In recent years, several human diseases have been linked to abnormal CRAC channel activity, including severe combined immunodeficiency disorders, allergy, inflammatory bowel disease, thrombosis, brain injury, breast cancer, and AP [22,23]. As a result, pharmaceutical company researchers have invested considerable time and effort into developing potent CRAC inhibitors [24–26]. Two such ORAI1 channel inhibitors, GSK‐7975A (containing a pyrazole core structure) and CM4620 (also known as CM‐128, with a pyrazine core structure) were developed independently by GlaxoSmithKline (GSK, Stevenage, UK) and CalciMedica (La Jolla, CA), respectively [18,27]. GSK‐7975A has been demonstrated to inhibit SOCE induced by thapsigargin in isolated murine pancreatic acinar cells with an IC50 of approximately 3.4 μM, inhibiting endocytic vacuole formation and reducing necrosis induced by toxins that cause AP [19]. The Liverpool Pancreatitis Research Group conducted a thorough preclinical validation study evaluating the effect of GSK‐7975A and CalciMedica’s CM4620 in ex vivo and in vivo experimental AP using three clinically relevant models [26]. Both GSK‐7975A and CM4620 showed concentration‐dependent inhibitory effects on SOCE and necrosis in murine and human pancreatic acinar cells induced by taurolithocholic acid 3‐sulfate (TLCS) or cholecystokinin (CCK)‐8. The effects of CM4620 on ORAI1 were substantiated by examination of its effect on CRAC currents in ORAI1/STIM1‐transfected HEK 293 cells. The in vitro work informed in vivo pharmacokinetic analysis. GSK‐7975A was given at selected doses after induction of AP with TLCS (TLCS‐AP) or seven injections of cerulein (CER‐AP) or ethanol and palmitoleic acid (FAEE‐AP) [26,28]. Because GSK‐7975A markedly reduced all pathological parameters in a dose‐dependent manner, a high dose of GSK‐7975A and separately CM4620 was begun at two different time points after disease induction, to determine the effect of early versus late drug administration. Drug administration that was begun one hour after disease induction was highly effective in reducing all pathological parameters, and significantly more effectively than drug administration begun six hours after disease induction, in all models.
On the basis of these strong preclinical data, an intravenous formulation of CM4620 went through preclinical toxicity assessment into clinical development. Following a successful Phase I study in early 2017 (NCT03709342), the drug was fast‐tracked in early 2018 by the US Food and Drug Administration (FDA) and was taken into a Phase IIA trial (NCT03401190) for predicted moderate to severe pancreatitis. The results remain eagerly awaited as inhibition of calcium toxicity is a strategy that may prove successful in the treatment of AP.
Mitochondrial Dysfunction
Mitochondria were the first intracellular organelles to be associated with calcium handling [29] and in 2004, the mitochondrial calcium uniporter located in the inner mitochondrial membrane was identified as the key means through which mitochondria regulate calcium uptake and accumulation in the mitochondrial matrix [30] (Figure 12.1). Pathologically, calcium overload within the mitochondrial matrix leads to opening of the mitochondrial permeability transition pore (MPTP) across the inner mitochondrial membrane, allowing unregulated entry and exit of particles up to 1.5 kDa into and out of the mitochondrial matrix [13]. This results in the loss of inner mitochondrial membrane potential, diminished ATP production, mitochondrial swelling and rupture of the outer mitochondrial membrane followed by necrotic cell death [31–34]. Mitochondrial dysfunction as a result of intracellular calcium overload induced by toxins that include bile acids and ethanol metabolites has become established as a key pathogenic mechanism in acute pancreatitis [9,13,35,36]. MPTP opening is physiological in low‐conductance mode, releasing calcium and reactive oxygen species to match metabolism with workload, but pathological in high‐conductance mode compromising ATP production and inducing cell death [33,34]; both functions are regulated by the mitochondrial matrix protein peptidyl‐prolyl cis‐trans isomerase (PPI) cyclophilin D [CypD, also known as cyclophilin F (ppif) in mice located on chromosome 14] [37]. Little is known about the physiological role of CypD; CypD knockout (ppif –/–) mice are born healthy at the expected Mendelian ratios [37], but display increased anxiety and adult‐onset obesity [38]. Beyond behavioral traits, the lack of significant phenotype suggests that CypD inhibition may carry a low risk of toxicity. The role of CypD and MPTP opening in disease derives from studies of ischemia–reperfusion injury in the heart, brain, lung and kidney, muscular dystrophies, neurodegeneration, osteoporosis, and AP [13,31,33,34,39–42].
Figure 12.1 Pancreatic acinar cell focused AP treatment strategies. Physiological acinar cell calcium (Ca2+) signaling occurs through muscarinic (M3R) or cholecystokinin (CCK1R) receptors coupled with the second messengers inositol trisphosphate (IP3) and nicotinic acid adenine dinucleotide phosphate (NAADP) acting on endoplasmic reticulum‐based IP3 receptors (IP3R) and ryanodine receptors (RyR), respectively, resulting in store‐operated Ca2+ entry (SOCE). Abnormal Ca2+ signaling in the pancreatic acinar cell initiated by pancreatitis toxins (e.g. bile acids, fatty acid ethyl esters, hyperstimulation) causes injury dependent on continued SOCE via ORAI channels. As a consequence mitochondria are overloaded with Ca2+ thought to be via the mitochondrial calcium uniporter (MCU), leading to induction of the mitochondrial permeability transition pore (MPTP), allowing the passage of solutes <1500 kDa across the mitochondrial membrane with subsequent loss of the mitochondrial membrane potential and reduction in ATP production, required to clear Ca2+ through sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) and plasma membrane Ca2+‐ATPase (PMCA) pumps and thus protect the cell. Protective acinar cell strategies that have shown significant beneficial effects in a variety of experimental pancreatitis models focus on the prevention of calcium overload, the reduction of mitochondrial injury, the modulation of autophagy, and serine protease or serine protein kinase inhibition.
Cyclosporin A (CsA), a macrocyclic oligopeptide and nonspecific inhibitor of cyclophilins, has played a significant role in studying MPTP in in vitro and in vivo experimental models of different diseases. Its large size, poor solubility, and immunosuppressive nature (from interaction with calcineurin [43]) are properties that hinder its use in AP. As a result of initial promising preclinical findings, some non‐immunosuppressive analogs of CsA have also been synthesized including Debiopharm’s DEB025 (alisporovir) and Novartis’s NIM‐811 [39]. Our research has demonstrated MPTP to be a valid target for AP treatment by inhibiting CypD genetically (ppif –/–) and pharmacologically using CsA, DEB025 and TRO40303 (Trophos) [13]. DEB025 proved effective in maintaining membrane potential and inhibiting necrosis in freshly isolated murine and human pancreatic acinar cells exposed to pancreatitis toxins. Moreover, DEB025 significantly reduced all the biochemical and histological parameters in five different experimental models of AP. Common to all ciclosporin analogs synthesized thus far,