CYCLOSPORIN ANALOG AND USE THEREOF

Abstract

The present invention provides a cyclosporin analog and use thereof, and in particular relates to a compound and use thereof as a mitochondrial protective agent for storing a donated organ. The compound is a compound of formula 1 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 can be linked together to form a C.sub.3-C.sub.5 heteroalkyl ring.

##STR00001##

Claims

1. (canceled)

2. (canceled)

3. A method of preserving an organ from an organ donor, the method comprising administering a mitochondrial protectant compound to the organ donor to protect the organ prior to removal of the organ from the organ donor, wherein the compound is a compound of Formula 1: ##STR00029## 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.

4. The method of claim 3 wherein the compound is Compound 1: ##STR00030## or a salt thereof.

5. The method of claim 3 wherein the organ is a kidney.

6. A method of preserving a kidney from a kidney donor comprising administering a compound to said donor prior to removal of said kidney from said donor, wherein the compound is Compound 1: ##STR00031## or a salt thereof.

7. The method of claim 3 wherein the donor is a live donor.

8. (canceled)

9. A The method of claim 3, wherein the dose of the compound is 0.1 to 10 mg/kg.

10. The method of claim 9, wherein the dose of the compound is 1 to 3 mg/kg.

11. The method of claim 3, wherein the compound is administered to a live organ donor prior to an organ transplantation.

12. The method of claim 3, wherein the compound is administered together with one or more further active substances.

13. The method of claim 3, further comprising administering the compound to the organ after removing the organ from the organ donor and prior to a transplantation.

14. The method of claim 3, further comprising administering the compound to an organ recipient after organ transplantation; or shortly before receiving the organ.

15. The method of claim 3, wherein the compound is administered systemically.

16. The method of claim 3, wherein the compound is administered shortly before organ removal surgery, up to 1 to 8 hours before surgery, or during organ removal surgery.

17. The method of claim 3, wherein the compound is administered to protect the organ against ischaemia-reperfusion injury.

18. The method of claim 3, wherein the compound is administered to protect the organ in a period of time between removing the organ from the donor's blood supply to reconnection to a donor recipient's blood supply.

19. The method of claim 3, wherein the compound is formulated for intravenous administration to the donor prior to removal of the organ, or wherein a fluid in which the organ is placed comprises the compound; and/or wherein the compound is a fluid that is adapted for recirculation in and/or through the organ.

20. The method of claim 3, wherein the organ donor is a non-human.

21. The method of claim 20, wherein the organ donor is a transgenic animal.

22. The method of claim 20, wherein the organ donor is a cat, dog, horse, or pig.

23. The method of claim 3, wherein the organ donor is human.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0075] 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.

[0076] 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.

[0077] FIG. 3 shows the inhibitory and/or protective effect of Compound 1 against LPS induced Acute Kidney injury.

[0078] FIG. 4 shows the effects of Compound 1 on kidney function. Lower creatinine and blood urea nitrogen levels for animals treated with Compound 1 are consistent with reduced levels of damage to the kidney.

[0079] FIGS. 5-7 show data showing kidney preservation ex-vivo after removal. 4 kidneys for control, without protectant compound i.v. (intravenous injection) dose before kidney perfusion, 5 kidneys received 5 mg/kg C4066 (compound 1) i.v. dose 1 hr before kidney perfusion. Data shows the average score across the studied kidneys at times after removal at 0, 6, 24 and 48 h. HE score criteria: according to the degree of inflammation from mild to severe, followed by semi-quantitative scoring, for very small or no lesion negative “−” 0; mild or small “+” 1; moderate or medium size “+” 2; severe or large “++” 3; extremely severe or large “+++” 4. FIG. 5 shows the Inflammation score, FIG. 6 the Dilation of the renal capsule and FIG. 7 the Renal tubular dilation.

CHINESE KEY TO DRAWINGS

[0080] FIG. 1: Creatinine: creatinine.

[0081] FIG. 3: LPS induced acute kidney injury: LPS induced acute kidney injury

[0082] FIG. 4: Control: control; Compound: compound; Creatinine: creatinine.

[0083] FIG. 5: Inflammation score: inflammation score.

[0084] FIG. 6: Dilatation of renal capsule: dilatation of renal capsule.

DETAILED DESCRIPTION

[0085] The invention will now be illustrated by the following examples.

[0086] Experimental Methods and Results

[0087] The skilled person will recognise that compounds of Formula 1 may be prepared in a variety of ways. The route below is merely one example 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.

[0088] 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 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 or 2-butanone and the like. Preferably the reducing agent is sodium triacetoxyborohydride or sodium cyanoborohydride.

##STR00014##

[0089] 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 yielding 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].SUP.3.-cyclosporine A

[0090] ##STR00016##

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

[0091] 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 THE (500 mL), the flask was then 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 THE (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 THE (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, OH), 7.45 (d, J=8.54 Hz, 1H), 7.35 (ddd, J=1.73, 6.97, 8.77 Hz, OH), 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, OH), 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, OH), 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].SUP.3.-cyclosporine A (Compound 1)

[0092] ##STR00017##

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

[0093] Copper triflate (0.291 g, 0.8 mmol) and 3 angstrom molecular sieves were added to a flask, dry THE (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 THE (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 layer 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 extract 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).

[0094] 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, butyl).

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

[0095] ##STR00018##

[0096] Copper triflate (4.95 g, 13.7 mmol) and 3 A molecular sieves were suspended in anhydrous THE (50 mL) and stirred under argon for 30 min. A solution of [2′-(2-thiopyridyl)-Sar].sup.3-cyclosporine A (1a) (5.0 g, 3.82 mmol) and N-Boc-ethanolamine (2.64 g, 16.4 mmol) in anhydrous THE (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 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 product 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

[0097] ##STR00019##

[0098] 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 product was purified on silica to deliver the title compound, 1.99 g.

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

[0099] ##STR00020##

[0100] [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.

[0101] Cyclophilin Inhibition Binding Measurements

[0102] The cyclophilin inhibition binding activity 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 dropwise 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 obtained 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 then calculated.

[0103] Microtiter Plates are coated with D-Lys.sup.8-Cs-BSA conjugate (2 μg/ml in PBS for 24 hours at 4° C.). Titration 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 plate 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 serum diluted in PBS containing 1% BSA and incubates overnight at 4° C. Titration Plates are washed as described above. Bound rabbit antibodies are then detected by goat anti-rabbit IgG conjugated to alkaline phosphatase and diluted in 1% BSA-PBS and allowed to incubate for 2 hours at 37° C. Titration 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.

[0104] PPlase Inhibition

[0105] 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 a solution of 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 Ki for an inhibitor was obtained from the rate constant plotted against the inhibitor concentration.

[0106] Mitochondrial Permeability Transition

[0107] 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 precipitate 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 μM.

##STR00021##

TABLE-US-00001 TABLE 1 Measurement results for Cyclophilin A inhibition, Cyclophilin D inhibition and Mitochondrial Permeability Transition (MPT). CypA EC.sub.50 CypD EC.sub.50 MPT Entry X (nM) (nM) (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

[0108] 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.

[0109] Protective Effects of Compound 1 in Animal Models of Organ Damage

[0110] Acute Kidney Iniury Induced by Renal Ischemia-Reperfusion Iniury

[0111] Compound 1 and Cyclosporin A formulations were prepared by mixing these compounds with Cremophor/saline/DMSO.

[0112] Sprague-Dawley rats were divided into six groups: Group (i) the sham group, dosed with Cremophor/saline/DMSO with no active component; Group (ii) the control group, dosed with Cremophor/saline/DMSO with no active component; 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 release of ligation.

[0113] 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.

[0114] The results of those experiments are shown below, and graphically in FIGS. 1 and 2.

TABLE-US-00002 TABLE 2 Measurement results for 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

Discussion of Results

[0115] 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 blood serum Creatinine from 25 μmol/ml (Group i) to 195 μmol/ml group (Group ii). Treating rats with induced AKI with 3 mg/kg of CsA (Group iv) results in the Creatinine levels dropping from 195 μmol/ml to 115 μmol/ml as compared to Group ii. Therefore, it is understood that CsA is acting to prevent the ischaemia-reperfusion injury.

[0116] Surprisingly, when rats with induced AKI are treated with 3 mg/kg of Compound 1 (Group iii), this gives a very marked reduction in Creatinine levels, dropping from 195 umol/ml to 60 umol/ml (as compared to Group ii), which is approaching the Creatinine levels seen in the ‘sham group’ (Group i), i.e. rats with no induced AKI. When the doses of Compound 1 and CsA are increased from 3 mg/ml (Groups iii and iv) to 10 mg/ml (Groups v and vi), it appears that the benefit of the CsA and Compound 1 are reduced, with Compound 1 still performing better than CsA.

[0117] In FIG. 2 the Blood Urea Nitrogen (BUN) concentration 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 iv compared to Group ii), whereas 3 mg/kg of Compound 1 shows a very marked reduction in BUN levels (Group iii compared to Group ii), getting towards the BUN level seen in the ‘sham group’ (Group i). Increasing the concentration of Compound 1 and CsA from 3 mg/kg (Groups iii and v) to 10 mg/kg (Groups v and vi) proves to be less effective. This result supports the result seen in FIG. 1.

[0118] Acute Kidney Injury Induced by Lipopolysaccharide (LPS) Challenge

[0119] 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 (FIG. 3) and by evaluation of markers of kidney function (FIG. 4).

TABLE-US-00003 Creatinine Blood Urea Nitrogen Survival (umol/L) (mmol/L) Control  40% 37 52 Compound 1 100% 26 38

Discussion of Results

[0120] In FIG. 3 the protective effects of Compound 1 are presented in terms of animal survival. At a dose level of 3 mpk, Compound 1 administration resulted in survival of all animals in the group compared to only 4 out of 10 animals in the control group which did not receive Compound 1.

[0121] FIG. 4 shows the effects of Compound 1 on kidney function in this experiment. Lower creatinine and blood urea nitrogen levels for animals treated with Compound 1 are consistent with reduced levels of damage to the kidney.

[0122] Organ Protection During Transplantation by Administration to an Organ Donor.

[0123] The protective effect of Compound 1 toward an organ subjected to conditions of transplantation was exemplified using pig kidneys. In the experiment conducted, a single dose of Compound 1 was administered to the pig at 5 mg/kg via intravenous delivery 1 hr before kidney resection. The kidney was resected, perfused with standard hypertonic citrate adenine (HCA) preservation fluid and then preserved in HCA solution at low temperature (0° C.-4° C.). The organ was monitored to record damage by histologic evaluation and measurement of inflammatory markers over several time points after the resection procedure: [0124] Zero point [0125] 6 h [0126] 24 h [0127] 48 h.

[0128] Data is shown in FIGS. 5-7.

[0129] Histologic evaluation was made following hemotoxalyn and eosin (HE) staining using score criteria according to the degree of inflammation. A semi-quantitative scoring system, “0 to 4” was employed in which very small or no lesion is assigned “0”; mild or small is assigned “1”; moderate is assigned “2”; severe is assigned “3”; extremely severe is assigned “4”.

[0130] The experiment was conducted with a total of 9 pig kidneys in which 4 kidneys were used as controls, without protectant compound, and 5 kidneys received 5 mg/kg Compound 1 i.v. dose 1 hr before kidney resection. Data shows the average across the studied kidneys at times after removal.

[0131] FIG. 5 shows the results of an averaged inflammation score;

[0132] FIG. 6 shows the effects on dilation of the renal capsule and

[0133] FIG. 7 shows the effects on renal tubular dilation.

Discussion of Results

[0134] 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 administered to an organ donor prior to removal of an organ (for subsequent implantation to a recipient). FIGS. 5 to 7 show that an organ can be preserved ex-vivo by administering the compound to a donor prior to organ removal.