CYCLOSPORIN ANALOGUES AND USES THEREOF

Abstract

A compound for use in the treatment or prevention of acute or chronic inflammatory disorders wherein the compound is a compound of Formula 1:

##STR00001##

or a salt thereof,

wherein n is 2-5, and

R.sub.1 and R.sub.2 are independently selected from H or C.sub.1-C.sub.4 alkyl, wherein R.sub.1 and R.sub.2 may be joined together to form a C.sub.3-C.sub.5 heteroalkyl ring.

Claims

1. A method for preserving an organ removed from a donor comprising exposing the organ to a compound which is Compound 1: ##STR00029## or a salt thereof.

2. The method according to claim 1 wherein the compound is administered to an organ donor.

3. The method according to claim 1 wherein the compound is administered shortly before or during surgery.

4. The method according to claim 1 wherein the compound is administered to transgenic animals having organs suitable for human transplantation.

5. The method according to claim 1 wherein the compound is administered to the organ after the removal of the organ from an organ donor.

6. The method according to claim 5 wherein the compound is included in a fluid in which the organ is placed.

7. The method according to claim 5 wherein the compound is included in a fluid that is recirculated through the organ.

8. The method according to claim 1 wherein the compound is administered to a recipient prior to receiving the organ.

9. The method according to claim 1 wherein the compound is administered to a recipient after organ transplantation.

10. The method according to claim 1 wherein the organ is kidney, pancreas, liver, heart, lungs or intestines.

11. The method according to claim 1 wherein the organ is a kidney.

12. The method according to claim 1 comprising administering to a kidney donor a dose of Compound 1: ##STR00030## or a salt thereof.

13. The method according to claim 12 wherein the dose of the compound is 0.1 to 10 mg/kg.

14. The method according to claim 13 wherein the dose of the compound is 1 to 3 mg/kg.

15. The method according to claim 12 wherein the compound is administered 1-8 hours before kidney removal surgery.

16. The method according to claim 1 comprising administering to a kidney recipient a dose of Compound 1: ##STR00031## or a salt thereof.

17. The method according to claim 16 wherein the dose of the compound is 0.1 to 10 mg/kg.

18. The method according to claim 17 wherein the dose of the compound is 1 to 3 mg/kg.

19. The method according to claim 1 wherein the organ is protected against ischaemia-reperfusion injury.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0074] FIG. 1 shows the inhibitory and/or protective effect of Compound 1, and of comparative compound CsA, on induced Acute Kidney Injury in rats, by measuring blood serum Creatinine concentrations.

[0075] FIG. 2 shows the inhibitory and/or protective effect of Compound 1, and of comparative compound CsA, on induced Acute Kidney Injury in rats by measuring Blood Urea Nitrogen (BUN) concentrations.

[0076] FIG. 3 shows the inhibitory and/or protective effect of Compound 1 against LPS induced Acute Kidney injury.

DETAILED DESCRIPTION

[0077] The invention will now be illustrated by the following examples.

[0078] Experimental Methods and Results

[0079] The skilled person will recognise that compounds of Formula 1 may be prepared in a variety of ways. The route below is merely illustrative of a way that could be employed for the synthesis of Compound 1. That said, the route used to prepare Compound 1 in U.S. Pat. No. 6,583,265 was not effective. Many attempts were made to replicate the methodology in U.S. Pat. No. 6,583,265 without great success. Without being bound by theory, it is believed that the dimethyl amino group (being basic) reacted preferentially with the acid catalyst. The acid catalyst was thereby prevented from activating the loss of the leaving group, inhibiting the progress of the reaction.

[0080] Compounds of general formula 1 can be conveniently prepared using several pathways. In one instance (Scheme 1), reaction of compound 2, in which R is lower alkyl, with a carbonyl compound and a reducing agent can perform a reductive amination procedure to a give the desired compounds. Preferably the carbonyl compound is a lower alkyl aldehyde or ketone and the reducing agent is a metal borohydride. More preferably the aldehyde is formaldehyde, acetaldehyde or propionaldehyde and the ketone is acetone, 2-butanone and the like. Preferably the reducing agent is sodium triacetoxyborohydride or sodium cyanoborohydride.

##STR00014##

[0081] The amine compound 2 can be conveniently prepared from a suitably protected ethanolamine compound such as 3, wherein R is hydrogen or lower alkyl, by treating said compound with conditions known to remove the protecting group and yield the free amine compound. Suitable protecting groups that can be removed in the presence of other functional groups in the molecule include tert-butoxycarbonyl (BOC), 9-fluorenylmethyloxycarbonyl (FMOC) and the like. Preferably the protecting group is tert-butoxycarbonyl (BOC) and the conditions employed for removal of the BOC group involve treatment with acid, such as trifluoroacetic acid.

##STR00015##

Step 1: Preparation of [2′-(2-Thiopyridyl)-Sar]3-cyclosporine A

[0082] ##STR00016##

[2′-(2-Thiopyridyl)-Sar].SUP.3.-cyclosporine A (1a

[0083] To a dry 1 L flask was added cyclosporine A (20 g, 16.6 mmol), anhydrous lithium chloride (21.1 g, 499 mmol) and dry THF (500 mL), the flask was flushed with argon and the mixture was cooled to −45 C. In a separate flask, diisopropylamine (13.5 g, 133 mmol) was dissolved in dry THF (120 mL) and cooled to −78 C. To this flask was added n-butyllithium (53.2 mL of a 2.5 M solution, 133 mmol) and the resulting solution was stirred at −78 C for 20 min. Using a canula, the solution of lithium diisopropylamide was transferred to the solution of cyclosporine and the resulting mixture was stirred at −45 C for 90 min. A solution of 2-pyridyldisulfide (11 g, 49.9 mmol) in dry THF (20 mL) was added dropwise and the resulting mixture was allowed to warm to room temperature overnight. The reaction was quenched by the careful addition of saturated NaCl solution (200 mL) and the resulting organic layer was separated. The aqueous layer was extracted with ethyl acetate (3×100 mL) and the combined organic fractions were washed with 3N NaOH (2×100 mL), saturated NH.sub.4Cl (100 mL) and saturated NaCl (100 mL) followed by drying over anhydrous Na.sub.2SO.sub.4 and evaporation. The title compound was isolated by silica gel chromatography as a solid, 7.18 g. .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 8.45 (ddd, J=0.88, 1.73, 4.90 Hz, 1H), 7.98 (d, J=9.66 Hz, 1H), 7.65-7.73 (m, 1H), 7.59 (dt, J=1.85, 7.71 Hz, 1H), 7.51 (ddd, J=0.76, 1.68, 6.44 Hz, 0H), 7.45 (d, J=8.54 Hz, 1H), 7.35 (ddd, J=1.73, 6.97, 8.77 Hz, 0H), 7.25 (s, OH), 7.17 (d, J=7.96 Hz, 1H), 7.09-7.15 (m, 2H), 6.72 (dt, J=1.17, 6.71 Hz, 0H), 5.70 (dd, J=4.29, 10.88 Hz, 1H), 5.50 (d, J=6.39 Hz, 1H), 5.32-5.38 (m, 1H), 5.28 (dd, J=3.88, 11.74 Hz, 1H), 5.13 (d, J=10.88 Hz, 1H), 4.97-5.11 (m, 2H), 4.84 (dq, J=7.03, 7.24 Hz, 1H), 4.69 (t, J=9.15 Hz, 1H), 4.54 (quin, J=7.31 Hz, 1H), 4.13 (q, J=7.16 Hz, 0H), 3.81 (dt, J=1.00, 5.75 Hz, 1H), 3.59-3.72 (m, 1H), 3.50 (s, 2H), 3.38 (s, 2H), 3.26 (s, 2H), 3.13 (s, 5H), 2.70 (d, J=1.07 Hz, 5H), 2.34-2.54 (m, 1H), 1.92-2.23 (m, 4H), 1.55-1.85 (m, 11H), 1.19-1.54 (m, 11H), 1.12 (d, J=6.54 Hz, 2H), 0.78-1.07 (m, 30H), 0.73 (d, 3H).

Step 2: Preparation of [2′-(2-Dimethylaminoethoxy)-Sar]3-cyclosporine A (Compound 1)

[0084] ##STR00017##

[2′(2-Dimethylaminoethoxy)-Sar].SUP.3.-cyclosporine A (1)

[0085] Copper triflate (0.291 g, 0.8 mmol) and 3 angstrom molecular sieves were added to a flask, dry THF (3 mL) was added and the flask was flushed with argon. In a separate flask, a mixture of [2′-(2-thiopyridyl)-Sar].sup.3-cyclosporine A (1a) (0.293 g, 0.223 mmol), dimethylaminoethanol (0.086 g, 0.96 mmol) and 3 A molecular sieves in dry THF (2 mL) was stirred for 30 minutes and then added to the copper triflate solution. The reaction was allowed to stir at room temperature overnight. A saturated solution of NaHCO.sub.3 (10 mL) was added and the mixture was filtered through celite. The celite was washed with ethyl acetate (3×25 mL) and added to the filtrate. The organic layer was separated; the aqueous was extracted with EtOAc (2×25 mL) and the combined organic fractions were dried over anhydrous Na.sub.2SO.sub.4 and evaporated. Purification of the crude material on silica gel afforded the title compound, 86.4 mg. .sup.1H NMR (400 MHz, CHLOROFORM-d) δ 7.92 (d, J=9.61 Hz, 1H), 7.75 (d, J=7.32 Hz, 1H), 7.22 (d, J=8.15 Hz), 7.15 (d, J=7.86 Hz), 6.01 (s, 1H), 5.70 (dd, J=4.22, 10.86 Hz, 1H), 5.46 (d, J=6.10 Hz, 1H), 5.35 (q, J=4.77 Hz, 1H), 5.27 (dd, J=4.15, 11.42 Hz, 1H), 5.14 (d, J=10.83 Hz, 1H), 5.05-5.11 (m, 1H), 4.94-5.04 (m, 1H), 4.77-4.90 (m, 1H), 4.73 (s), 4.66 (t, J=8.83 Hz, 1H), 4.46-4.57 (m, 1H), 3.71-3.81 (m, 1H), 3.58-3.67 (m, J=5.15, 5.64, 5.64, 5.83 Hz, 1H), 3.53-3.58 (m, 1H), 3.51 (s, 2H), 3.24 (s, 2H), 3.20 (s, 2H), 3.13 (d, J=2.10 Hz, 3H), 2.71 (d, J=6.54 Hz, 3H), 2.49-2.67 (m, 2H), 2.33-2.46 (m, 1H), 2.27 (s, 4H), 1.88-2.20 (m, 4H), 1.74 (d, J=0.29 Hz, 6H), 1.57-1.68 (m, 5H), 1.38-1.52 (m, 2H), 1.35 (d, J=7.27 Hz, 3H), 1.26 (d, J=2.88 Hz, 4H), 0.77-1.12 (m, 30H), 0.70 (d, 2H).

[0086] The skilled person will for example appreciate that analogues of Compound 1 can be made by using different amino alcohol reagents. For example, the number of carbon atoms between the alcohol and amine group could be increased or decreased (examples of linking groups include, methylene, ethylene, propylene, butylene, pentalene, and may include branched versions thereof, such as iso-propylene, sec-butylene, tert-butylene, 2-methylbutylene, 2,2-dimethylpropylene). Alternatively, or in addition, the N-amino substituents on the amino alcohol could also be changed to give further analogues of Compound 1 (examples of N-amino substituents include, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl).

Preparation of [2′-(2-N-Boc-aminoethoxy)-Sar]3-cyclosporine A

[0087] ##STR00018##

[0088] Copper triflate (4.95 g, 13.7 mmol) and 3 A molecular sieves were suspended in anhydrous THF (50 mL) and stirred under argon for 30 min. A solution of [2′-(2-thiopyridyl)-Sar].sup.3-cyclosporine A (1) (5.0 g, 3.82 mmol) and N-Boc-ethanolamine (2.64 g, 16.4 mmol) in anhydrous THF (10 mL) was dried over 3 A molecular sieves for 30 min and then added to the copper triflate suspension. The resulting mixture was stirred at room temperature overnight. Saturated NaHCO.sub.3 (2×50 mL) was added and the mixture was filtered through Celite. The Celite pad was washed with EtOAc (4×100 mL) and the organic layer was separated. The aqueous phase was extracted with EtOAc (2×50 mL) and the combined organic fractions were washed with saturated NaCl (50 mL), dried over anhydrous Na.sub.2SO.sub.4 and evaporated. The crude material was purified on silica to yield the title compound, 4.18 g. .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 0.72 (ddd, 2H) 0.91 (m, 31H) 1.32 (m, 8H) 1.48 (dddd, J=3.95, 3.07, 2.23, 0.95 Hz, 2H) 1.69 (m, 10H) 2.10 (m, 4H) 2.39 (m, 1H) 2.70 (m, 4H) 2.95 (m, 2H) 3.12 (d, J=7.42 Hz, 4H) 3.17 (d, J=9.37 Hz, 1H) 3.20 (s, 2H) 3.25 (s, 2H) 3.29 (m, J=6.69, 3.02, 1.45, 0.76, 0.63 Hz, 1H) 3.41 (m, 1H) 3.51 (s, 2H) 3.61 (m, 1H) 3.75 (dddd, J=7.73, 1.54, 1.02, 0.73 Hz, 1H) 4.13 (q, J=7.11 Hz, 1H) 4.50 (m, 1H) 4.65 (dd, J=18.06, 0.44 Hz, 1H) 4.98 (m, 4H) 5.30 (m, 2H) 5.47 (m, 1H) 5.70 (m, 1H) 5.93 (d, J=0.34 Hz) 7.21 (m, 1H) 7.71 (m) 8.03 (m).

Preparation of [2′-(2-aminoethoxy)-Sar].SUP.3.-cyclosporine A

[0089] ##STR00019##

[0090] A solution of [2′-(2-N-Boc-aminoethoxy)-Sar].sup.3-cyclosporine A (3) (3.0 g, 2.2 mmol) in dry CH.sub.2Cl.sub.2 (30 mL) was cooled to 0 C and trifluoroacetic acid (6.54 mL, 10.03 g, 88 mmol) was added dropwise and the mixture was stirred for 30 min. The solvent was evaporated and the crude material was purified on silica to deliver the title compound, 1.99 g.

Preparation of [2′-(2-Dimethylaminoethoxy)-Sar].SUP.3.-cyclosporine A (2)

[0091] ##STR00020##

[0092] [2′-(2-Aminoethoxy)-Sar].sup.3-cyclosporine A (0.273 g, 0.216 mmol) was dissolved in CH.sub.2Cl.sub.2 (5 mL) and formaldehyde (37% aqueous sol., 0.048 mL, 0.69 mmol) was added followed by NaB(OAc).sub.3H (0.138 g, 0.649 mmol) and the reaction was allowed to stir at about room temperature for 18 h. The reaction mixture was filtered through a small pad of silica gel which was washed with 90:9:1 CH.sub.2Cl.sub.2:MeOH:conc.NH.sub.4OH (5×100 mL). The solvent was evaporated and the product was isolated by chromatography on silica gel to afford the title compound, 0.214 g.

[0093] Cyclophilin Inhibition Binding Measurements

[0094] The cyclophilin inhibition binding of compounds disclosed herein was determined using a competitive ELISA adapted from the methods described by Quesniaux et al. (Eur. J Immunol., 1987, 17:1359-1365). Activated ester of succinyl spacers bound to D-Lys.sup.8-cylosporine A (D-Lys.sup.8-Cs) are coupled to bovine serum albumin (BSA) through D-lysyl residue in position 8. BSA is dissolved in 0.1 M borate buffer, pH 9.0 (4 mg in 1.4 ml). A hundredfold molar excess of D-Lys.sup.8-Cs dissolved in dimethyl formamide (0.6 ml) is added drop wise to the BSA under vigorous stirring. The coupling reaction is performed for 2 to 3 hours at room temperature under mild stirring and the conjugate is extensively dialyzed against phosphate-buffered saline (PBS, pH 7.4). After acetone precipitation of an aliquot of the conjugated protein, no covalently bound D-Lys.sup.8-Cs remains in the acetone solution and the extent of cyclosporine covalent binding is calculated.

[0095] Microtiter Plates are coated with D-Lys.sup.8-Cs-BSA conjugate (2 μg/ml in PBS for 24 hours at 4° C.). Plates are washed with Tween®/PBS and with PBS alone. To block nonspecific binding, 2% BSA/PBS (pH 7.4) is added to the wells and allowed to incubate for 2 hours at 37° C. A five-fold dilution series of the compound to be tested is made in ethanol in a separate microtiter plate. The starting concentration is 0.1 mg/mL for assays with human recombinant cyclophilin. 198 μL of 0.1 μg/mL cyclophilin solution is added to the microtiter immediately followed by 2 μL of diluted cyclosporine A (used as a reference compound) or the compound of the invention. The reaction between coated BSA-Cs conjugate, free cyclosporine A and cyclophilin is allowed to equilibrate overnight at 4° C. Cyclophilin is detected with anti-cyclophilin rabbit antiserum diluted in 1% BSA containing PBS and incubates overnight at 4° C. Plates are washed as described above. Bound rabbit antibodies are then detected by goat anti-rabbit IgG conjugated to alkaline phosphatase diluted in 1% BSA-PBS and allowed to incubate for 2 hours at 37° C. Plates are washed as described above. After incubation with 4-nitrophenyl phosphate (1 g/I in diethanolamine buffer, pH 9.8) for 1 to 2 hours at 37° C., the enzymatic reaction is measured spectrophotometrically at 405 nm using a spectrophotometer. The results are expressed as an EC.sub.50, which is the concentration of the compound of the invention required to achieve 50% inhibition. Compound 1 had EC.sub.50 values of less than 100 nM against cyclophilins A, B and D.

[0096] PPIase Inhibition

[0097] The assay was performed using an Agilent 8453 spectrophotometer essentially as described as the ‘uncoupled assay’ by Janowski et al. {Jankowski et al. Anal. Biochem. (1997), 252:299-307}. Assay buffer consisting of 35 mM HEPES pH 7.8 and 50 μM DTT was cooled to 10° C. (with stirring) in a precision glass cuvette and inhibitor was added from a 100% DMSO stock solution. A blank spectrum was obtained and then purified His tagged recombinant human cyclophilin enzyme (f/c 2 nM) and tetra peptide substrate, Suc-Ala-Ala-Pro-Phe-para-nitroanilide dissolved in 0.5 M LiCl in trifluoroethanol (Bachem, f/c 60 μM) were added and the change in absorbance measured over 5 min at 330 nM. A first order rate equation was fitted to the absorbance data to obtain a rate constant (first 10 to 15 s were eliminated due to mixing). The catalytic rate was calculated from the enzymatic rate minus the background rate. The K.sub.i for an inhibitor was obtained from the rate constant plotted against the inhibitor concentration.

[0098] Mitochondrial Permeability Transition

[0099] Mitochondrial Permeability Transition (MPT) is determined by measuring swelling of the mitochondria induced by Ca.sup.2+. The procedure is adapted from the method described by Blattner et al., 2001, Analytical Biochem, 295:220. Mitochondria are prepared from rat livers, which have been perfused with phosphate-buffered saline (PBS) to remove blood, using standard methods that utilize gentle homogenization in sucrose based buffer and then differential centrifugation to first remove cellular debris and then to pellet the mitochondria. Swelling is induced by 150 micro molar Ca.sup.2+ (added from a concentrated solution of CaCl.sub.2)) and is monitored by measuring the scattering at 535-540 nm. Representative compounds are added 5 minutes before swelling is induced. EC.sub.50 is determined by comparing swelling with and without the compounds disclosed herein. Compound 1 inhibited mitochondrial swelling with an EC.sub.50 of less than 0.2 uM.

[0100] Acute Kidney Injury

[0101] Compound 1 and Cyclosporin A formulations were prepared by mixing these compounds with Cremophor/saline/DMSO.

[0102] Sprague-Dawley rats were divided into six groups: Group (i) the sham group, dosed with Cremophor/saline/DMSO with no active; Group (ii) the control group, dosed with Cremophor/saline/DMSO with no active; Group (iii) dosed with Compound 1 (3 mg/kg); Group (iv) dosed with CsA (3 mg/kg); Group (v) dosed with Compound 1 (10 mg/kg); Group (vi) dosed with CsA (10 mg/kg). With the exception of Group (i), i.e. the ‘sham group’, renal Ischemia-Reperfusion Induced Acute Kidney Injury (AKI) was induced in the rats by ligation of bilateral renal arteries for 30 min and then ligation was released.

[0103] Animals in the control and treatment groups were administered intraperitoneal injections three times (1 h before ligation, 4 h and 8 h after ligation). Blood was taken from the animals 24 hours after the ligation/release procedure and analyzed for serum Creatinine and Blood Urea Nitrogen (BUN) concentrations, as a measure of kidney injury.

[0104] The results of those experiments are shown below, and graphically in FIGS. 1 and 2.

##STR00021##

TABLE-US-00001 TABLE 1 Measurement of Cyclophilin A inhibition, Cyclophilin D inhibition and Mitochondrial Permeability Transition (MPT). CypA EC.sub.50 CypD EC.sub.50 Entry X (nM) (nM) MPT (μM) 1 [00022] 61 2170 10 2 [00023] 202 3550 7.6 3 [00024] 14 ND 2.69 4 Compound 1 [00025] 60 24 0.1 5 [00026] 66 ND 10 6 [00027] 118 2500 7.5 7 [00028] 12 1200 10

[0105] The results displayed in Table 1 demonstrate the unexpectedly high Cyclophilin D inhibition and MPT of Compound 1 (entry 4) relative to similar analogues (entries 1-3 and 5-7). A 100 fold improvement in MPT was observed relative to three other compounds (entries 1, 5 and 7) and an over 25 fold improvement was observed relative to the next best performing analogue (entry 3). Compound 1 also displayed superior Cyclophilin D inhibition, with at least a 50 fold improvement relative to all other analogues tested.

TABLE-US-00002 TABLE 2 Measurement of serum Creatinine and BUN concentrations of Groups (i) to (vi) Creatinine (umol/L) Blood Urea Nitrogen (mmol/L) Group (i) 25 5 Group (ii) 195 38 Group (iii) 60 14 Group (iv) 115 22 Group (v) 250 40 Group (vi) 290 42

[0106] LPS induced Acute Kidney Injury (AKI) was induced in mice (C57) by intraperitoneal injection of LPS (15 mg/kg). Twenty mice were randomly divided into two groups. Animals in the control group received vehicle (Cremophor/saline/DMSO) and the treatment group received Compound 1 (3 mg/kg in Cremophor/saline/DMSO) each dosed intraperitoneally. The animals were dosed with vehicle or Compound 1 three times (1 h before LPS injection and 4 h and 8 h after LPS injection) and blood was taken from the animals 12 h after LPS injection. The activity of the compound was determined by increased survival rate and by evaluation of markers of kidney function.

[0107] Discussion of Results

[0108] In FIG. 1 the blood serum Creatinine concentration is indicative of kidney damage. The ‘sham group’ are rats without induced AKI. The ‘control group’ represents rats with induced AKI, and which are untreated. Therefore, it can be seen that induced AKI results in increased levels of Creatinine from 25 umol/ml (Group 1) to 195 umol/ml (Group 2).

[0109] Treating rats with induced AKI with 3 mg/kg of CsA (Group 4) results in the Creatinine levels dropping from 195 umol/ml to 115 umol/ml as compared to Group 2. Therefore, it is understood that CsA is acting to prevent the ischaemia-reperfusion injury.

[0110] Surprisingly, when rats with induced AKI are treated with 3 mg/kg of Compound 1 (Group 3), this gives a very marked reduction in Creatinine levels, dropping from 195 umol/ml to 60 umol/ml (as compared to Group 2), which is approaching the Creatinine levels seen in the ‘sham group’ (Group 1), i.e. rats with no induced AKI. When the dose of Compound 1 and CsA are increased from 3 mg/ml (Groups 3 and 4) to 10 mg/ml (Groups 5 and 6), it appears that the benefit of the CsA and Compound 1 are reduced, with Compound 1 still performing better than CsA.

[0111] In FIG. 2 the Blood Urea Nitrogen (BUN) concentrations is indicative of kidney damage. FIG. 2 follows the same trend as seen in FIG. 1. That is, 3 mg/kg of CsA results in a drop in BUN levels (Group 4 compared to Group 2), whereas 3 mg/kg of Compound 1 shows a very marked reduction in BUN levels (Group 3 compared to Group 2), getting towards the BUN level seen in the ‘sham group’ (Group 1). Increasing the concentration of Compound 1 and CsA from 3 mg/kg (Groups 3 and 4) to 10 mg/kg (Groups 5 and 6) proves to be less effective. This result supports the result seen in FIG. 1.

[0112] Necrotic Cell Death of Murine Pancreatic Acinar Cells by Propidium Iodide

[0113] In order to assess activation of necrotic cell death pathway, 1 μM propidium iodide (PI; λex: 543 nm, λem: 610-690 nm) was used to evaluate plasma membrane rupture. In order to evaluate the inhibition of necrosis by a potential drug, freshly isolated pancreatic acinar cells (PAC) from a CD1 mouse were divided into the four following groups:

[0114] 1. Control group; PACs incubated with sodium HEPES and vehicle (0.5% DMSO).

[0115] 2. Taurolithocholate acid sulphate (TLCS) group; PACs incubated with 500 μM TLCS and vehicle.

[0116] 3. Potential drug only group (for cytotoxicity test); PACs incubated with the potential drug.

[0117] 4. Potential drug+TLCS group; PACs incubated with the potential drug in the presence of 500 μM TLCS.

[0118] For each group, PACs were incubated for 30 minutes at room temperature with gentle shaking. For each group, 8 randomly selected fields (each field contained mostly over 100 cells) of view under confocal microscope were taken of each mouse isolate, and the total number of cells displaying PI uptake were counted per field to give a percentage ratio for each field, averaged across fields, and converted to a mean and standard error of mean for a minimum of three mice per experimental group. The whole assay was performed in a blinded fashion in such a way that the observer choosing the fields and the observer undertaking image analysis were unaware of the treatment groups.

[0119] In Vivo Mouse TLCS Acute Pancreatitis Model

[0120] The experiment measures the ability of a compound to rescue bile acid, taurolithocholylsulphate (TLCS) induced acute pancreatitis in a mouse model. In vivo animal protocols are approved by the UK Home Office. C57BL6/J mice (Charles River UK Ltd) are acclimated for at least 1 week before in vivo experiments. Compounds are made up in the appropriate formulation. TLCS-AP is induced by retrograde pancreatic duct injection of 3 mM TLCS as described previously [Laukkarinen et al., 2007]. Compound is typically administered by i.p. injection, and then animals sacrificed 24 h later. Histology was visualised and serum amylase, interleukin 6 and myeloperoxidase activity were measured as described previously [Mukherjee et al, 2016].

[0121] Serum amylase levels were determined using a Roche automated clinical chemistry analyzer (Roche).

[0122] Serum IL-6 levels were determined by Quantikine mouse ELISA kit (R&D systems).

[0123] Myeloperoxidase (MPO) activity was used as a marker of neutrophil infiltration and determined as described. Pancreatic and lung tissue were first homogenized and resuspended in 100 mM potassium phosphate buffer (pH 7.4) containing protease inhibitor, after repeating centrifugation and resuspension 2-3 times, then further resuspended in 100 mM potassium phosphate buffer (pH 5.4) containing 0.5% hexadecyltrimethyl ammonium bromide, 10 mM EDTA and protease inhibitors, then freeze-thawed three times, sonicated for 30 sec and finally centrifuged for 15 min at 16,000×g. The supernatant was collected and MPO activity with general peroxidase substrates 3,3,5,5-tetramethylbenzidine was measured using H.sub.2O.sub.2. Absorbance was measured at 655 nm. The homogenate protein level was detected using BCA assay kit. MPO activity was normalized based on the protein level of each sample and mean value of TLCS group.

[0124] Histology

[0125] For morphological examination, pancreatic tissues were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E). Histopathological evaluation was assessed blindly on 10 random fields (×10 high power fields) of each slide by two independent investigators, grading the degree and extent of oedema, inflammatory infiltration and necrosis from 0 to 3 [5], calculating summated mean±s.e.m.

[0126] Discussion of Results

[0127] In summary, compound 1 was found to be surprisingly efficacious in the treatment or prevention of pancreatitis when compared to similar compounds.

[0128] In overall summary, Compound 1 was found to be surprisingly efficacious in the treatment or prevention of ischaemia-reperfusion injury, in particular at lower concentration levels. The compound is also particularly efficacious against acute kidney injury and pancreatitis.