Background & AimsSustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice.MethodsMouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects.ResultsGSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca2+ currents after Ca2+ release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis.ConclusionsCytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis.

Calcium-release activated calcium (CRAC) channel inhibitors represent a new class of oral immunomodulatory agents with a potential safety profile suitable for chronic dosing to treat autoimmune disorders. Calcium entry through CRAC channels is a critical step in the functional responses of T cells and mast cells. Patients with mutations in CRAC channels are known to have a form of severe combined immunodeficiency, wherein the adaptive immune response is suppressed without major impairment of other organ systems. CalciMedica has discovered several novel small molecules that selectively inhibit CRAC channels, and is currently developing the first of these inhibitors, CM2489, for the treatment of psoriasis. We describe here the properties of CM3457, another selective CRAC channel inhibitor that is structurally distinct from CM2489. CM3457 potently inhibits CRAC channels, Th1, Th2 and Th17-derived cytokine production, T cell proliferation, and mast cell degranulation. Once-daily oral dosing of CM3457 significantly inhibits inflammation and joint histopathology in a rat collagen-induced arthritis model. CM3457 also inhibits lung inflammation and eosinophilia, as well as improves lung function, in a rat OVA-induced asthma model. These data provide evidence that CM3457 may be an effective therapeutic in the treatment of autoimmune disorders and asthma.

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Urinary excretion of piperazine has not been widely studied owing to the lack of a quantitative method of analysis in biological fluids. A solution of potassium bismuth iodide gives a red precipitate with piperazine, and was used by Zimmermann (1901) for the qualitative demonstration of the drug. Nessler's reagent gives a heat-soluble precipitate; the picrate, chloroaurate, and platinate are also characteristic. Piperazine is precipitated by tannic acid, and can also be identified in urine by shaking a strongly alkaline sample with benzoyl chloride and determining the melting-point of the final product after crystallization in alcohol. Valseth and Wickstr6m (1954) The present paper describes a colorimetric method, using 1: 2-naphthoquinone-4-sulphonic acid (Folin's amino-acid reagent) for the quantitative determination of piperazine in urine. It is a modification of a procedure originally devised by Dr. Eileen Short (private communication), and, although it is not specific for piperazine, the method gives accurate results, and is suitable as a practical laboratory procedure involving the minimum number of manipulations. Amino-acids and other substances present in the urine also give coloured derivatives with the reagent, but the effect of interfering substances can be eliminated by using internal standards in a manner similar to that of Harris and Raymond (1939) for the determination of nicotinic acid. The method has been employed to study the excretion rates in human subjects after oral doses of piperazine. The results include a small number of excretion values from Africans infected with ascarides. 2-.