q Ca2+ channels (Ca(v)2.1), is associated with familial hemiplegic migraine-1. We... Show moreBackground: The R192Q mutation of the CACNA1A gene, encoding for the alpha 1 subunit of voltage-gated P/Q Ca2+ channels (Ca(v)2.1), is associated with familial hemiplegic migraine-1. We investigated whether this gain-of-function mutation changed the structure and function of trigeminal neuron P2X(3) receptors that are thought to be important contributors to migraine pain. Results: Using in vitro trigeminal sensory neurons of a mouse genetic model knockin for the CACNA1A R192Q mutation, we performed patch clamp recording and intracellular Ca2+ imaging that showed how these knockin ganglion neurons generated P2X(3) receptor-mediated responses significantly larger than wt neurons. These enhanced effects were reversed by the Cav2.1 blocker.-agatoxin. We, thus, explored intracellular signalling dependent on kinases and phosphatases to understand the molecular regulation of P2X(3) receptors of knockin neurons. In such cells we observed strong activation of CaMKII reversed by.-agatoxin treatment. The CaMKII inhibitor KN-93 blocked CaMKII phosphorylation and the hyperesponsive P2X(3) phenotype. Although no significant difference in membrane expression of knockin receptors was found, serine phosphorylation of knockin P2X(3) receptors was constitutively decreased and restored by KN-93. No change in threonine or tyrosine phosphorylation was detected. Finally, pharmacological inhibitors of the phosphatase calcineurin normalized the enhanced P2X(3) receptor responses of knockin neurons and increased their serine phosphorylation. Conclusions: The present results suggest that the CACNA1A mutation conferred a novel molecular phenotype to P2X3 receptors of trigeminal ganglion neurons via CaMKII-dependent activation of calcineurin that selectively impaired the serine phosphorylation state of such receptors, thus potentiating their effects in transducing trigeminal nociception. Show less
