COMBINATION THERAPY COMPRISING A POLYUNSATURATED KETONE AND A CORTICOSTEROID

Abstract

A synergistic pharmaceutical composition for simultaneous, parallel, sequential or separate use comprising a polyunsaturated ketone, a corticosteroid and, optionally, a secosteroid partner calciptriol. The composition has utility in the treatment and prevention of skin disorders.

Claims

1. A pharmaceutical composition comprising: (A) at least one compound of formula (I):
R-L-COX(I) wherein R is a C.sub.10-24 unsaturated hydrocarbon group optionally interrupted by one or more heteroatoms or groups of heteroatoms selected from S, O, N, SO, SO.sub.2, said hydrocarbon group comprising at least 4 non-conjugated double bonds; L is a linking group forming a bridge of 1 to 5 atoms between the R group and the carbonyl CO wherein L comprises at least one heteroatom in the backbone of the linking group; and X is an electron withdrawing group; or a pharmaceutically acceptable salt, or a hydrate or solvate thereof; and (B) one or more corticosteroid partners, preferably selected from the group consisting of betamethasone, clobetasol, halometasone, dexamethasone, fluocortolone, desoximetasone, diflorasone, fluocinonide, flurandrenolide, halobetasol, amcinonide, halocinonide, triamcinolone, hydrocortisone, aclometasone, fluticasone, mometasone, clocortolone, fluocinolone, desonide, prednisone, prednisolone, and prednicarbate or a pharmaceutically acceptable salt, or a hydrate or solvate thereof, especially betamethasone or a pharmaceutically acceptable salt, or a hydrate or solvate thereof.

2. The pharmaceutical composition of claim 1 wherein the composition is a fixed combination or non-fixed combination.

3. A pharmaceutical composition as claimed in claim 1 for simultaneous, parallel, sequential or separate use comprising a kit comprising a first composition comprising at least one compound (I) and a pharmaceutically-acceptable diluent or carrier, and a second composition comprising at least one compound (B) and a pharmaceutically-acceptable diluent or carrier.

4. A composition as claimed in claim 1 wherein the compound (B) is betamethasone, dexamethasone or fluocortolone or a pharmaceutically acceptable salt, or a hydrate or solvate thereof.

5. A composition as claimed in claim 1 wherein the compound (B) is betamethasone or a pharmaceutically acceptable salt, or a hydrate or solvate thereof.

6. A composition as claimed in claim 1 wherein the compound (B) is betamethasone diproprionate, betamethasone valerate, betamethasone acetate or betamethasone sodium phosphate.

7. A composition as claimed in claim 1 wherein in formula (I), the group X is CHal.sub.3, preferably CF.sub.3.

8. A composition as claimed in claim 1 wherein in formula (I), the group R is a linear unsubstituted C.sub.10-24 unsaturated alkylene group comprising at least 4 non-conjugated double bonds.

9. A composition as claimed in claim 1 wherein L is SCH.sub.2.

10. A composition as claimed in claim 1 wherein said compound of formula (I) has the formula: ##STR00007## wherein X is as defined in claim 1, e.g. CF.sub.3.

11. A composition as claimed in claim 1 wherein the compound of formula (I) is Compound A or Compound A2: ##STR00008## especially when compound (B) is betamethasone or a salt thereof.

12. A composition as claimed in claim 1 wherein the molar ratio of compound (A) to (B) in the composition is 1:1 to 1:20.

13. A composition as claimed in claim 1 further comprising clotrimazole, gentamicin, salicylic acid or calcipotriol or a pharmaceutically acceptable salt, or a hydrate or solvate thereof.

14. A composition as claimed in claim 13 further comprising calcipotriol or a pharmaceutically acceptable salt, or a hydrate or solvate thereof.

15. A composition as claimed in claim 14 comprising compound A, betamethasone or a pharmaceutically acceptable salt, or a hydrate or solvate thereof and calcipotriol or a pharmaceutically acceptable salt, or a hydrate or solvate thereof.

16. (canceled)

17. A method of treating, such as reducing symptoms of, or preventing a skin disorder such as psoriasis or dermatitis in a patient in need thereof comprising administering to said patient, preferably a human, an effective amount of a composition as claimed in claim 1.

18. A method of treating, such as reducing symptoms of, or preventing a skin disorder such as psoriasis or dermatitis in a patient in need thereof comprising administering to said patient, preferably a human, an effective amount of at least one compound of formula (I) and simultaneously, in parallel, separately or sequentially administering to said patient at least one compound (B), wherein formula (I) and compound (B) are defined in claim 1.

19. A method of treating such as, reducing symptoms of, or preventing a skin disorder such as psoriasis or dermatitis, in a patient in need thereof comprising: (i) identifying a patient who has received either a compound of formula (I) as or a compound (B) respectively; and (ii) administering to said patient an effective amount of either at least one compound (B) or at least one compound of formula (I) so that said patient is administered with both a compound of formula (I) and a compound (B), wherein formula (I) and compound (B) are as defined in claim 1.

20. A method of treating, such as reducing symptoms of, or preventing a skin disorder such as psoriasis or dermatitis in an animal subject in need thereof comprising administering to said animal an effective amount of a composition as claimed in claim 1.

21. A method of treating, such as reducing symptoms of, or preventing a skin disorder such as psoriasis or dermatitis in an animal subject in need thereof comprising administering to said animal an effective amount of at least one compound of formula (I) and simultaneously, in parallel, separately or sequentially administering to said animal at least one compound (B), wherein formula (I) and compound (B) are defined in claim 1.

22. The method of claim 20, wherein the animal subject is a rodent, monkey, or a pig.

23. The method of claim 21, wherein the pharmaceutical composition or the effective amount of compound of formula (I) and compound (B) is used as a positive control.

24. (canceled)

25. The pharmaceutical composition of claim 1 comprising betamethasone or a pharmaceutically acceptable salt, or a hydrate or solvate thereof optionally in combination with one or more additional corticosteroids or a pharmaceutically acceptable salt, or a hydrate or solvate thereof.

26. The pharmaceutical composition as claimed in 25, wherein the additional corticosteroid is selected from the group consisting of clobetasol, halometasone, dexamethasone, fluocortolone, desoximetasone, diflorasone, fluocinonide, flurandrenolide, halobetasol, amcinonide, halocinonide, triamcinolone, hydrocortisone, aclometasone, fluticasone, mometasone, clocortolone, fluocinolone, desonide, prednisone, prednisolone, and prednicarbate or a pharmaceutically acceptable salt, or a hydrate or solvate thereof.

27. A pharmaceutical composition as claimed in claim 1 in a form suitable for topical administration, e.g. a cream, gel, foam or ointment.

Description

DESCRIPTION OF FIGURES

[0123] FIG. 1 shows the results of the combination therapy of the invention. Co-treatment with cPLA2a inhibitor Compound A and corticosteroid Betamethasone 17, 21-dipropionate shows synergistic effects on decreasing keratinocyte cell proliferation and viability compared to each inhibitor alone. Average and standard deviation of 2-4 independent experiments performed in series of 8 technical replicates per treatment.

[0124] FIG. 2 shows co-treatment with corticosteroid betamethasone and vitamin D analogue calcipotriol shows synergistic effects on keratinocyte cell proliferation and viability compared to each inhibitor alone. Average and standard deviation of 2-4 independent experiments performed in series of 8 technical replicates per treatment. The use of betamethasone and calcipotriolis a known synergistic psoriasis treatment. FIG. 2 is added to show that the results of the present invention are comparable to the results in FIG. 2, proving the presence of synergy.

[0125] FIG. 3 shows a dose response of Compound A on immortalized keratinocyte cell line HaCat cell viability. Data presented are average and standard deviation of 3 independent experiments performed in series of 8 technical replicates per treatment. Star (*) represent significant difference compare to control (100%) (*P0.05; **P0.01; ***P0.001; ****P0,0001).

[0126] FIG. 4 shows a dose response of betamethasone on immortalized keratinocyte cell line HaCat cell viability. Data presented are average and standard deviation of 3 independent experiments performed in series of 8 technical replicates per treatment. Star (*) represent significant difference compare to control (100%) (*P0.05; **P0.01; ***P0.001; ****P0,0001).

[0127] FIG. 5 shows co-treatment with compound A and betamethasone has synergistic effects on human Keratinocyte cell viability compared to each inhibitor alone. Data presented are average and standard deviation of 3 independent experiments performed in series of 8 technical replicates per treatment. Star (*) represent significant difference in compare to control (100%) and in between inhibitors indicated with bars (*P0.05; **P0.01; ***P0.001; ****P0,0001).

[0128] FIG. 6 shows co-treatment of Compound A with Calcipotriol and Betamethasone has a synergistic effect on human Keratinocyte cell viability. Data presented are average and standard deviation of 1 independent experiments performed in series of 8 technical replicates per treatment. Star (*) represent significant difference in compare to control (100%) and in between inhibitors indicated with bars (*P0.05; **P0.01; ***P0.001; ****P0,0001).

EXAMPLE 1

[0129] The following compounds were used in the Experiments:

##STR00006##

Co-Treatment Compound A & Betamethasone:

Methods:

Cell Culture:

[0130] The spontaneously immortalized, nontumorigenic skin keratinocyte cell line HaCaT was maintained in DMEM supplemented with 5% (v/v) FBS, 0.3 mg/ml glutamine and 0.1 mg/ml gentamicin at 37 C. with 5% CO.sub.2 in a humidified atmosphere. Subculture using trypsin-EDTA was performed every 3-4 days with split ratio of 1:3-1:4 to ensure actively proliferating cells.

Resazurin Assay:

[0131] Cells were seeded in 96 well plates in fully supplemented medium at a density of 2500 cells per well. Following 72 hours of cultivation, the cells were starved of serum in 0.25% FBS/DMEM overnight to halt proliferation, synchronize the cells and to increase cell sensitivity to treatment. On day 4, the cells were treated with cPLA2a inhibitor Compound A and corticosteroid Betamethasone 17, 21-dipropionate (Sigma Aldrich # B1152) and left to incubate for 2 hour in incubator at 37 C. with 5% CO.sub.2 in a humidified atmosphere before fluorescence was read at 544 nm excitation and 590 nm emission wavelength. The cells were observed under the microscope to evaluate possible morphology changes and signs of stress before addition of resazurin. The experiments were performed in series of 8 wells per treatment and repeated 2-3 times.

Results:

[0132] Co-treatment with cPLA2a inhibitor Compound A and corticosteroid betamethasone shows synergistic effects on decreasing keratinocyte cell proliferation and viability compared to each inhibitor alone.

[0133] Initial experiments were performed to determine dose response of Compound A alone. The inhibitor slightly reduced cell proliferation and viability at 10 M, whereas 5 M did not show any affect (FIG. 1). On this basis, combination treatment experiments were designed in which sub-effective doses of the Compound A inhibitor and Betamethasone were combined.

[0134] Following 24 hours of treatment, 50 M of Betamethasone and 5 or 10 M Compound A alone showed little or no effect on reducing proliferation and viability of HaCaT cells, whereas 15 M Compound A clearly reduced viability by 70%. However, when combining the sub-effective 5 and 10 M doses of Compound A and Betamethasone, a significant 40% and 80% reduction of proliferation and viability was observed (FIG. 1). This observed trend of synergistic effects on cell proliferation and viability indicates beneficial effects of co-treatment of on skin disorders Several key pathways are dysregulated in skin disorders such as psoriasis and atopic dermatitis. cPLA2a inhibitors represent a promising adjuvant treatment to other drugs in treatment of the inflammation and itching caused by a number of skin conditions such as psoriasis and dermatitis.

EXAMPLE 2

Co-Treat Betamethasone & Calcipotriol

Methods:

Cell Culture:

[0135] The spontaneously immortalized, nontumorigenic skin keratinocyte cell line HaCaT was maintained in DMEM supplemented with 5% (v/v) FBS, 0.3 mg/ml glutamine and 0.1 mg/ml gentamicin at 37 C. with 5% CO.sub.2 in a humidified atmosphere. Subculture using trypsin-EDTA was performed every 3-4 days with split ratio of 1:3-1:4 to ensure actively proliferating cells.

Resazurin Assay:

[0136] Cells were seeded in 96 well plates in fully supplemented medium at a density of 2500 cells per well. Following 72 hour of cultivation, the cells were starved of serum in 0.25% FBS/DMEM overnight to halt proliferation, synchronize the cells and to increase cell sensitivity to treatment. On day 4, the cells were treated with corticosteroid Betamethasone 17, 21-dipropionate (Sigma Aldrich # B1152) and vitamin Danalogue Calcipotriol hydrate (Sigma Aldrich # C4369) for 24 hours. On day 5, resazurin was added according to the manufacturer's instruction (RnD Systems, UK) and left to incubate for 2 hour incubator at 37 C. with 5% CO in a humidified atmosphere before fluorescence was read at 544 nm excitation and 590 nm emission wavelength. The cells were observed under the microscope to evaluate possible morphology changes and signs of stress before addition of resazurin. The experiments were performed in series of 8 wells per treatment and repeated 2-3 times.

Results:

[0137] Co-treatment with corticosteroid Betamethasone and vitamin Danalogue Calcipotriol shows synergistic effects on decreasing keratinocyte cell proliferation and viability compared to each inhibitor alone.

[0138] Betamethasone and Calcipotriol combination has already been established in treatment of Psoriasis. We here tested this established co-treatment to verify our methodology. Following 24 hours of treatment, 50 M of Betamethasone and 10 M Calcipotriol alone showed a 10% and 20% reduction of cell proliferation respectively which increased to a 35% reduction when given in combination (FIG. 2). This observed trend of synergistic effects on cell proliferation and viability show the relevance of the rezasurin assay and validate the previously reported beneficial effects of Betamethasone and Calcipotriol co-treatment on skin disorders.

EXAMPLE 3

[0139] Compound A and betamethasone show dose response on immortalized keratinocyte cell line HaCat cell viability.

Cell Culture:

[0140] The spontaneously immortalized, nontumorigenic skin keratinocyte cell line HaCaT was maintained in DMEM supplemented with 5% (v/v) FBS, 0.3 mg/ml glutamine and 0.1 mg/ml gentamicin at 37 C. with 5% CO.sub.2 in a humidified atmosphere. Subculture using trypsin-EDTA was performed every 3-4 days with split ratio of 1:3-1:4 to ensure actively proliferating cells.

Resazurin Assay:

[0141] Cells were seeded in 96 well plates in fully supplemented medium at a density of 3000 cells per well. Following 48-72 hour of cultivation, the cells were starved of serum in 0.25% FBS/DMEM overnight to halt proliferation, synchronize the cells and to increase cell sensitivity to treatment. Next day, the cells were treated with compound A and betamethasone dipropionate for 24 hours. Resazurin was added next day according to the manufacturer's instruction (RnD Systems, UK) and left to incubate for 2 hour in incubator at 37 C. with 5% CO.sub.2 in a humidified atmosphere before fluorescence was read at 544 nm excitation and 590 nm emission wavelength. The cells were observed under the microscope to evaluate possible morphology changes and signs of stress before addition of resazurin. The experiments were performed in series of 8 wells per treatment and repeated 2-3 times.

Results

[0142] In this study, experiments were performed to determine dose response of Betamethasone dipropionate and compound A. Compound A was found to affect cell viability at 15 M, whereas at doses 1-10 M no signs of impairment in cell viability were observed (FIG. 3).

[0143] On the other hand, Hacat keratinocytes show resistance to betamethasone up to 200 M (FIG. 4). The little effect seen is rather because of higher concentration of solvent DMS than Betamethasone (FIG. 4).

EXAMPLE 4

[0144] Co-treatment with compound A and Betamethasone shows synergistic effects on immortalized keratinocyte cell line HaCat cell viability compared to each inhibitor alone. Example 4 employs the same assay as example 3.

[0145] As noted in FIG. 3/4, the suboptimal dose of viability effect found in compound A and betamethasone was 10 M and 50 M (FIG. 3/4). Combination of the compound A and betamethasone were also compared with already established combination of betamethasone and calcipotriol. Following 24 hours of treatment, doses of betamethasone (50 M) and calcitoriol (10 M) shows 45% reduction in proliferation of HaCat cells. But the combination of compound A (10 M) with Betamethasone (50 M) modestly reduced an additional 25% more viability which is nearly 70% (FIG. 5). This observed trend of synergistic effects on cell viability indicate beneficial effects of co-treatment of compound A and betamethasone on skin disorders.

EXAMPLE 5

[0146] Co-treatment of compound A with vitamin D analogue Calcipotriol and corticosteroid hormone receptor agonist Betamethasone shows synergistic effects on immortalized keratinocyte cell line HaCat viability both in dual and triple combination in compared to each inhibitor alone.

Cell Culture:

[0147] The spontaneously immortalized, nontumorigenic skin keratinocyte cell line HaCaT was maintained in DMEM supplemented with 5% (v/v) FBS, 0.3 mg/ml glutamine and 0.1 mg/ml gentamicin at 37 C. with 5% CO.sub.2 in a humidified atmosphere. Subculture using trypsin-EDTA was performed every 3-4 days with split ratio of 1:4 to ensure actively proliferating cells.

Resazurin Assay:

[0148] Cells were seeded in 96 well plates in fully supplemented medium at a density of 3000 cells per well. Following 72 hour of cultivation, the cells were starved of serum in 0.25% FBS/DMEM overnight to halt proliferation, synchronize the cells and to increase cell sensitivity to treatment. Next day, the cells were treated with Compound A, vitamin D analogue Calcipotriol and corticosteroid hormone receptor agonist Betamethasone dipropionate for 24 hours. Resazurin was added next day according to the manufacturer's instruction (RnD Systems, UK) and left to incubate for 2 hour in incubator at 37 C. with 5% CO.sub.2 in a humidified atmosphere before fluorescence was read at 544 nm excitation and 590 nm emission wavelength. The cells were observed under the microscope to evaluate possible morphology changes and signs of stress before addition of resazurin. The experiments were performed in series of 8 wells per treatment and repeated 2-3 times.

Results:

[0149] Initial experiments were performed to determine dose response of Compound A and Calcipotriol and Betamethasone alone. Combination treatment was designed in which sub-optimal doses of the inhibitor Compound A and Calcipotriol and Betamethasone were combined. Combination of Compound A with Calcipotriol and Betamethasone were compared with already established combo of Betamethasone and Calcipotriol. Following 24 hours of treatment, 12 M of Calcipotriol and 50 M of Betamethasone shows 45% reduction of cell viability which increased to nearly 80% when same concentration of Calcipotriol is given with Compound A 7 M. In addition, combination of Compound A 7 M with 50 M of Betamethasone cause 60% reduction.

[0150] In the same way, Calcipotriol 8M and Betamethasone 30 M does not have any effect on cell viability. Nevertheless, addition of 7M to that dual combination cause almost 80% reduction, which is far better than the dual combination of same doses of Calcipotriol and Betamethasone with similar dose of Compound A.

[0151] These results show that Compound A may be used as adjuvant treatment to other drugs in treatment of the inflammation and itching caused by a number of skin conditions such as psoriasis.