Combination of the CRAC Channel Inhibitor CM4620 and Galactose as a Potential Therapy for Acute Pancreatitis

Abstract Acute pancreatitis (AP) is a life-threatening inflammatory disease with no specific therapy. Excessive cytoplasmic Ca2+ elevation and intracellular ATP depletion are responsible for the initiation of AP. Inhibition of Ca2+ release–activated Ca2+ (CRAC) channels has been proposed as a potential treatment, and currently, a novel selective CRAC channel inhibitor CM4620 (Auxora, CalciMedica) is in Phase 2b human trials. While CM4620 is on track to become the first effective treatment for AP, it does not produce complete protection in animal models. Recently, an alternative approach has suggested reducing ATP depletion with a natural carbohydrate galactose. Here, we have investigated the possibility of using the smallest effective concentration of CM4620 in combination with galactose. Protective effects of CM4620, in the range of 1-100 n m, have been studied against necrosis induced by bile acids, palmitoleic acid, or l-asparaginase. CM4620 markedly protected against necrosis induced by bile acids or asparaginase starting from 50 n m and palmitoleic acid starting from 1 n m. Combining CM4620 and galactose (1 m m) significantly reduced the extent of necrosis to near-control levels. In the palmitoleic acid-alcohol–induced experimental mouse model of AP, CM4620 at a concentration of 0.1 mg/kg alone significantly reduced edema, necrosis, inflammation, and the total histopathological score. A combination of 0.1 mg/kg CM4620 with galactose (100 m m) significantly reduced further necrosis, inflammation, and histopathological score. Our data show that CM4620 can be used at much lower concentrations than reported previously, reducing potential side effects. The novel combination of CM4620 with galactose synergistically targets complementary pathological mechanisms of AP.


Introduction
Acute pancreatitis (AP) is a life-threatening, inflammatory human disease with incidence rates of up to 100 people per 100 , 000 per annum and increasing numbers of pediatric cases. 1 , 2 The general mortality rate is typically up to 5%; howev er, adv anced forms of AP dev elop in appr oximatel y 20% of patients with prolonged hospitalization and mor e sev er e complications c har acterized by significant pancr eatic necr osis, a systemic inflammator y r esponse, m ultiple-organ failur e, and an increased mortality of 30%. 1 , 3 -6 Without a specific therapeutic av aila b le in clinics, this devastating disease r e pr esents an increasing burden for society and healthcare services.
Gallstone biliary disease and excessive alcohol consumption are the leading causative factors of AP, responsible for appr oximatel y 70%-80% of cases. 7 -10Gallstone obstruction of the bile duct can result in bile reflux into the pancreatic duct or an increase in pressure, exposing the pancreas to biliary components that induce pancreatic acinar cell (PAC) injury. 11 -13ther known causes of AP are either her editar y 13 or due to side effects of some drugs, such as cancer drugs based on l -asparaginase (ASNase), an essential treatment received by patients suffering from acute lymphoblastic leukemia (ALL), the most common type of cancer affecting children. 14The development of ASNase-induced AP (AAP) in up to 10% of cases becomes the most common reason for ending this life-saving treatment. 15 -20t has been esta b lished pr eviousl y that AP-eliciting agents, including bile acids, alcohol metabolites, and ASNase, cause c ytosolic Ca 2 + o verload in PACs as a result of excessi v e Ca 2 + r elease fr om the internal stor es followed by Ca 2 + entr y mainl y thr ough Orai1/Ca 2 + r elease-acti v ated Ca 2 + (CRAC) channels. 21he aberrant Ca 2 + signaling leads to pr ematur e intracellular acti v ation of digesti v e pr oenzymes (as opposed to normal activation occurring when they are secreted into the pancreatic acinar lumen) and loss of cellular ATP due to mitochondrial dysfunction. 22 -25This results in PAC necrosis and tissue inflammation. 26n recent years, various human diseases have been associated with abnormal CRAC channel activity, including severe disorders of the immune system, allergies, m y ocardial infar ction, thrombosis, Alzheimer's disease, cancer, and AP. 27 -33The recognition of store-operated Ca 2 + entry (SOCE) as a potential therapeutic target for AP dates back to as early as 2000. 34The pharmacological development of specific CRAC channel inhibitors for AP treatment has significantly expanded over recent years. 35The substantial therapeutic appeal of targeting CRAC channels is due to the dependence of intracellular protease activation on cytosolic Ca 2 + overload, which occurs after abnormal Ca 2 + depletion of the endoplasmic r eticulum (ER) and excessi v e Orai1/CRAC channel-mediated Ca 2 + entry. 21 , 29 , 36 -38M4620 (ze gocr actin), a novel small molecule Or ai1/CRAC channel inhibitor developed by CalciMedica, has completed a Phase 2 clinical trial for treating moderate to sev er e AP and is the most advanced pharmaceutical in clinical development for the treatment of AP. 39 , 40 The effectiveness of CM4620 at inhibiting SOCE in PACs, immune cells, and pancreatic stellate cells (PSCs) of a mouse, rat, and human origin, as well as in in vivo mouse and rat models of cerulein-induced AP, was recently demonstr ated.42 Intr avenous infusion of Auxora, the intravenous (IV) em ulsion form ulation of CM4620, in an in vivo rat model of pancr eatitis significantl y diminished pancr eatic edema, acinar cell v acuolization, intrapancr eatic tr ypsin acti vity, and acinar cell necrosis.The expression of inflammatory cytokines in pancreas and lung tissues and cytokine generation in human peripheral blood mononuclear cells and rodent PSCs were markedly decreased, thus revealing a role for Orai1/STIM1 in the cellular inflammator y pathw a ys in v olv ed in AP.Howev er, the efficacy of CM4620 on pancreatic histopathology was not 100%, and higher doses or long-term application of this compound is potentially pr ob lematic due to the inhibitory effects of CM4620 on immune and other cells that could lead to unwanted immunological, muscular, or intestinal consequences.32 , 41 , 42 In in vitro experiments performed by Waldron et al., 42 the reduction of cerulein-induced Ca 2 + entry in mouse PACs in the presence of 1 μM CM6420 was up to 70%, leaving room for a potential reduction of an effective CM4620 concentration to lower levels.However, reduced inhibition of calcium entry could also affect the effecti v eness of the drug treatment.
To enhance the treatment efficacy of reduced concentrations of CM4620, it might be possible to use it in combination with other proposed treatments.Recently, we have shown that energy supplements such as galactose and pyruvate can provide a high degree of protection against pancreatic necrosis in PACs by restoring ATP production , 25 as discussed in detail in several re vie ws. 1 , 5 , 43 ATP metabolism plays a major role in Ca 2 + homeostasis and regulation of PAC function; therefore, maintaining cytoplasmic ATP levels is an ultimate condition of cell survi v al. 1 , 5 Due to cytosolic and mitochondrial Ca 2 + overload during the initial stages of AP, the ATP production by mitochondria is seriously affected causing ATP depletion at a cellular level.For the first time, we have provided detailed evidence of the role of gl ycol ysis in AP in vitro and in vivo . 24 , 25We have demonstrated that a potential mechanism inv olv es the inhibition of hexokinases (HKs), the enzymes that convert glucose into glucose-6phosphate, b y se veral well-known AP-inducing a gents, namel y bile acids, alcohol, and aspar aginase . 25Addition of pyruvate or galactose as a source of energy that acts inde pendentl y of HKs significantly reduced sustained Ca 2 + elevations, ATP loss, and PAC necrosis induced by alcohol metabolites, bile acids, or ASNase. 25Galactose markedl y r educed all main histological parameters of the damage to pancreatic tissue in experimentally induced fatty acid ethyl ester (FAEE)-AP 1 , 33 and AAP 25 in vivo models of AP.The safety of galactose administration in humans, even at high m M concentrations, has also been shown. 48At relati v el y high concentrations (ie, up to 70 m M ), galactose is present in a variety of lactose-free dairy products 44 and is regularly consumed by large proportions of the population.Free galactose is also a component of breast milk at m M concentrations as well as existing in formula milk at concentrations of 2-4 m M . 45 , 46Galactose has been used in a number of clinical trials 47 , 59 with a maximum dose of 1.5 g/kg for up to 18 wk. 48 , 57 , 59 , 60her efor e, a combination of such treatments that target v er y different mechanisms would be highly appropriate and have a high chance of success.
We have aimed our study at investigating the possibility of finding the lowest effecti v e concentration of CM4620 and testing whether its pr otecti v e effects in AP could be enhanced by using a combination of CM4620 and galactose.Such a combination could provide effective protection against pathological effects elicited by AP-inducing agents and at the same time minimize potential adverse effects of CM4620.

Materials
l -Aspara ginase w as purchased fr om Abcam, Cambridge, UK.CM4620 was a gift from CalciMedica, La Jolla, California.Cyclopiazonic acid (CPA) was obtained from Tocris, Bristol, UK.Fluo-4 AM and propidium iodide (PI) were purchased from Thermo Fisher Scientific, Paisley, UK.All other r ea gents wer e fr om Sigma-Aldrich, UK.

Experimental Mouse Model of AP
All r egulated pr ocedur es inv olving animals wer e performed in compliance with the UK Home Office regulations under the Animal (Scientific Pr ocedur es) Act, 1986.C57BL6/J male mice (6-8 wk old, 23 ± 3 g) were obtained from Charles River Laboratories (UK).They were housed with corn cob bedding and an enriched environment, which included nesting material and car dboar d tunnels.Mice were randomly and blindly allocated per experimental group from the available stock.
For the induction of experimental alcohol-induced AP, the mice r ecei v ed 2-hourl y intraperitoneal (IP) injections of palmitoleic acid (POA; 150 mg/kg) combined with ethanol (1.35 g/kg) to induce AP (FAEE-AP) (positi v e contr ol). 25 , 33 In order to reduce potential damage to peritoneal organs at the injection site, 200 μL sterile phosphate-buffered saline (PBS) was injected immediatel y befor e the ethanol/POA injection.Contr ol mice (negati v e contr ol) r ecei v ed 2-hourl y IP injections of PBS alone.Twenty-four hours prior to FAEE-AP induction, analgesia was gi v en for compassionate reasons by oral administration of 2.5 μg/mL bupr enorphine hydr oc hloride .In the CM4620 treatment group, mice were co-administered IP injections of 0.1 mg/kg CM4620 (dissolved in a mixture of dimethylsulfoxide (DMSO) and PBS) together with the first ethanol/POA injections, which wer e gi v en 2 times at 1-h interv als. 25 , 33 In the galactose CM4620 tr eatment gr oup, the drinking w ater w as supplemented with 100 m m galactose 24 h before and during co-administration of IP injections of 0.1 mg/kg CM4620 together with the first ethanol/POA injections, which wer e gi v en 2 times at 1-h intervals.Animals were sacrificed 24 h after the first injection and pancreatic tissue was extracted for histological analysis, to assess the severity of FAEE-AP.Treatment groups consisted of ≥4 mice/group.Pancreatic tissues were fixed in 4% formaldehyde, 24 h before pr ocessing.Fixed pancr eatic tissues wer e then embedded in paraffin and stained with hematoxylin and eosin (H&E).A total of 15 or more random fields (magnification, x200) per slide were assessed for edema, acinar cell necrosis, and inflammatory cell infiltration by 2 independent investigators in a blinded manner using a 0-3 grading scale, as pr eviousl y described. 49

Isolation of PACs
PAC isolation was performed as described pr eviousl y. 50riefly, the pancreas was rapidly dissected from a mouse and washed twice in standard 4-(2-Hydroxy ethyl)piper azine-1-ethanesulfonic acid sodium salt (NaHEPES) buffer solution (140 m M NaCl; 4.7 m M KCl; 10 m M HEPES; 1 m M MgCl 2 ; 10 m M d ( + )glucose, 1 m M CaCl 2 , pH 7.2).The pancreas was injected with collagenase solution and incubated for 5-6 min at 37 • C.After incubation, the tissue was manually agitated by pipetting in NaHEPES buffer.PACs were collected and centrifuged at 200 x g for 1 min.The supernatant was discarded, and the cell pellet was resuspended in fresh NaHEPES buffer solution and centrifuged a second time at 200 x g for 1 min.The final cell pellet was suspended in fresh NaHEPES buffer and used for experiments.All experiments were conducted at room temperature (22 • C).

Cytosolic Ca 2 + Measurements
Fr eshl y isolated PACs were loaded for 45 min with the Ca 2 +sensiti v e fluor escent pr obe Fluo-4 AM (5 μM ).The cells were adhered to glass coverslips and continuously perfused, in a flow chamber, with an NaHEPES-based extracellular solution. 50Fluor escence w as ima ged ov er time using a Leica SP5 2-photon or Leica TCS SPE confocal micr oscopes (Leica Micr osystems, Milton Keynes, UK; 40x oil objective; excitation, 488 nm; emission, 510-560 nm).A Scientifica imaging system based on an inverted Olympus IX71 system (Tokyo, Japan; 40x oil objective; excitation 470 nm; emission 515-560 nm; WinFluo softw ar e w as used for data recording) was also used.

Necr osis Measur ements
Propidium iodide (PI) was used to visualize and count cell necrosis levels with the help of a Lecia confocal microscope TCS SPE.Positi v e PI staining (excitation 532 nm, emission: 585-705 nm), r e pr esented by intense red nucleus staining due to plasma membr ane rupture , allow ed for the detection of necrotic cells.A total of 20-25 images, per condition, were taken and the total number of cells was calculated by counting the number of necr otic (PI positi v e staining) and via b le (PI negati v e staining) cells.At least 3 independent experiments ( N = 3) for each condition were performed ( > 100 cells per condition).This enabled the av era ge percenta ge of necrotic cells of the total number of cells ± SEM to be calculated and presented as a bar chart. 21

Statistical Analysis
Outcome measures (mean ± SEM) were analyzed using the Pearson test to determine the normality of data distribution.Statistical significance and P -v alues wer e calculated using a one-way ANOVA or Kruskal-Wallis tests, with the significance threshold set at .05 and asterisks r e pr esenting the range ( * P < .05,* * P < .01,* * * P < .001,* * * * P < .0001).

Low Submicromolar Concentrations of CM4620 Provide Significant Inhibition of Ca 2 + Entry in PACs
To investigate a potential pr otecti v e effect of concentrations of CM4620 lower than r e ported befor e a gainst excessi v e Ca 2 + entry in AP, we have compared Ca 2 + influx levels in freshly isolated contr ol (untr eated) PACs and cells pr etr eated with 100 n M , 1 μM, or 10 μM CM4620 ( Figure 1 A).Fr eshl y isolated PACs loaded with Fluo-4 AM wer e initiall y perfused with standard buffer in the absence of external Ca 2 + and then perfused in the presence of the specific Sarcoendoplasmic Reticulum Calcium ATPase (SERCA) pump inhibitor CPA (10 μM ) to deplete ER Ca 2 + stor es.Ther eafter, to observ e Ca 2 + entr y, 5 m M CaCl 2 w as added, r esulting in a considera b le rise of [Ca 2 + ] i r e pr esenting Ca 2 + influx ( Figure 1 A).After 500 s when a sta b le [Ca 2 + ] i plateau was reached, cells were perfused with a solution of nominall y fr ee Ca 2 + , causing [Ca 2 + ] i r ecov er y to the baseline.In other experiments, cells were preincubated for 30 min with different concentrations of CM4620 prior to administration of the solution with 5 m M Ca 2 + .CM4620 at 1 or 10 μM CM4620 significantly inhibited the amplitude of Ca 2 + entry as compared to untreated control cells ( P < .0001, Figure 1 A and B).These data are in line with previously published results. 33 , 42However, we have found that the muc h low er concentr ation of 100 n M CM4620 was also able to cause a significant reduction of Ca 2 + influx ( P < .0001, Figure 1 A and B), suggesting that it might be possib le to r educe effecti v e concentrations of CM4620 without a substantial loss of its ability to suppress Ca 2 + entry.

Effects of Low Concentrations of CM4620 on Physiological and Supramaximal Ca 2 + Responses Elicited by ACh in PACs
The secr eta gogue acetylcholine (ACh) plays an important role as a physiological stimulus that controls Ca 2 + signaling in PACs.
Ther efor e , w e ha ve in v estigated a possib le effect of 100 n M and 1 μM ( Figure 2 A-F) of CM4620 on cytosolic Ca 2 + signals evoked  in untreated control cells (141.6 s ± 7.67) ( Figure 2 F).However, in cells treated with 100 n M CM4620, the rate of Ca 2 + recover y w as not significantl y differ ent ( P > .05)fr om contr ol cells ( Figure 2 F).

Effects of Submicromolar Concentrations of CM4620 in Combination with Galactose on Necrosis in PACs
The ability of micromolar concentrations of CM4620 to significantl y r educe mouse PAC necr osis elicited by AP-inducing a gents w as demonstrated pr eviousl y. 33 , 42 In the present study, we extended previous results by testing the effectiveness of nanomolar concentrations of CM4620 alone or in combination with 1 m m galactose to reduce PAC necrosis ( Figure 3 ).Concentrations of CM4620 (1-100 n M ) have been tested under the pathological conditions induced by the nonoxidati v e ethanol metabolite POA or a mixture of bile acids (sodium c holeate , BA) or ASNase. 25In our experiments, the level of PAC necrosis elicited by BA (0.06%) was very significantly reduced by either 50 or 100 n M CM4620 ( P < .0001)( Figure 3 A).Treatment with 10 n M CM4620 did not significantly affect the BA-induced levels of necrosis ( P > .05)( Figure 3 A).However, the mixture of 10 n M CM4620 and 1 m M galactose pr ovided highl y significant pr otection a gainst BA-elicited necr otic cell death ( P < .0001)as compared to cell death levels in the presence of 10 n M CM4620 alone ( Figure 3 A).There was no significant difference between BA and galactose and the combination of BA, galactose, and 10 n M CM4620 ( P > .05), in line with the first observation of insignificant inhibition by 10 n M CM4620 alone ( Figure 3 A).Combining galactose with either 50 or 100 n M CM4620 v er y effecti v el y inhibits BA-induced necrosis ( P < .01 and P < .05,respecti v el y) ( Figur e 3 A) .In line with our pr evious data, 25 1 m m galactose v er y significantl y inhibited necr osis induced b y B A alone ( P < .0001).
We have also tested the protective effect of CM4620 against POA-induced necr osis ( Figur e 3 B).In comparison to the avera ge necr osis lev el of untr eated contr ol cells (gray column), tr eatment with 30 μM POA substantially increased the number of necrotic cells (red column) ( P < .0001)( Figure 3 B).Pretreatment of PACs with 1, 10, or 50 n M CM4620 (green columns) significantly r educed lev els of necr osis compar ed to POA alone ( P < .0001).Furthermore, 50 n M CM4620 reduced necrosis to a level that w as not significantl y differ ent fr om necr osis in contr ol cells ( P > .05).Ther efor e, the application of 50 n M CM4620 together with 1 m M galactose had a similar significant effect on POAev oked necr osis ( P > .05) to that of 50 n M CM4620 alone.Both 1 n M CM4620 and 10 n M CM4620 alone highly significantly reduced POA-induced cell death rates compared to necrosis due to POA treatment ( P < .0001, Figure 3 B).Addition of galactose significantl y decr eased necr osis lev els as compar ed to 1 or 10 n M CM4620 alone ( P < .001and P < .01,r especti v el y, Figur e 3 B).In fact, a combination of treatments almost entirely inhibited POA-ev oked necr osis: CM4620 at all 3 concentrations with galactose decr eased necr osis lev els induced by POA to the control level ( P > .05).Galactose alone also significantl y pr otected cells a gainst POA-ev oked necr osis ( P < .0001),similar to our previous r esults.Howev er, adding 10 or 50 n M CM4620 significantly r educed necr osis further ( P < .001) as compared to galactose alone.These results demonstrate a synergy between CM4620 and galactose that allows us to use CM4620 at significantly lower concentrations than have been used previously.
While the pr otecti v e effects of CRAC channel blockers against alcohol or bile-induced PAC injur y hav e been well documented pr eviousl y, 21 , 33 their effecti v eness in r educing ASNase-elicited toxicity r equir es further inv estigation.Ther efor e , w e studied changes in PAC necrosis levels induced by ASNase (200 U/mL) in the presence of 50 n M CM4620, a concentration that has been shown to have a highly significant pr otecti v e effect in our experiments with POA-induced necrosis ( Figure 3 B).The results shown in Figure 3 C demonstrate that ASNase-elicited necrosis in PACs w as v er y significantl y r educed following PAC tr eatment with 50 n M CM4620 alone ( P < .0001)and further reduced when used in combination with 1 m M galactose ( P < .001).Treatment of cells with galactose alone ( Figure 3 C) decreased ASNase-induced necrosis to a level that is similar to the effect of 50 n M CM4620 tr eatment.Howev er, using 50 n M CM4620 with 1 m M galactose significantl y r educed necr osis ( P < .001) as compar ed to galactose alone.

Effectiveness of Low Concentrations of CM4620 in Combination with Galactose in In Vivo Alcohol Mouse Model of AP
We have now investigated the protective effect of CM4620 at a muc h low er dose (0.1 mg/kg) than has been pr eviousl y published 25 , 33 in an in vivo experimental mouse model of AP induced by a mixture of alcohol and POA (FAEE-AP) as described previously.Results shown in F igure 4 A-E demonstr ate that the injection of 0.1 mg/kg CM4620 alone at the time of FAEE-AP induction significantl y r educed the total histological scores ( P < .0001),including edema ( P < .0001),necrosis ( P < .0001),and inflammatory cell infiltration ( P < .0001), in pancreatic tissue as compared to a positive control (FAEE-AP).A combination of CM4620 with pr etr eatment of mice with 100 m M galactose in the drinking water significantly improved the histological score ( P < .05),necrosis ( P < .01),and inflammation ( P < .001),whereas it did not significantl y impr ov e pancreatic tissue edema ( P > .05)as compared to CM4620 treatment alone.A combination of treatments also reduced both necrosis and inflammation to control levels ( P > .05 as compared to untreated control).We believe that such combinational therapy is the way forward in developing potential treatments for AP.

Discussion
It has been accepted that AP is initiated by intracellular Ca 2 + overload, causing mitochondrial malfunction, ATP loss, and prematur e intracellular acti v ation of digesti v e enzymes, leading to necrosis. 1 , 2 In physiological Ca 2 + signaling in PACs, the classical secr eta gogue ACh and the hormone cholecystokinin (CCK) evoke r e petiti v e cytosolic Ca 2 + oscillations at physiologically relevant concentrations. 1 , 50 These oscillations are required for the normal physiological functioning of PACs, namely, the secretion of digesti v e enzymes and fluid.However, our data show that micromolar concentrations of CM4620 inhibited physiological calcium spikes induced by ACh in PACs ( Figure 2 A), prompting a need for the reduction of the CM4620 concentration in the treatment of pancreatitis.Our data ( Figure 2 A-C) show that concentrations of CM4620 at least 10 times lower ( < 100 n M ) are capable of preserving the physiological function of PACs.Although 100 n M of CM4620 displayed only partial inhibition of the calcium entry in PACs, the effect w as highl y significant ( Figur e 1 ), allowing the use of substantially lower concentrations of CM4620.
Recent resear c h for potential AP treatments has been largely focussed on reducing the enhanced Ca 2 + entry through Or ai1/CRAC c hannels in PACs acti v ated as a result of excessi v e Ca 2 + r elease fr om internal stores. 1 , 5 , 21 , 33 , 34 , 42 We have demonstrated pr eviousl y that pr ev ention of alcohol meta bolite-ev oked excessi v e Ca 2 + entr y by b locka ge of Or ai1/CRAC c hannels with GSK-7975A (GlaxoSmithKline) in vitro is beneficial for cell survi v al. 21 Our findings have been strengthened later by in vivo studies that have demonstrated the protective effect of GSK-7975A and a novel potent Or ai1/CRAC c hannel bloc ker CM4620 (ze gocractin, CalciMedica) against toxicity in AP. 33 , 42 As a result, the nanoem ulsion form ulation of CM4620, Auxora, is curr entl y used in several clinical trials in patients with sev er e AP and COVID-19-induced pneumonia. 39 , 40 , 51 , 52It has been demonstrated that Auxora is rapidly distributed to the pancreas and lungs, providing effecti v e inhibition of Or ai1/CRAC c hannels in these tissues. 39Recent r esults fr om the current Phase 2b, which is a randomized, doub le-b lind, placebo-contr olled dose-ranging clinical trial (NCT04681066) of Auxora in patients with AP and associated systemic inflammator y r esponse syndr ome and hypoxemia (CARPO) 40 , r e ported a fav ora b le safety pr ofile of the drug with a significant reduction in the pr oinflammator y cytokines and the disease severity. 39 , 40Pr eviousl y, it has been shown that the efficacy of CM4620 in pr ev enting histopatholog ical chang es of the mouse pancreas in experimental models of AP was potent but incomplete. 33Mor eov er, higher doses or long-term application of this compound could be challenging due to unwanted imm unological, m uscular, or intestinal side effects. 32 , 41 , 42The function of immune cells, as nonexcitable cells similar to pancreatic acinar cells, relies on Ca 2 + entry mechanisms that inv olv e Or ai1/CRAC c hannels.Ther efor e, the b locka ge of these channels by CM4620 would result in a profound inhibition of imm une cell r esponse affecting the patients' r ecov er y after AP and other inflammatory conditions.Previous studies have also observ ed sev er e bacterial dysbiosis and the r eduction in antimicr obial secr etion in Orai1 KO mice within the first 3 wk resulting in up to 70% mortality. 41Furthermore, loss-of-function mutations of Or ai1 w ere linked to the increased risk of immunological and muscle disease in humans. 32However, current clinical trials of CM4620 for the treatment of severe AP and COVID-19 pneumonia r e port pr omising r esults demonstrating the effecti v eness and safety of the drug for patients. 39 , 40At present, experimental and clinical data have demonstrated that targeting SOCE by CM4620 is an effecti v e and pr omising therapeutic av en ue for combatting AP.
Recently another Or ai1/CRAC c hannel inhibitor, CM5480, was also successfully used to inhibit the pr ogr ession of r ecurr ent AP to chronic pancreatitis (CP) by protecting against Ca 2 + overload in pancreatic acinar and duct cells. 53 , 54However, it is also paramount to consider the risks and the benefits of prolonged treatments with Or ai1/CRAC c hannel bloc kers for patients with AP or CP.It has been demonstrated pr eviousl y that genetic deletion of Orai1 in PACs in mice caused bacterial outgrowth, dysbiosis, systemic inflammation, and significant mortality. 41At the same time, a recent paper suggested that a partial (70%) knockout of Orai1 in the pancreas has protected it against AP but failed to protect against associated lung injury. 61On the other hand, specific knockout of Orai1 in neutrophils protected against lung injury but failed to protect against pancreatic damage in AP. 61 Extension of this work and, in particular, a combination of approaches could provide better results. 62n humans, CRAC channelopathies with loss-of-function mutations in Orai1 predispose to severe immunodeficiencies, autoimm unity, m uscular hypotonia, and other a bnormalities. 32Waldron et al. have demonstrated the profound effect of CM4620 on the immune cells in the pancreas. 42Therefore, the longterm inhibition of Or ai1/CRAC c hannels with Auxor a should be considered with caution.One way to reduce such risks is to reduce the inhibitor concentration to the effecti v e minim um.We hav e mana ged to substantiall y r educe the r equir ed CM4620 concentrations to inhibit pancreatic pathology in vitro .The lowest effecti v e concentration of CM4620 that significantly inhibited PAC necrosis induced by the POA (30 μM ) was 1 n M ( Figure 1 B).Higher levels of CM4620 were required to significantl y decr ease lev els of cell necr osis induced by bile acids ( Figure 3 A).Nevertheless, it seems that 50 n M of CM4620 was highly effective for all 3 pathological stimulations in vitro ( Figure 3 A-C).Therefore , w e suggest that it is possible to use much lower doses of the inhibitor for effecti v e pr otection a gainst toxicity induced by the main AP-inducing agents.
Our in vitro findings have been used to investigate the potential pr otecti v e effects of a reduced dose of CM4620 in an experimental in vivo mouse model of alcohol-induced AP (F AEE-AP) ( Figure 4 A-E).W e injected mice with 0.1 mg/kg CM4620, which is 20 times lower than the doses r e ported pr eviousl y. 33 , 42 While we did not measure levels of the drug in the pancreas, linear dose dependence was demonstrated in a previous study with CM4620 in mice, allowing us to extrapolate estimated levels of this compound in the pancreas after a single IP dose of 0.1 mg/kg of around 70 ng/mL, which is still a bov e the lower limit of detection of the bioanalytical assay (10 ng/mL) (CalciMedica, unpublished).In addition, because the formulation of the compound in our study used DMSO and PBS , w e w ould expect gr eater a bsorption and, ther efor e , higher concentr ations within the tissue than in the previous experiment, so the pancreatic concentration could be higher than 70 ng/mL.Our results demonstrate that treatment with 0.1 mg/kg of CM4620 significantl y impr ov ed patholo gical histolo gy scor es r elev ant to AP such as pancreatic tissue edema, necrosis, and inflammation, demonstrating the r emarka b le potency of CM4620 ( Figure 4 A-E).
Low er concentr ations of CM4620 inevita b l y r educed the pr otecti v e effects against AP both in vitro ( Figure 3 A-C) and in vivo ( Figure 4 A-E) as compared to results in earlier r e ports. 33 , 42Nevertheless, our data show that even a r elati v el y small r eduction of Ca 2 + influx into acinar cells was already beneficial for cell survi v al in AP.Howev er, r educed ATP pr oduction r emains to be central in the development of the disease.Ther efor e, boosting ATP production and cellular metabolism with the help of energy supplements such as galactose has synergistically supported cytoplasmic Ca 2 + clearance by the plasma membrane Ca 2 + ATPase the plasma membrane Ca 2 + ATPase (PMCA) and SERCA pumps while restoring Ca 2 + handling mechanisms and pr ev enting necr otic cell death. 1 , 25 Ther efor e , w e hav e consider ed using a combination of CM4620 with another proposed AP treatment.
We have previously shown that the application of galactose to stimulate ATP production by mitochondria has a significant pr otecti v e effect a gainst AP in vitro and in vivo. 25Ther efore , w e have used the addition of 1 m M galactose with low concentrations of CM4620 and found a significant impr ov ement in PAC survi v al challenged b y B A, POA, or ASNase, bringing the degr ee of necr osis to the contr ol lev el (untr eated cell death rate) ( Figure 3 C).These results suggest that simultaneous restriction of Ca 2 + entry by CM4620 and stimulation of ATP production by the energy supplement galactose can allow full compensation for the reduced effect of a low dose of CM4620 for PAC survival.Inter estingl y, galactose alone also showed a significant reduction of cell necrosis induced b y B A, POA, or ASNase .How ever, a combination with low doses of CM4620 synergistically increased the pr otecti v e effect of galactose, emphasizing the major role of ATP in PAC survi v al under pathological conditions.
Similarly to the in vitro results, we have shown that the combination of CM4620 with galactose synergistically reduced both necrosis and inflammation parameters to the control levels ( Figure 4 C and D), as well as significantly reduced the histology score, in an in viv o e xperimental model of alcohol-induced AP in mice.This compr ehensi v e in vivo evaluation confirmed the adv anta ge of using Or ai1/CRAC c hannel inhibition and an energy supplement as a novel therapeutic strategy.
Energy supplements 55 and stimulation of intracellular energy pr oduction 56 hav e been suggested pr eviousl y as a tr eatment for AP.High energy administration in the early phase of AP is being tested in a multicenter, randomized, double-blind clinical trial. 55Our recent findings 25 suggest using the carbohydrate galactose to compensate for ATP in AP.Galactose has a clear adv anta ge for use in clinical studies since it is a natural fr eel y av aila b le substance and part of some food products (lactose-fr ee dair y), including ba by milk mixtur es. 46Galactose can be administered by IV injection of up to 0.5 g/kg 57 or by feeding (drink) protocols. 58Clinical trials have shown that oral galactose at a dose of up to 50 g/d can be safely consumed and w ell toler ated by patients, 47 , 48 exce pt in v er y rar e cases of galactosemia . 63Our new results suggest a need for clinical trials with galactose in combination with low submicromolar doses of CM4620 for patients in the early phase of AP.Such a combination would allow synergistic inhibition of calcium overload, using two independent mechanisms, while reducing the potential unwanted effects of the Or ai1/CRAC c hannel inhibitor.Since galactose works synergistically with the Orai1/CRAC channel inhibitor, it could also help in combination with other inhibitors of Ca 2 + entry or Ca 2 + release to reduce Ca 2 + overload in other pathologies.

Ac kno wledgments
Authors express their gratitude to Prof. Ole H. Petersen for his help in the pr e paration of this manuscript.

Figure 1 .
Figure 1.Submicromolar concentrations of CM4620 significantly inhibit Ca 2 + entry in PACs in a dose-dependent manner.(A) Representative traces depict Ca 2 + entry in PACs in the presence or absence of treatment with different concentrations of CM4620 (100 n M , 1 μM, or 10 μM ).To observ e Ca 2 + entr y, cells were treated with CPA (10 μM ) in nominally Ca 2 + -free standard buffer to deplete the ER Ca 2 + followed by the addition of 5 m M Ca 2 + .(B) Effect of CM4620 on cytosolic Ca 2 + ([Ca 2 + ] i ) amplitude change ( F / F o ) as a result of Ca 2 + entry in PACs.CM4620 significantl y r educed the av era ge amplitudes of Ca 2 + signals r e pr esenting the extent of Ca 2 + entry in PACs at 100 n M (red bar, n = 24), 1 μm (blue bar, n = 23), or 10 μM (green bar, n = 16) as compared to control cells (untreated with CM4620, dark gr ay tr ace , n = 39) ( P < .0001).Cells were loaded with Fluo-4 AM.Bars presented as mean ± SEM.

Figure 2 .
Figure 2. CRAC channel inhibitor CM4620 at a concentration of 1 μM significantly reduces cytosolic Ca 2 + signals induced by physiologicall y r elev ant concentrations of ACh.(A) Re pr esentati v e traces depict [Ca 2 + ] i oscillations induced by 20 n M ACh in control cells (green tr ace , n = 33), cells preincubated with 100 n M CM4620 (blue tr ace , n = 21), or in cells preincubated with 1 μM CM4620 (red tr ace , n = 9).(B) Quantitative analysis of inte gr ated [Ca 2 + ] i signals shown in (A) by calculation of av era ged areas under the curve of ACh-elicited [Ca 2 + ] i responses (time interval 800 s from the addition of ACh) in the presence of 100 n M CM4620 (blue bar) as compared to control ( P > .05)or in the presence of 1 μM CM4620 (red bar, * P < .05)as compared to control (green bar).(C) Comparison of the maximal amplitudes of the oscillations shown in (A).Av era ged maximal amplitudes of ACh-induced [Ca 2 + ] i signals in cells preincubated with 100 n M CM4620 (blue bar) compared to control ( P > .05)or in the presence of 1 μM CM4620 (red bar, * * * P < .0001)as compared to control (green bar).(D) Representative traces demonstrate [Ca 2 + ] i global signals evoked by 1 μM ACh in control cells (green tr ace , n = 60), cells preincubated with 100 n M CM4620 (blue tr ace , n = 19), or cells preincubated with 1 μM CM4620 (red tr ace , n = 27).(E) Comparison of the av era ge ar eas under the curve of [Ca 2 + ] i changes induced by a high concentration of ACh (1 μM ) shown in (D) in the presence of 100 n M CM4620 (blue bar) or 1 μM ACh (red bar).The responses to ACh in the presence of 100 n M CM4620 were significantly lower ( * P < .05)than in control and highly significantly lower in the presence of 1 μM CM4620 ( * * * P < .001).(F) Comparison of the half-time of [Ca 2 + ] i r ecov er y following maximal stimulation with 1 μM ACh shown in (D) in the presence of either 100 n M CM4620 ( P > .05,blue bar) or 1 μM ACh ( * P < .05,red bar).Data represent mean ± SEM.Cells were loaded with Fluo-4 AM.Experiments were performed in a standard buffer containing 1 m M CaCl 2 .

Figure 3 .
Figure 3.A combination of galactose and reduced concentrations of CM4620 provides significant protection against necrosis in PACs induced by sodium c holeate , POA, or ASNase .(A) The treatment of cells with 10 n M CM4620 did not protect against sodium choleate (B A) (0.06%)-e voked necrosis as compared to the necr osis lev el pr oduced b y B A alone ( P > .05).Howe ver, a combination of 10 n M CM4620 and 1 m M galactose does markedly diminish cell death induced by BA as compared to a combination of 10 n M CM4620 and BA ( P < .0001).CM4620 at concentrations of 50 and 100 n M significantly reduced PAC necrosis induced b y B A ( P < .0001).Galactose (1 m M ) supplement has also significantl y r educed BA-elicited necrosis further to nearly the control level in cells treated with 50 or 100 n M CM4620 ( P < .05 and P > .05,r especti v el y, when compared to control).The combination of galactose with CM4620 (50 and 100 n M ) has significantl y incr eased the pr otection pr ovided by CM4620 alone ( P < .01 and P < .05,r especti v el y).Tr eatment of cells with 1 m M galactose alone v er y highl y significantl y decr eased necr osis induced b y B A ( P < .0001).At least 3 experiments per gr oup wer e performed with mor e than 150 cells in eac h sample .Data presented as mean ± SEM. (B) POA (30 μM )-evoked necrosis is significantly reduced by treatment of cells with CM4620 in a dose-dependent manner (1, 10, and 50 n M ) ( P < .0001).The addition of 1 m M galactose to cells treated with 1 and 10 n M CM4620 was effective in significantly reducing POA-induced necrosis in cells, as compared to CM4620 treatments alone ( P < .001and P < .01,respectively).Also, 50 n M CM4620 w as a b le to reduce necrosis to the control level ( P > .05).The combination of galactose (1 m M ) with CM4620 (1 and 10 n M ) significantly increased the pr otection pr ovided by CM4620 alone ( P < .001and P < .01,r especti v el y).No further significant reduction was found between 50 n M CM4620 alone and in combination with 1 m M galactose ( P > .05).Treatment of cells with 1 m M galactose alone v er y highl y significantl y decr eased necr osis induced by POA ( P < .0001).At least 3 experiments per gr oup wer e performed with more than 150 cells in each sample.Data presented as mean ± SEM. (C) CM4620 (50 n M ) significantl y r educed the extent of necr osis ( P < .0001)induced by ASNase (200 U/mL).Using a combinational appr oach, appl ying a mixtur e of galactose (1 m M ) and 50 n M CM4620 reduced ASNase-evoked necrosis further ( P < .05 as compared to 50 n M CM4620 alone).A combination of CM4620 and galactose reduced ASNaseinduced necrosis to the control level ( P > .05).Galactose (1 m M ) alone was able to significantly reduce ASNase-elicited cell necrosis ( P < 0001).At least 3 experiments per group were performed with more than 150 cells in eac h sample .Data presented as mean ± SEM.

Figure 4 .
Figure 4.A combination of galactose and a reduced dose of CM4620 markedly diminishes AP development in an in viv o e xperimental mouse model of alcohol-induced AP (FAEE-AP).(A) Re pr esentati v e ima ges of H&E-stained pancr eatic acinar tissue sections demonstrate a normal pancr eatic tissue histology in contr ol, in FAEE-AP, and in tissue from FAEE-AP mice with CM4620 (0.1 mg/kg) treatment in the absence or presence of galactose (100 m M ) supplement in drinking water (CM4620 + FAEE-AP or Gal + CM4620 + FAEE-AP, r especti v el y).Ma gnification x200, scale bar: 50 μm.(B-E) Significant increases ( P < .0001) in inflammation (B), necrosis (C), edema (D), and total histology score (E) have been observed in pancreatic tissue of mice with FAEE-AP as compared to control.Administration of 0.1 mg/kg CM4620 via intraperitoneal injections markedl y pr otected a gainst all pathological changes evoked by POA and ethanol (FAEE-AP) in vivo ( P < .0001).Combinational treatment of mice by addition of 100 m M galactose to the drinking water before and during the induction of FAEE-AP with CM4620 injection reduced further necrosis and inflammation ( P < .01 and P < .001,r especti v el y) as well as histological score ( P < .05).Both necrosis and inflammation have been reduced to untreated control levels ( P > .05)by the combination of CM4620 and galactose.Experimental groups consisted of ≥4 mice/group.Data are shown as mean ± SEM.