Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models

Abstract

Background & aims: Sustained 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 Ca(2+) 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.

Methods: Mouse 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.

Results: GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca(2+) currents after Ca(2+) 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.

Conclusions: Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis.

Keywords: Calcium Entry Inhibition; Drug Development; Experimental Pancreatitis; SOCE; STIM1.

Figure 1

GSK-7975A and CM_128 inhibit CRAC entry (Fura-2 340:380 normalized at 1200 or 2000 s) and necrosis (PI uptake) in human pancreatic acinar cells and CM_128 concentration-dependently inhibits I_CRAC in hORAI1/hSTIM1 HEK 293 cells. (A) Typical trace showing the inhibitory effect of GSK-7975A (50 μmol/L) on thapsigargin-induced Ca²⁺ influx. (B) Mean (±SEM) [Ca²⁺]_C at 1200 and 1400 s from thapsigargin and thapsigargin plus GSK-7975A traces, showing a marked reduction with GSK-7975A (≥20 cells/group; *P < .001; thapsigargin vs thapsigargin plus GSK-7975A at 1400 s). (C) Changes in human pancreatic acinar [Ca²⁺]_C induced by thapsigargin (Fura-2 340:380 normalized at 2000 s), showing the inhibitory effect of 1 μmol/L CM_128. (D) GSK-7975A and CM_128 protected isolated human pancreatic acinar cells from necrotic cell death pathway activation induced by TLCS (500 μmol/L) (mean ± SEM; 3 experiments/group for GSK-7975A; *P < .05, TLCS vs TLCS plus GSK-7975A and 1 experiment/group [4 wells and 16 high-power fields each; total. 172 control cells, 97 TLCS, 110 TLCS, and CM_128] for CM_128; *P < .05, TLCS vs TLCS plus CM_128). (E) Typical trace showing I_CRAC current in response to Ca²⁺-depletion protocol with 1 μmol/L CM_128 in hORAI1/hSTIM1 HEK 293 cells. (F) Concentration-dependent inhibitory effects of CM_128 on I_CRAC current.

Figure 2

GSK-7975A concentration-dependently inhibits CRAC entry (Fura-2 340:380 normalized at 700 s) and necrosis (PI uptake). Changes in mouse pancreatic acinar [Ca²⁺]_C induced by (A) TLCS (500 μmol/L) and (B) CCK (1 nmol/L) showing effects of GSK-7975A from 700 s, expanded. (C and D) Mean (±SEM) [Ca²⁺]_C at 700, 1200, and 2000 s from panels A and B, showing progressive reduction with increasing GSK-7975A (≥19 cells/group; *P < .001, toxin vs toxin plus GSK-7975A at 1200 s; ^†P < .001 at 2000 s). (E) GSK-7975A protected isolated murine pancreatic acinar cells from necrotic cell death pathway activation induced by TLCS (500 μmol/L) (mean ± SEM, normalized to TLCS at 100; ≥3 experiments/group; *P < .001, control vs TLCS; ^†P < .001, TLCS vs TLCS plus GSK-7975A).

Figure 3

CM_128 concentration-dependently inhibits CRAC entry and necrosis (PI uptake). (A) Changes in mouse pancreatic acinar [Ca²⁺]_C induced by thapsigargin (Fura-2 340:380 normalized at 2000 s), showing effect of 1 μmol/L CM_128. (B) Mean (±SEM) [Ca²⁺]_C at 2000 and 3000 s from panel A, showing a marked reduction with 1 μmol/L CM_128 (≥62 cells/group; *P < .001, thapsigargin vs thapsigargin plus CM_128 at 3000 s). (C) CM_128 protected isolated murine pancreatic acinar cells from necrotic cell death pathway activation induced by TLCS (500 μmol/L) (mean ± SEM; ≥3 experiments/group; *P < .001, TLCS vs control; ^†P < .05, TLCS vs TLCS plus CM_128). (D) Concentration-dependent inhibitory effects of CM_128 on cyclopiazonic acid–induced Ca²⁺ influx, showing a progressive reduction of the initial rate of Ca²⁺ entry and plateau with increasing CM_128, with complete inhibition of Ca²⁺ entry at 10 μmol/L (≥17 cells/group). (E) Concentration-dependent inhibitory effects of CM_128 on the initial rate of Ca²⁺ entry.

Figure 4

GSK-7975A markedly reduces all biochemical responses of TLCS-AP, CER-AP, and FAEE-AP. All models resulted in substantial increases of (A) serum amylase, (B) IL6, (C) pancreatic trypsin activity, (D) pancreatic activity, and (E) lung MPO activity. Subcutaneous osmotic minipump administration of GSK-7975A given as prodrug GSK-6288B at low (L) or high (H) doses significantly reduced all parameters, with a more marked reduction of serum amylase and IL6, pancreatic trypsin at the high dose (mean ± SEM ≥6 mice/group; *P < .05, control vs 3 models; ^†P < .05 TLCS-AP vs TLCS-AP plus GSK-7975A; ^‡P < .05, CER-AP vs CER-AP plus GSK-7975A; and ^§P < .05, FAEE-AP vs FAEE-AP plus GSK-7975A).

Figure 5

GSK-7975A markedly reduces pancreatic histopathology in TLCS-AP, CER-AP, and FAEE-AP. All models resulted in substantial increases in (A) edema, (B) inflammation, (C) necrosis, and (D) total histology score. Subcutaneous osmotic minipump administration of GSK-7975A given as prodrug GSK-6288B at low (L) or high (H) doses markedly reduced pancreatic damage, with more marked reduction at high dose (mean ± SEM ≥6 mice/group; *P < .05 control vs 3 models; ^†P < .05, TLCS-AP vs TLCS-AP plus GSK-7975A; ^‡P < .05, CER-AP vs CER-AP plus GSK-7975A; and ^§P < .05, FAEE-AP vs FAEE-AP plus GSK-7975A). (E) Representative images showing normal pancreatic histology, typical histopathology from all 3 models, and typical histopathology from all 3 models after treatment with GSK-7975A at low (L) or high (H) doses (H&E; scale bar: 50 μm).

Figure 6

CM_128 markedly reduces all biochemical responses of TLCS-AP and FAEE-AP. Two models resulted in substantial increases of (A) serum amylase, (B) IL6, (C) pancreatic trypsin activity, (D) pancreatic activity, and (E) lung MPO activity. Intraperitoneal administration of CM_128 at 20 mg/kg given at 1 hour after disease induction (early) and 6 hours after (late) significantly reduced all parameters, with more marked reduction of IL6, pancreatic activity, and lung MPO activity when CM_128 was administered early (mean ± SEM ≥6 mice/group; *P < .05, control vs 2 models; ^†P < .05 TLCS-AP vs TLCS-AP plus CM_128; ^‡P < .05, FAEE-AP vs FAEE-AP plus CM_128; and ^§P < .05, CM_128 early vs late).

Figure 7

CM_128 markedly reduces pancreatic histopathology in TLCS-AP and FAEE-AP. Both models resulted in substantial increases in (A) edema, (B) inflammation, (C) necrosis, and (D) total histology score. Intraperitoneal administration of CM_128 at 20 mg/kg given at 1 hour after disease induction (early) and 6 hours after (late) significantly reduced all parameters, with a more marked reduction when CM_128 was administered early (mean ± SEM ≥6 mice/group; *P < .05, control vs 2 models; ^†P < .05 TLCS-AP vs TLCS-AP plus CM_128; ^‡P < .05, FAEE-AP vs FAEE-AP plus CM_128; and ^§P < .05, CM_128 early vs late). (E) Representative images showing normal pancreatic histology, typical histopathology from 2 models, and typical histopathology from 2 models after treatment with CM_128 early and late after disease induction (H&E; scale bar: 50 μm).

Supplementary Figure 1

GSK-7975A inhibits CRAC entry (Fura-2 340:380 normalized at 700 s). (A) Changes in mouse pancreatic acinar [Ca²⁺]_C induced by CCK (1 nmol/L) with external physiological [Ca²⁺] (1.8 mmol/L) applied, showing the effect of 15 μmol/L GSK-7975A from 700 seconds, expanded (≥79 cells/group). Changes in mouse pancreatic acinar [Ca²⁺]_C induced by (B) TLCS (500 μmol/L) and (C) CCK (1 nmol/L), showing effects of 50 and 100 μmol/L GSK-7975A from 700 seconds, expanded. (D and E) Mean (±SEM) [Ca²⁺]_C at 700, 1200, and 2000 seconds from panels B and C, showing a marked reduction with 50 μmol/L GSK-7975A, but not 100 μmol/L GSK-7975A (≥27 cells/group; *P < .001, toxin vs toxin plus GSK-7975A at 1200 s; ^†P < .001, at 2000 s).

Supplementary Figure 2

GSK-7975A given as prodrug GSK-6288B administered by a subcutaneous osmotic minipump can be delivered consistently to each mouse and maintained throughout the experimental period. There was no detectable GSK-6288B in the blood or pancreas, suggesting complete conversion into GSK-7975A. (A) Blood and pancreas levels of GSK-7975A after administration of 2 mg/kg/h GSK-6288B showing a steady state 4 hours after minipump implantation, when the mean concentrations in blood and pancreas were approximately 0.4 and approximately 0.6 μmol/L respectively. (B) Blood and pancreas levels of GSK-7975A at the (lower) dose of 28 mg/kg/h GSK 6288B (L) reached a steady state 1 hour after minipump implantation, when the mean concentrations in blood and pancreas were approximately 5 and approximately 10 μmol/L, respectively. (C) Blood and pancreas levels of GSK-7975A at the (higher) dose of 110 mg/kg/h GSK-6288B (H) reached a steady state 4 hours after minipump implantation, when the mean concentrations in blood and pancreas were approximately 15 and approximately 50 μmol/L, respectively. (D) Mean (±SEM) plasma and pancreas levels of CM_128 at 20 mg/kg sampling at the time point when drug efficacy was assessed, were approximately 10 and approximately 50 μmol/L, respectively.

Supplementary Figure 3

CM_128 administered from 6 hours after disease induction (late) markedly reduced biochemical responses in TLCS-AP and FAEE-AP. Two models at 6 and 24 hours resulted in substantial increase of (A) serum amylase, (B) IL6, (C) pancreatic trypsin activity, (D) pancreatic activity, and (E) lung MPO activity, with more marked increase of IL6 and lung MPO activity at 6 hours, but of pancreatic trypsin and MPO activity at 24 hours. Intraperitoneal administration of CM_128 at 20 mg/kg late significantly reduced all parameters from levels at 6 hours (mean ± SEM ≥6 mice/group; *P < .05, control vs 2 models at 6 h; ^†P < .05 2 models at 6 vs 24 h; ^‡P < .05, TLCS-AP at 6 h vs TLCS-AP plus CM_128; ^§P < .05, FAEE-AP at 6 h vs FAEE-AP plus CM_128).

Supplementary Figure 4

CM_128 administered from 6 hours after disease induction (late) markedly reduced pancreatic histopathology in TLCS-AP and FAEE-AP. Two models at 6 and 24 hours resulted in substantially progressive increases in (A) edema, (B) inflammation, (C) necrosis, and (D) total histology score, with more marked increase of all scores at 24 hours. Intraperitoneal administration of CM_128 at 20 mg/kg from 6 hours after disease induction significantly reduced edema, but not inflammation, necrosis, or total histology scores at 6 hours (mean ± SEM ≥6 mice/group; *P < .05, control vs 2 models at 6 hours; ^†P < .05 2 models at 6 vs 24 hours; ^‡P < .05, TLCS-AP at 6 h vs TLCS-AP plus CM_128; ^§P < .05, FAEE-AP at 6 h vs FAEE-AP plus CM_128). (E) Representative images showing normal pancreatic histology, typical histopathology from 2 models at 6 and 24 hours, and typical histopathology from 2 models after treatment with CM_128 administered late after disease induction (H&E; scale bar: 50 μm).

Supplementary Figure 5

GSK-7975A administered from 6 hours after disease induction (late) less effectively reduced biochemical responses of TLCS-AP and FAEE-AP. Two models resulted in substantial increases of (A) serum amylase, (B) IL6, (C) pancreatic trypsin activity, (D) pancreatic activity, and (E) lung MPO activity. Subcutaneous osmotic minipump administration of GSK-7975A given as prodrug GSK-6288B at high dose administered from a late time point was less protective than when given early (mean ± SEM ≥6 mice/group; *P < .05, control vs 2 models; ^†P < .05 TLCS-AP vs TLCS-AP plus GSK-7975A; ^‡P < .05, FAEE-AP vs FAEE-AP plus GSK-7975A; and ^§P < .05, GSK-7975A early vs GSK-7975A late).

Supplementary Figure 6

GSK-7975A administered from 6 hours after disease induction (late) less effectively reduced pancreatic histopathology in TLCS-AP and FAEE-AP. Two models resulted in substantial increases in (A) edema, (B) inflammation, (C) necrosis, and (D) total histology score. Subcutaneous osmotic minipump administration of GSK-7975A given as prodrug GSK-6288B at high dose administered from a late time point was less effective that when begun early (mean ± SEM ≥6 mice/group; *P < .05, control vs 2 models; ^†P < .05 TLCS-AP vs TLCS-AP plus GSK-7975A; ^‡P < .05, FAEE-AP vs FAEE-AP plus GSK-7975A, and ^§P < .05, GSK-7975A early vs GSK-7975A late). (E) Representative images showing normal pancreatic histology, typical histopathology from 2 models, and typical histopathology from 2 models after treatment with GSK-7975A administered early and late after disease induction (H&E; scale bar: 50 μm).