COMBINATION

Abstract

The present invention provides a combination comprising (a) a sulfonylurea; and (b) at least one of the following components: (i) an insulin modulator, and (ii) an aldosterone antagonist, for instance a combination comprising (a) glibenclamide, or a structural or functional analogue thereof; and (b) at least one of the following components: (i) exenatide, or a structural or functional analogue thereof, or a pharmaceutically acceptable salt thereof; and (ii) potassium canrenoate, or a structural or functional analogue thereof. Said combinations are suitable for the treatment of stroke and other neurodegenerative disorders and for treating and/or preventing ischemia and/or reperfusion injury in various vital organs, including the brain and the heart. Further aspects of the invention relate to pharmaceutical products and pharmaceutical compositions comprising said combinations according to the invention, and methods of treatment using the same.

Claims

1. A combination comprising: (a) a sulfonylurea; and (b) at least one of the following components: (i) an insulin modulator, and (ii) an aldosterone antagonist.

2. A combination according to claim 1 which comprises a sulfonylurea and an aldosterone antagonist.

3. A combination according to claim 1 which comprises a sulfonylurea and an insulin modulator.

4. A combination according to claim 1 which comprises a sulfonylurea, an insulin modulator, and an aldosterone antagonist.

5. A combination according to any preceding claim wherein the insulin modulator is selected from exenatide, and structural and functional analogues thereof, and pharmaceutically acceptable salts thereof.

6. A combination according to any preceding claim wherein the sulfonylurea is selected from glibenclamide, and structural and functional analogues thereof.

7. A combination according to claim 5 wherein the exenatide structural or functional analogue is a GLP-1 receptor agonist.

8. A combination according to claim 5 wherein the exenatide structural or functional analogue is selected from lixisenatide, albiglutide, liraglutide, taspoglutide and dulaglutide (LY2189265).

9. A combination according to claim 6 wherein the glibenclamide structural or functional analogue is selected from acylhydrazone, sulfonamide and sulfonylthiourea derivatives of glibenclamide, glimepiride, glipizide and gliclazide, preferably gliclazide.

10. A combination according to any preceding claim wherein the aldosterone antagonist is selected from spironolactone, eplerenone, canrenone, potassium canrenoate, finerenone and prorenone, and pharmaceutically acceptable salts thereof where applicable.

11. A combination according to claim 10 wherein the aldosterone antagonist is potassium canrenoate or a structural or functional analogue thereof.

12. A combination according to any preceding claim which comprises at least one further active pharmaceutical ingredient (API) selected from a beta blocker, a renin-angiotensin inhibitor, a statin (HMG-CoA reductase inhibitor), an inhibitor of platelet activation or aggregation, a phosphodiesterase-3 inhibitor, a calcium sensitizer, an antioxidant and an anti-inflammatory agent.

13. A pharmaceutical composition comprising a combination according to any preceding claim and a pharmaceutically acceptable carrier, diluent or excipient.

14. A pharmaceutical composition according to claim 13 in a form suitable for parenteral administration, preferably intravenous administration.

15. A pharmaceutical product comprising: (a) a sulfonylurea; and (b) at least one of the following components: (i) an insulin modulator, and (ii) an aldosterone antagonist.

16. A pharmaceutical product according to claim 15 comprising: (a) glibenclamide, or a structural or functional analogue thereof; and (b) at least one of the following components: (i) exenatide, or a structural or functional analogue thereof, or a pharmaceutically acceptable salt thereof; and (ii) potassium canrenoate, or a structural or functional analogue thereof.

17. A pharmaceutical product according to claim 16 which comprises glibenclamide and exenatide, or a pharmaceutically acceptable salt thereof.

18. A pharmaceutical product according to claim 16 which comprises glibenclamide and potassium canrenoate.

19. A pharmaceutical product according to claim 16 which comprises glibenclamide, potassium canrenoate and exenatide, or a pharmaceutically acceptable salt thereof.

20. A combination according to any one of claims 1 to 12 or a pharmaceutical composition according to any one of claims 13 and 14 for use in the treatment and/or prevention of one or more of ischemia and/or reperfusion injury, stroke, neurodegenerative diseases, neonatal asphyxia, cardiac arrest, cardiogenic shock and acute myocardial infarction, or for use in providing cardioprotection against cardiotoxic drugs, or for use in providing neuroprotection.

21. A combination or a pharmaceutical composition for use according to claim 20 wherein the ischemia and/or reperfusion injury is ischemia and/or reperfusion injury of the brain, heart, lung, kidney, preferably cerebral ischemia, cerebral reperfusion injury or stroke.

22. A combination or a pharmaceutical composition for use according to any one of claims 20 to 21 wherein the components are for administration intravenously.

23. A combination or a pharmaceutical composition for use according to any one of claims 20 to 22 wherein the components are for administration during reperfusion.

24. A combination or a pharmaceutical composition for use according to any one of claims 20 to 22 wherein the components are for administration before reperfusion.

25. A combination or a pharmaceutical composition for use according to any one of claims 20 to 22 wherein the components are for administration after reperfusion.

26. A pharmaceutical product according to any one of claims 15 to 19 for use in the treatment and/or prevention of one or more of the following: ischemia and/or reperfusion injury, stroke, neurodegenerative diseases, neonatal asphyxia, cardiac arrest, cardiogenic shock and acute myocardial infarction, or for use in providing cardioprotection against cardiotoxic drugs, or for use in providing neuroprotection, wherein the components are for administration simultaneously, sequentially or separately.

27. A pharmaceutical product for use according to claim 26 wherein the ischemia and/or reperfusion injury is ischemia and/or reperfusion injury of the brain, heart, lung, kidney, preferably cerebral ischemia, cerebral reperfusion injury or stroke.

28. A pharmaceutical product for use according to any one of claims 26 and 27 wherein the components are for parenteral administration, preferably intravenous administration.

29. A pharmaceutical product for use according to any one of claims 26 to 28 wherein the components are for administration during reperfusion.

30. A pharmaceutical product for use according to any one of claims 26 to 28 wherein the components are for administration before reperfusion.

31. A pharmaceutical product for use according to any one of claims 26 to 28 wherein the components are for administration after reperfusion.

32. A pharmaceutical product for use according to any one of claims 26 to 31 wherein the components are for simultaneous administration.

33. A method of treating and/or preventing one or more of ischemia and/or reperfusion injury, stroke, neurodegenerative diseases, neonatal asphyxia, cardiac arrest, cardiogenic shock and acute myocardial infarction, or for providing cardioprotection against cardiotoxic drugs, or for providing neuroprotection, said method comprising simultaneously, sequentially or separately administering to a subject in need thereof: (a) a sulfonylurea; and (b) at least one of the following components: (i) an insulin modulator, and (ii) an aldosterone antagonist.

34. A method according to claim 33 which comprises simultaneously, sequentially or separately administering to a subject in need thereof: (a) glibenclamide, or a structural or functional analogue thereof; and (b) at least one of the following components: (i) exenatide, or a structural or functional analogue thereof, or a pharmaceutically acceptable salt thereof; and (ii) potassium canrenoate, or a structural or functional analogue thereof.

35. A method according to claim 34 wherein the exenatide structural or functional analogue is a GLP-1 receptor agonist.

36. A method according to any one claims 34 and 35 wherein the exenatide structural or functional analogue is selected from lixisenatide, albiglutide, liraglutide, taspoglutide and dulaglutide (LY2189265).

37. A method according to any one claims 34 to 36 wherein the glibenclamide structural or functional analogue is selected from acylhydrazone, sulfonamide and sulfonylthiourea derivatives of glibenclamide, glimepiride, glipizide and gliclazide, preferably glicazide.

38. A method according to any one claims 33 to 37 wherein the ischemia and/or reperfusion injury is ischemia and/or reperfusion injury of the brain, heart, lung, kidney, preferably cerebral ischemia, cerebral reperfusion injury or stroke.

39. A method according to any one of claims 33 to 38 wherein the components are administered parenterally, preferably intravenously.

40. A method according to any one of claims 34 to 39 wherein the glibenclamide is administered at a dosage of about 0.001 to about 30 μg/kg body weight of the subject.

41. A method according to any one of claims 34 to 39 wherein the exenatide, or pharmaceutically acceptable salt thereof, is administered at a dosage of about 0.001 to about 1.5 μg/kg body weight of the subject.

42. A method according to any one of claims 34 to 39 wherein the potassium canrenoate is administered at a dosage of about 0.03 to about 10 mg/kg body weight of the subject.

43. A method according to any one of claims 33 to 42 which comprises simultaneously administering the components to said subject.

44. Use of: (a) a sulfonylurea; and (b) at least one of the following components: (i) an insulin modulator, and (ii) an aldosterone antagonist; in the manufacture of a medicament for the treatment and/or prevention of one or more of ischemia and/or reperfusion injury, stroke, neurodegenerative diseases, neonatal asphyxia, cardiac arrest, cardiogenic shock and acute myocardial infarction, or for providing cardioprotection against cardiotoxic drugs, or for providing neuroprotection.

45. Use of: (a) glibenclamide, or a structural or functional analogue thereof; and (b) at least two of the following components: (i) exenatide, or a structural or functional analogue, or a pharmaceutically acceptable salt thereof; and (ii) potassium canrenoate, or a structural or functional analogue thereof; in the manufacture of a medicament for the treatment and/or prevention of one or more of ischemia and/or reperfusion injury, stroke, neurodegenerative diseases, neonatal asphyxia, cardiac arrest, cardiogenic shock and acute myocardial infarction, or for providing cardioprotection against cardiotoxic drugs, or for providing neuroprotection.

46. A use according to claim 45 wherein the exenatide structural or functional analogue is a GLP-1 receptor agonist.

47. A use according to claim 45 or claim 46 wherein the exenatide structural or functional analogue is selected from lixisenatide, albiglutide, liraglutide, taspoglutide and dulaglutide (LY2189265).

48. A use according to any one claims 45 to 47 wherein the glibenclamide structural or functional analogue is selected from acylhydrazone, sulfonamide and sulfonylthiourea derivatives of glibenclamide, glimepiride, glipizide and gliclazide, preferably glicazide.

49. A use according to any one claims 44 to 48 wherein the ischemia and/or reperfusion injury is ischemia and/or reperfusion injury of the brain, heart, lung, kidney, preferably cerebral ischemia, cerebral reperfusion injury or stroke.

50. A use according to any one of claims 44 to 49 wherein the components are administered parenterally, preferably intravenously.

51. A use according to any one of claims 44 to 50 wherein the components are administered during reperfusion.

52. A use according to any one of claims 44 to 50 wherein the components are administered before reperfusion.

53. A use according to any one of claims 44 to 50 wherein the components are administered after reperfusion.

54. A use according to any one of claims 44 to 53 which comprises simultaneously administering the components to said subject.

55. Use of a combination comprising: (a) a sulfonylurea; and (b) at least one of the following components: (i) an insulin modulator, and (ii) an aldosterone antagonist; for treating and/or preventing ischemia and/or reperfusion injury in an ex vivo organ prior to or during transplantation.

56. Use of a combination comprising: (a) glibenclamide, or a structural or functional analogue thereof; and (b) at least two of the following components: (i) exenatide, or a structural or functional analogue thereof, or a pharmaceutically acceptable salt thereof; and (ii) potassium canrenoate, or a structural or functional analogue thereof; for treating and/or preventing ischemia and/or reperfusion injury in an ex vivo organ prior to or during transplantation.

57. A use according to claim 56 wherein the exenatide structural or functional analogue is a GLP-1 receptor agonist.

58. A use according to claim 56 or claim 57 wherein the exenatide structural or functional analogue is selected from lixisenatide, albiglutide, liraglutide, taspoglutide and dulaglutide (LY2189265).

59. A use according to any one claims 56 to 58 wherein the glibenclamide structural or functional analogue is selected from acylhydrazone, sulfonamide and sulfonylthiourea derivatives of glibenclamide, glimepiride and gliclazide, preferably glicazide.

60. A use according to any one claims 55 to 59 wherein the ischemia and/or reperfusion injury is cerebral ischemia, cerebral reperfusion injury or stroke.

61. A use according to any one of claims 55 to 60 wherein the components are administered prior to transplantation.

62. A combination according to any one of claims 1 to 12 or a pharmaceutical composition according to any one of claims 13 and 14 for use in the treatment and/or prevention of stroke.

63. A combination according to any one of claims 1 to 12 or a pharmaceutical composition according to any one of claims 13 and 14 for use in the treatment and/or prevention of a neurodegenerative disease, preferably selected from Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis and vascular dementia.

64. A combination according to any one of claims 1 to 12 or a pharmaceutical composition according to any one of claims 13 and 14 for use in providing neuroprotection.

65. A method according to any one of claims 33 to 43 wherein the neurodegenerative disease is selected from Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis and vascular dementia.

Description

[0411] The invention is further described with reference to the accompanying non-limiting examples, and the following figures wherein:

[0412] FIG. 1 shows relative brain infarct volume (%) on Day 7 following repeated administration (7 days) of the low dose triple combination (treatment S) versus that in control animals in Part I and Part II and in animals that received the corresponding monotherapies (treatments A, E, and I) and the corresponding double combinations (treatments M, N, and O) in a rat model of transient middle cerebral artery occlusion. Treatment A: Exenatide 0.05 μg/kg (n=4); Treatment E: Potassium canrenoate 0.33 mg/kg (n=3); Treatment I: Glibenclamide 1 μg/kg (n=4); Treatment M: Exenatide 0.05 μg/kg and Potassium canrenoate 0.33 mg/kg (n=13); Treatment N: Exenatide 0.05 μg/kg and Glibenclamide 1 μg/kg (n=12); Treatment O: Potassium canrenoate 0.33 mg/kg and Glibenclamide 1 μg/kg (n=11); Treatment S: Exenatide 0.05 μg/kg and Potassium canrenoate 0.33 mg/kg and Glibenclamide 1 μg/kg (n=7). Control Part I (n=6); Control animals Part II (n=5).

[0413] FIG. 2 shows the Modified Neurological Severity Score on Day 2 following repeated administration (7 days) of the low dose triple combination (treatment S) versus that in control animals in Part I and Part II and in animals that received the corresponding monotherapies (treatments A, E, and I) and the corresponding double combinations (treatments M, N, and O) in a rat model of transient middle cerebral artery occlusion. Treatment A: Exenatide 0.05 μg/kg (n=4); Treatment E: Potassium canrenoate 0.33 mg/kg (n=3); Treatment I: Glibenclamide 1 μg/kg (n=4); Treatment M: Exenatide 0.05 μg/kg and Potassium canrenoate 0.33 mg/kg (n=13); Treatment N: Exenatide 0.05 μg/kg and Glibenclamide 1 μg/kg (n=12); Treatment O: Potassium canrenoate 0.33 mg/kg and Glibenclamide 1 μg/kg (n=11); Treatment S: Exenatide 0.05 μg/kg and Potassium canrenoate 0.33 mg/kg and Glibenclamide 1 μg/kg (n=7). Control Part I (n=6); Control animals Part II (n=5).

[0414] FIG. 3 shows the Modified Neurological Severity Score on Day 7 following repeated administration (7 days) of the low dose triple combination (treatment S) versus that in control animals in Part I and Part II and in animals that received the corresponding monotherapies (treatments A, E, and I) and the corresponding double combinations (treatments M, N, and O) in a rat model of transient middle cerebral artery occlusion. Treatment A: Exenatide 0.05 μg/kg (n=4); Treatment E: Potassium canrenoate 0.33 mg/kg (n=3); Treatment I: Glibenclamide 1 μg/kg (n=4); Treatment M: Exenatide 0.05 μg/kg and Potassium canrenoate 0.33 mg/kg (n=13); Treatment N: Exenatide 0.05 μg/kg and Glibenclamide 1 μg/kg (n=12); Treatment O: Potassium canrenoate 0.33 mg/kg and Glibenclamide 1 μg/kg (n=11); Treatment S: Exenatide 0.05 μg/kg and Potassium canrenoate 0.33 mg/kg and Glibenclamide 1 μg/kg (n=7). Control Part I (n=6); Control animals Part II (n=5).

[0415] FIG. 4 shows the relative brain infarct volume (%) on Day 7 following repeated administration (7 days) of the higher dose triple combination (treatment T) versus that in control animals in Part I and Part II and in animals that received the corresponding monotherapies (treatments B, F, and K) and the corresponding double combinations (treatments Q, and R) in a rat model of transient middle cerebral artery occlusion. Treatment B: Exenatide 0.15 μg/kg (n=8); Treatment F: Potassium canrenoate 1 mg/kg (n=8); Treatment K: Glibenclamide 10 μg/kg (n=8); Treatment Q: Exenatide 0.15 μg/kg and Glibenclamide 10 μg/kg (n=4); Treatment R: Potassium canrenoate 1 mg/kg and Glibenclamide 10 μg/kg (n=5); Treatment T: Exenatide 0.15 μg/kg and Potassium canrenoate 1 mg/kg and Glibenclamide 10 μg/kg (n=7). Control Part I (n=6); Control animals Part II (n=5).

[0416] FIG. 5 shows the Modified Neurological Severity Score on Day 2 following repeated administration (7 days) of the higher dose triple combination (treatment T) versus that in control animals in Part I and Part II and in animals that received the corresponding monotherapies (treatments B, F, and K) and the corresponding double combinations (treatments Q, and R) in a rat model of transient middle cerebral artery occlusion. Treatment B: Exenatide 0.15 μg/kg (n=8); Treatment F: Potassium canrenoate 1 mg/kg (n=8); Treatment K: Glibenclamide 10 μg/kg (n=8); Treatment Q: Exenatide 0.15 μg/kg and Glibenclamide 10 μg/kg (n=4); Treatment R: Potassium canrenoate 1 mg/kg and Glibenclamide 10 μg/kg (n=5); Treatment T: Exenatide 0.15 μg/kg and Potassium canrenoate 1 mg/kg and Glibenclamide 10 μg/kg (n=7). Control Part I (n=6); Control animals Part II (n=5).

[0417] FIG. 6 shows the Modified Neurological Severity Score on Day 7 following repeated administration (7 days) of the higher dose triple combination (treatment T) versus that in control animals in Part I and Part II and in animals that received the corresponding monotherapies (treatments B, F, and K) and the corresponding double combinations (treatments Q, and R) in a rat model of transient middle cerebral artery occlusion. Treatment B: Exenatide 0.15 μg/kg (n=8); Treatment F: Potassium canrenoate 1 mg/kg (n=8); Treatment K: Glibenclamide 10 μg/kg (n=8); Treatment Q: Exenatide 0.15 μg/kg and Glibenclamide 10 μg/kg (n=4); Treatment R: Potassium canrenoate 1 mg/kg and Glibenclamide 10 μg/kg (n=5); Treatment T: Exenatide 0.15 μg/kg and Potassium canrenoate 1 mg/kg and Glibenclamide 10 μg/kg (n=7). Control Part I (n=6); Control animals Part II (n=5).

[0418] FIG. 7 shows the results of histology TUNEL staining for apoptosis in the hippocampal area of rats treated with the triple combination of exenatide/potassium canrenoate/glibenclamide in a rat model for vascular dementia. More specifically, FIG. 7 shows the percentage of apoptotic cells (average±SEM) for rats treated with exenatide 0.05 μg/kg+potassium canrenoate 0.33 mg/kg+glibenclamide 1 μg/kg (Group 2M; 13 animals; intravenous administration), compared to the vehicle treated control (Group 1M; 9 animals).

EXAMPLES

[0419] The present invention is further illustrated by the following examples, which should not be construed as limiting in any way.

Example 1. Dose-Response Study of the Efficacy of Exenatide, Potassium Canrenoate and Glibenclamide and their Combinations in a Rat Model of Cerebral Ischemia and Reperfusion Injury

[0420] The aim of this study was to assess (a) the dose-response of the neuroprotective effect of glibenclamide, exenatide, and potassium canrenoate in a rat model of cerebral ischemia and reperfusion injury following repeated intravenous administration as monotherapies (Study Part I) and (b) the effect of a combination of the compounds vs the corresponding monotherapies (Study Part II).

[0421] The transient middle artery occlusion (t-MCAO) was performed according to the method described by R. Schmid-Elsaesser et al. (Stroke. 1998; 29(10): 2162-70). Test compounds were administrated intravenously 20 minutes before reperfusion and then twice a day thereafter for six consecutive days. The modified neurological severity score (NSS) was graded on a scale of 0 to 18 (in which normal score was 0 and maximal deficit score was represented by 18), on Study Day 2 (one day following surgery) and on Study Day 7 (7 days post-surgery and before study termination); it included a set of clinical-neurological tests (composite of motor, sensory, reflex and balance tests). At study termination brains were harvested, sliced into five 2 mm thick coronal sections and stained with triphenyl tetrazolium chloride (TTC) for infarct size measurement using ImageJ program. Morbidity, mortality, body weight and clinical observation were also recorded. Numerical results are shown as means±standard deviation of the mean. The statistical significance (P) of treated groups vs the untreated control group was determined using two-way ANOVA followed by Bonferroni post hoc test, using GraphPad Prism 5 program.

[0422] In study Part I, a total of 112 SD male rats (270-320 gr upon arrival) were divided into 13 groups (5 or 10 rats per treated group and 8 rats in the control group). Due to mortality in some of the groups, the numbers within the groups were adjusted to have sufficient animals in all the groups. Groups were as follows:

CONTROL (saline)
EXENATIDE administered at 0.05 μg/kg, 0.15 μg/kg, 0.5 μg/kg and 1.5 μg/kg;
POTASSIUM CANRENOATE administered at 0.33 mg/kg, 1 mg/kg, 3 mg/kg and 10 mg/kg;
GLIBENCLAMIDE administered at 1 μg/kg, 3 μg/kg, 10 μg/kg and 30 μg/kg.

[0423] The results of the efficacy endpoints obtained in Part I are summarised in Table 1, and are also expressed as percentage change vs the corresponding controls including the results of the statistical comparison of each treatment vs the corresponding control groups. Twenty-seven animals died during the study across all groups (1 during the operation, 5 after the occlusion, 1 was euthanised on Day 2, 7 shortly after reperfusion and 13 were found dead in their cages within one-five days after surgery). There was no statistically significant differences in body weight between all animals' groups.

[0424] All monotherapies, with the exception of the lowest doses of exenatide, potassium canrenoate and glibenclamide (treatments A, E and I), showed a statistically significant decrease in the brain infarct size when compared with the control group. There was no clear indication of a dose-response.

[0425] In respect of the modified neurological severity score (NSS), only the lowest doses of exenatide, potassium canrenoate and glibenclamide (treatments A, E and I) and the 3 μg/kg dose of glibenclamide (treatment J) did not show a statistically significant decrease when compared with the control group on Day 2, while on Day 7, only the lowest doses of exenatide, potassium canrenoate and glibenclamide (treatments A, E and I) and the 0.5 μg/kg dose of exenatide (treatment C) did not show a statistically significant decrease when compared with the control group. As in the case of the brain infarct size, there was no clear indication of a dose-response on either day for the modified NSS.

[0426] In study Part II, a total of 86 SD male rats (270-320 gr upon arrival) were divided into 9 groups (7 or 15 rats per treated group and 6 rats in the control group). Due to mortality in some of the groups, the numbers within the groups were adjusted to have sufficient animals in all the groups. Groups were as follows:

CONTROL (saline)
EXENATIDE administered at 0.05 μg/kg, and POTASSIUM CANRENOATE administered at 0.33 mg/kg;
EXENATIDE administered at 0.05 μg/kg, and GLIBENCLAMIDE administered at 1 μg/kg;
POTASSIUM CANRENOATE administered at 0.33 mg/kg and GLIBENCLAMIDE administered at 1 μg/kg;
EXENATIDE administered at 0.15 μg/kg, and POTASSIUM CANRENOATE administered at 0.33 mg/kg;
EXENATIDE administered at 0.15 μg/kg, and GLIBENCLAMIDE administered at 10 μg/kg;
POTASSIUM CANRENOATE administered at 1 mg/kg, and GLIBENCLAMIDE administered at 10 μg/kg;
EXENATIDE administered at 0.05 μg/kg, and POTASSIUM CANRENOATE administered at 0.33 mg/kg and GLIBENCLAMIDE administered at 1 μg/kg;
EXENATIDE administered at 0.15 μg/kg, and POTASSIUM CANRENOATE administered at 1 mg/kg, and GLIBENCLAMIDE administered at 10 μg/kg.

[0427] The results of the efficacy endpoints obtained in Part II are summarised in Table 2, and are also expressed as percentage change vs the corresponding controls including the results of the statistical comparison of each treatment vs the corresponding control groups. Nineteen animals died during the study across all groups (2 were euthanised on Day 6, 4 died shortly after reperfusion and 13 were found dead in their cages within one-five days after surgery). There was no statistically significant differences in body weight between all animals' groups.

[0428] All double combinations (Groups M, N, O, P, Q and R) as well as both triple combinations (Groups S and T) showed a statistically significant change in the brain infarct size when compared with the control group. There was no difference between the double dose (Groups M, N, O, P, Q and R) or the triple dose combinations (Groups S and T). The decrease in brain infarct size following administration of the double combination of the lowest doses of exenatide and potassium canrenoate (Group M) was statistically different from that of the corresponding monotherapies (Groups A and E). The decrease in brain infarct size following administration of the lowest doses of the double combination of exenatide and glibenclamide (Group N) was statistically different only from that of the corresponding glibenclamide monotherapy (Group I). Furthermore, the triple combination with the lowest doses (Group S), but not that of the higher doses (Group T), was shown to be statistically significant in terms of the decrease in the brain infarct size compared to the corresponding monotherapies.

[0429] All double combinations (Groups M, N, O, P, Q, and R) and both triple combinations (Groups S and T) showed a statistically significant decrease in the modified neurological severity score (NSS) when compared with the control group on Day 2 and on Day 7. Furthermore, the decrease in the modified NSS with the triple combination of the lowest doses of exenatide, potassium canrenoate and glibenclamide (Group S) was statistically significantly different to the corresponding potassium canrenoate and glibenclamide monotherapies (Groups E and I) on Day 2 and to all three corresponding monotherapies (Groups A, E and I) on Day 7, but not to any of the corresponding double combinations (Groups M, N and O). The triple combination of the higher doses of exenatide, potassium canrenoate and glibenclamide (Group T) was not statistically significantly different to any of the corresponding double combinations (Groups Q, and R).

[0430] To appreciate the effect of the combination therapies, the triple combination of the lowest doses (treatment “S”) is shown in comparison with the corresponding monotherapies and double combinations in FIGS. 1-3, for the relative brain infarct volume, and the modified neurological severity score on Days 2 and 7, respectively. Similarly, the results of the comparison of the triple combination of the higher doses (treatment “T”) vs the corresponding monotherapies and double combinations are shown in FIGS. 4-6.

[0431] From the results obtained it is clear that low doses of exenatide, potassium canrenoate and glibenclamide, which are ineffective when administered as monotherapies, show a statistically significant efficacy when they are combined (both as double or triple combinations), indicative of a synergistic effect. The present results provide strong evidence that the combination therapy of glibenclamide with exenatide and/or potassium canrenoate [0432] reduced the extent of cerebral infarction and/or [0433] improved the neurological severity score and/or [0434] improved the motor performance score in a synergistic manner,
since the obtained combination effect exceeded the sum of the respective monotherapies' effects. Furthermore, the surprising finding was that the dose of glibenclamide, which produced this synergistic effect in the present study (i.e. 1 μg/kg twice daily that is 0.66 μg in the rats weighing 330 g used in the study), was significantly lower than the dose previously reported in the literature for stroke, i.e. daily infusions of 200 ng/h, that is 4.8 μg (Simard et al, Transl Stroke Res. 2012). Importantly, such very low doses of glibenclamide when used in the context of the present invention correspond to a dose (i.e. 70 μg/day) that is 100 times less than the defined daily dose (7 mg—orally of the micronized formulation) or ˜20 to 285-fold less than the recommended maintenance dose of glibenclamide (micronized formulation), and are therefore expected to be devoid of any effects on blood glucose levels or of any adverse effects. The above clinically effective dose of glibenclamide as a double or triple combination with low doses of exenatide and/or potassium carbonate is also significantly lower than the dose of glibenclamide shown to be neuroprotective (continuous infusions of 0.16 or 0.11 mg/h, that is 3.84 mg or 2.64 mg daily) in the clinical studies published in the literature (see King Z A et al).

Example 2. Study of the Efficacy of Exenatide, Potassium Canrenoate and Glibenclamide Combination in a Rat Model of Vascular Dementia

[0435] Chronic cerebral hypoperfusion model in Wistar rat causes cerebral lesions in the rat brain by permanent occlusion of both common carotid arteries which can also affect cognitive functional deficit. This model is similar to that of Vascular Dementia and the technique can decrease the blood flow in the cerebral cortex and hippocampus by up to 40-80% for several months, which induces certain learning disorders.

Study Objective

[0436] The purpose of the study was to evaluate the neuroprotective efficacy of a combination of exenatide, potassium canrenoate and glibenclamide, given intravenously 24 h after both common carotid arteries permanent ligation and then administered twice daily for three weeks, using the Wistar rat Vascular Dementia model.

Treatment Groups

[0437] Treatment Groups were follows:

Group 1M: Vehicle treated controls (9 animals, intravenous administration);
Group 2M: Exenatide 0.05 μg/kg+potassium canrenoate 0.33 mg/kg+glibenclamide 1 μg/kg (13 animals; intravenous administration).

[0438] Exenatide acetate salt was obtained from Bachem AG, Switzerland. Potassium canrenoate was obtained from Pfizer, Switzerland. Glibenclamide was obtained from Tocris Bioscience.

Study Design and Timeline

[0439] This study evaluated the neuroprotective effect of the combination administered at a low dose intravenously twice a day during three weeks in the Wistar rat Vascular Dementia model. Test compounds were administrated 24 hours after common carotid arteries ligation twice a day for three weeks. On Day 1 both common carotid arteries were permanently ligated. Morris water maze tests were performed before common carotid arteries ligation as training for baseline and on Week 4 and Week 8 thereafter. At study termination brains were harvested. Histological analysis was performed for the tissues.

[0440] The study timeline was as follows:

TABLE-US-00002 Study Day/Week CCAO BW Clinical Observation MWM Treatment Termination On the week ✓ ✓ ✓ before surgery (training) D 1 Surgery ✓ ✓ 4 hr post-surgery, 24 hours after common Week 4 (W 4) Twice twice a day during ✓ carotid arteries ligation, a week first two days, twice a day for three weeks Week 8 (W 8) then twice a week ✓ ✓ CCAO = Common Carotid Arteries Occlusion; D = Day; W = Week; BW = Body Weight; MWM = Morris Water Maze

[0441] First dosing day was assigned “Day 1” and termination was “Day 56”, eight weeks following common carotid arteries ligation.

Histology Analysis

[0442] Tissue preparation and trimming (affected hemisphere), X3 accurate cross sections of the striatum (Corpus Callosum) dorsal hippocampus and optical trac per brain. Paraffin block preparation H&E and TUNEL staining, IHC: Double Cortin for neuro-regeneration in brain sub-ventricular zone. MBP, myelin in white matter, lba-1 for microglia and GFAP for astrocytes. Olig-2 for all Oligodendrocytes, NG2 for young Oligodendrocytes. Slides evaluation analysis; cell bodies counting at hippocampal CA1 and CA3 regions—three sections per brain, three fields per section Morphometric analysis of neuronal death count and MBP.

Animals

[0443] Male Wistar rats were used in the study, weighing 290-390 g at study initiation.

Animal Management

Housing

[0444] Animal handling was performed according to guideline of the National Institute of Health (NIH) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Animals were housed in polyethylene cages (maximum 3 rats/cage) measuring 42.5×26.5×18.5 cm, with stainless steel top grill facilitating pelleted food and drinking water in plastic bottle; bedding: steam sterilized clean paddy husk (Envigo, Sani-chips cat #7090C). Bedding material was changed along with the cage at least twice a week.

Diet

[0445] Animals were fed ad libitum a commercial rodent diet (Teklad Certified Global 18% Protein Diet, Envigo cat #2018SC). Animals had free access to standard tap drinking water obtained from the municipality supply and treated according to Pharmaseed's SOP No. 214: “Water system”. Animal feed arrived with a certificate of analysis and the water was autoclaved prior to use.

Environment Conditions

[0446] Animals were singly housed for the period following surgery in a climate-controlled environment. Air was filtered (HEPA F6/6) with adequate fresh supply (minimum of 15 air changes/hour). Temperatures were maintained at 18-24° C. and relative humidity 30-70%. Animals were exposed to 12-hour light and 12-hour dark cycles (6 AM/6 PM).

Randomization

[0447] Animals were randomly allocated into cages according to Pharmaseed's SOP #027 “Random allocation of animals”.

Procedures and Evaluations

Surgical Procedure: Two Common Carotid Arteries Ligation

[0448] On the day of surgery anesthesia was induced on a heating pad with 4% isoflurane in a mixture of 70% N.sub.2O and 30% O.sub.2 and maintained with 1.5-2% isoflurane. Buprenorphine at 0.1 mg/kg was injected subcutaneously. Two common carotid artery occlusions were performed according to the method described by Hyun Joon Lee et al (Citicoline Protects Against Cognitive Impairment in a Rat Model of Chronic Cerebral Hypoperfusion, J Clin Neurol. 2009; 5(1):33-38). Both Common Carotid Arteries (CCA) were exposed through a midline neck incision and carefully dissected free from surrounding nerves and fascia. Both arteries were double ligated with a 4-0 silk suture at 8-10 mm below the visible region of the external carotid artery. The surgical wound was closed and the animals returned to their cages to recover from anesthesia. Analgesic treatment with Buprenorphine was given again by the end of the day and twice a day during the next four days.

Administration of Combination

[0449] Treatment started 24 hours after arteries ligation, via intravenous (IV) injection. Treatment was performed twice a day for three consecutive weeks.

Body Weight

[0450] Animals' body weight was monitored during acclimation, before common carotid artery ligation and twice a week thereafter. Animals were weighed according to Pharmaseed's SOP No. 010: “Weighing laboratory animals”. Individual body weight changes were calculated.

Clinical Observation

[0451] Clinical signs were monitored once during acclimation, for the first 4 h post-surgery, twice a day during the first two days following surgery, then twice a week.

Morris Water Maze Test

[0452] The Morris water maze (MWM) test is designed to assess cognitive deficits following common carotid arteries ligation. The test was performed according to Pharmaseed's SOP 100 (Morris Water Maze Testing V6) and related publications (e.g. Brandeis R, Brandys Y and Yehuda S, “The use of the Morris Water Maze in the study of memory and learning”, Int J Neurosci. 1989; 48(1-2):29-69).

Pre Surgery Training

[0453] Animals were trained and conditioned for one week in the Morris Water Maze, according to Pharmaseed's SOP 100 and the scientific publications (e.g. see Brandeis R et al). Before MWM, rats' cages were transferred from the animal housing to the behaviour testing room for an acclimation of about one hour.

[0454] The training results on the last day were considered as baseline data for comparison. MWM test had exclusion criteria as follows: failing to escape to the platform in 90 sec. (on Day 3 of training).

Post Common Carotid Arteries Ligation Testing

[0455] Before MWM, rats' cages were transferred from the animal housing to the behaviour testing room for an acclimation of about one hour.

[0456] The MWM test was performed on Week 4 and 8 after common carotid arteries ligation.

Statistical Analysis

[0457] Numerical results are given as means and standard deviations or standard errors. Descriptive statistics and group comparisons of data were performed, whenever possible, using statistical analysis program (GraphPad Prism version 5.02 for Windows, GraphPad Software, San Diego Calif. USA). The appropriate parametric or non-parametric test was performed followed by the appropriate post-hoc analysis. A probability of 5% (p≤0.05) is regarded as statistically significant.

Results

[0458] Preliminary results for the MWM test indicate that animals treated with the triple combination of exenatide/potassium/glibenclamide (treatment Group 2M; 13 animals) performed better than vehicle treated animals (Group 1M; 9 animals) both in terms of the average time taken to reach the platform (sec), and the average distance swim to the platform (cm). Furthermore, quantitative evaluation of histological TUNEL staining for apoptosis in the hippocampal area exhibited statistically significant neuroprotection (at p=0.03 according to the Mann Whitney test) for treatment Group 2M over Group 1M (vehicle). FIG. 7 shows the percentage apoptotic cells (average±SEM) for each group.

[0459] Thus, it can be concluded that the triple combination of exenatide/potassium/glibenclamide when administered at a low dose intravenously twice a day for three weeks exhibited a neuroprotective effect in the Wistar rat Vascular Dementia model, showing both improved cognitive behavior and reduced apoptosis in the hippocampal brain area.

[0460] Various modifications and variations of the described aspects of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

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TABLE-US-00003 TABLE 1 Effects of exenatide, potassium canrenoate and glibenclamide on cerebral infarction on Day 7 and on the modified neurological severity score (NSS) on Days 2 and 7, following repeated administration (7 days) as monotherapies in a rat model of transient middle cerebral artery occlusion. Treatment & Dose Relative brain infarct Modified NSS Modified NSS (number of animals at volume on Day 7 (%) on Day 2 on Day 7 start/on Day 2/on Day 7) (mean + SD) (mean + SD) (mean + SD) CONTROL (n = 8/6/6) 42 ± 4 14 ± 1 13 ± 2 A. EXENATIDE 0.05 μg/Kg 28 ± 5 13 ± 2 13 ± 2 (n = 5/4/4) [−33%; NS]  .sup. [−7%; NS] .sup. [0%; NS] B. EXENATIDE 0.15 μg/Kg 17 ± 6 11 ± 2  9 ± 2 (n = 10/9/8) [−60%; ***] [−21%; **] [−31%; ***] C. EXENATIDE 0.5 μg/Kg  19 ± 12 11 ± 2 11 ± 3 (n = 10/7/6) [−55%; ***] [−21%; *]  [−15%; NS]  D. EXENATIDE 1.5 μg/Kg 16 ± 7 10 ± 1 10 ± 2 (n = 10/10/10) [−62%; ***]  [−29%; ***] [−23%; **]  E. POTASSIUM CANRENOATE 36 ± 6 15 ± 1 12 ± 1 0.33 mg/Kg (n = 5/4/3) [−14%; NS]   .sup.  [7%; NS]  [−8%; NS] F. POTASSIUM CANRENOATE 15 ± 6 12 ± 3  8 ± 1 1 mg/Kg (n = 10/8/8) [−64%; ***] [−14%; *]  [−38%; ***] G. POTASSIUM CANRENOATE  28 ± 11 11 ± 2 10 ± 2 3 mg/Kg (n = 9/8/7) [−33%; **]  [−21%; *]  [−23%; *]  H. POTASSIUM CANRENOATE 12 ± 3 10 ± 1  8 ± 1 10 mg/Kg (n = 10/8/7) [−71%; ***] [−29%; **] [−38%; ***] I. GLIBENCLAMIDE 1 μg/Kg  33 ± 10 14 ± 1 12 ± 1 (n = 5/4/4) [−21%; NS]   .sup.  [0%; NS]  [−8%; NS] J. GLIBENCLAMIDE 3 μg/Kg 19 ± 9 13 ± 3 10 ± 1 (n = 10/7/7) [−55%; ***] .sup. [−7%; NS] [−23%; *]  K. GLIBENCLAMIDE 10 μg/Kg 16 ± 9 11 ± 3  8 ± 2 (n = 10/8/8) [−62%; ***] [−21%; **] [−38%; ***] L. GLIBENCLAMIDE 30 μg/Kg 17 ± 8 11 ± 3  8 ± 1 (n = 10/9/7) [−60%; ***] [−21%; **] [−38%; ***] In brackets is shown the percentage change of the effect in the treatment groups vs the effect in the control group, and the result of the statistical comparison of the effect in the treatment groups vs the control group (NS: not statistically significant; * p < 0.05; ** p < 0.01; *** p < 0.001)

TABLE-US-00004 TABLE 2 Effects of exenatide, potassium canrenoate and glibenclamide on cerebral infarction on Day 7 and on the modified neurological severity score (NSS) on Days 2 and 7, following repeated administration (7 days) as double or triple combinations in a rat model of transient middle cerebral artery occlusion. Treatment & Dose Relative brain infarct Modified NSS Modified NSS (number of animals at volume on Day 7 (%) on Day 2 on Day 7 start/on Day 2/on Day 7) (mean + SD) (mean + SD) (mean + SD) CONTROL (n = 6/5/5) 38 ± 8 14 ± 1 12 ± 1  M. EXENATIDE 0.05 μg/Kg & 13 ± 2 11 ± 1 9 ± 2 POTASSIUM CANRENOATE [−66%; ***] [−21%; ***] [−25%; ***] 0.33 mg/Kg (n = 15/15/13) N. EXENATIDE 0.05 μg/Kg & 17 ± 4 11 ± 1 9 ± 1 GLIBENCLAMIDE 1 μg/Kg [−55%; ***] [−21%; ***] [−25%; ***] (n = 15/12/12) O. POTASSIUM CANRENOATE 21 ± 8 11 ± 2 9 ± 1 0.33 mg/Kg & [−45%; **]  [−21%; ***] [−25%; ***] GLIBENCLAMIDE 1 μg/Kg (n = 15/14/11) P. EXENATIDE 0.15 μg/Kg & 11 ± 1 11 ± 1 9 ± 1 POTASSIUM CANRENOATE [−71%; ***] [−21%; **]  [−25%; **]  0.33 mg/Kg (n = 7/4/4) Q. EXENATIDE 0.15 μg/Kg & 13 ± 5 11 ± 2 8 ± 1 GLIBENCLAMIDE 10 μg/Kg [−66%; ***] [−21%; **]  [−33%; ***] (n = 7/5/4) R. POTASSIUM CANRENOATE 13 ± 5 10 ± 1 8 ± 1 1 mg/Kg & [−66%; ***] [−29%; ***] [−33%; ***] GLIBENCLAMIDE 10 μg/Kg (n = 7/5/5) S. EXENATIDE 0.05 μg/Kg & 12 ± 3 11 ± 1 9 ± 1 POTASSIUM CANRENOATE [−68%; ***] [−21%; **]  [−25%; **]  0.33 mg/Kg & GLIBENCLAMIDE 1 μg/Kg (n = 7/7/7) T. EXENATIDE 0.15 μg/Kg & 16 ± 7 12 ± 2 9 ± 1 POTASSIUM CANRENOATE [−58%; ***] [−14%; *]  [−25%; ***] 1 mg/Kg & GLIBENCLAMIDE 10 μg/Kg (n = 7/7/6) In brackets is shown the percentage change of the effect in the treatment groups vs the effect in the control group, and the result of the statistical comparison of the effect in the treatment groups vs the control group (NS: not statistically significant; * p < 0.05; ** p < 0.01; *** p < 0.001)