COMBINATION THERAPY COMPRISING A POLYUNSATURATED KETONE AND A SECOSTEROID

Abstract

A synergistic pharmaceutical composition for simultaneous, parallel, sequential or separate use comprising a polyunsaturated ketone, a secosteroid and, optionally, the corticosteroid partner betamethasone. 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 secosteroid partners, preferably selected from the group consisting of calcipotriol, alfacalcidol, calcifediol, calcitriol, calcitroic acid, cholecalciferol, dihydrotachysterol, 24,25-dihydroxycholecalciferol, eldecalcitol, ergocalciferol, falecalcitriol, paricalcitol, previtamin D3, tacalcitol, 22-dihydroergocalciferol, sitocalciferol or a pharmaceutically acceptable salt, or a hydrate or solvate thereof, especially calcipotriol or tacalcitol or a pharmaceutically acceptable salt, or a hydrate or solvate thereof.

2. A composition as claimed in 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 calcipotriol, tacalcitol, calcitriol or falecalcitriol, preferably calcipotriol or tacalcitol or a pharmaceutically acceptable salt, or a hydrate or solvate thereof.

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

6. A composition as claimed in claim 1 wherein the compound (B) is calcipotriol hydrate.

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: ##STR00008## 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: ##STR00009## especially when compound (B) is calcipotriol or tacalcitol or a salt, hydrate or solvate thereof.

12. A composition as claimed in claim 1 wherein the molar ratio of compound (A) to (B) in the composition is 10:1 to 1:10, preferably 1:5 to 5:1.

13. A composition as claimed in claim 1 further comprising betamethasone or a pharmaceutically acceptable salt, or a hydrate or solvate thereof.

14. A composition as claimed in claim 13 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.

15. (canceled)

16. 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.

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 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.

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: (i) identifying a patient who has received either a compound of formula (I) 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 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 an animal subject in need thereof comprising administering to said animal an effective amount of a composition as claimed 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 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.

21. The method of claim 19, wherein the animal subject is a rodent, monkey, or a pig.

22. The method of claim 20, wherein the pharmaceutical composition or the effective amount of compound of formula (I) and compound (B) is used as a positive control.

23. (canceled)

24. The pharmaceutical composition of claim 1 comprising calcipotriol or tacalcitol or a salt, hydrate or solvate thereof optionally in combination with one or more additional secosteroids.

25. The pharmaceutical composition as claimed in 24, wherein the additional secosteroid is selected from the group consisting of alfacalcidol, calcifediol, calcitriol, calcitroic acid, cholecalciferol, dihydrotachysterol, 24,25-dihydroxycholecalciferol, eldecalcitol, ergocalciferol, falecalcitriol, paricalcitol, previtamin D3, tacalcitol/calcipotriol, 22-dihydroergocalciferol, sitocalciferol or pharmaceutically acceptable salts, or hydrates or solvates thereof.

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

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

28. The method of claim 27, comprising the step of reducing the expression one or more of prostaglandin E2 (PGE2), interleukin-1beta (IL-1), tumor necrosis factor (TNF) or interleukin-6 (IL-6).

29. The method of claim 27, wherein the animal subject has or is suspected of having psoriasis or dermatitis.

Description

DESCRIPTION OF FIGURES

[0127] FIG. 1 shows the results of the combination therapy of the invention. Co-treatment with cPLA2 inhibitor Compound A and Calcipotriol hydrate 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.

[0128] 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 calcipotriol is 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.

[0129] 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).

[0130] FIG. 4 shows dose response of calcipotriol 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).

[0131] FIG. 5 shows that co-treatment with compound A and calcipotriol 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).

[0132] 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).

[0133] FIG. 7 shows that LPS induces PGE2 production in PBMC challenged with LPS. Compound A dose-dependently reduce PGE2 production. When suboptimal doses of Compound A are combined with a non-effective dose of calcipotriol, synergistic reduction of PGE2 levels are seen. Ctrl; untreated PBMC (no LPS, no inhibitor), Compound A, CAL: calcipotriol. Results shown for one blood donor experiment.

[0134] FIG. 8 shows that LPS induces cytokine production in PBMC challenged with LPS. Compound A dose-dependently reduce cytokine production, including IL-1, TNF and IL-6.

EXAMPLE 1

[0135] The following compounds were used in the Experiments:

##STR00007##

Co-Treatment Compound A & Calcipotriol:

Methods:

Cell Culture:

[0136] 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:

[0137] 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 and to increase cell sensitivity to treatment. On day 4, the cells were treated with cPLA2 inhibitor Compound A and vitamin D analogue Calcipotriol hydrate (Sigma Aldrich #C4369) 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:

[0138] Co-treatment with cPLA2 inhibitor Compound A and vitamin D analogue Calcipotriol shows synergistic effects on decreasing keratinocyte cell proliferation and viability compared to each inhibitor alone.

[0139] Initial experiments were performed to determine dose response of Compound A alone. The inhibitor reduced cell proliferation and viability slightly 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 Calcipotriol were combined.

[0140] Following 24 hours of treatment, 10 M of Calcipotriol 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 Calcipotriol, a significant 70% and 90% 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.

[0141] Several key pathways are dysregulated in skin disorders such as psoriasis and atopic dermatitis. With this preliminary result, cPLA2 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:

[0142] 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:

[0143] 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 D analogue 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 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:

[0144] Co-treatment with corticosteroid Betamethasone and vitamin D analogue Calcipotriol shows synergistic effects on decreasing keratinocyte cell proliferation and viability compared to each inhibitor alone.

[0145] 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 and viability 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

[0146] Compound A and Calcipotriol show dose response on immortalized keratinocyte cell line HaCat cell viability.

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 1:3-1:4 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 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 or calcipotriol 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] In this study, experiments were performed to determine dose response of Calcipotriol and compound A. Compound A and calcipotriol were found to affect cell viability at 15 M, whereas at doses 1-10 M no signs of impairment in cell viability were observed (FIG. 3/4).

EXAMPLE 4

[0150] Co-treatment with compound A and calcipotriol shows synergistic effects on immortalized keratinocyte cell line HaCat cell viability compared to each inhibitor alone.

[0151] As noted in FIG. 3/4, initial experiments were performed to determine dose response of compound A and calcipotriol alone (FIG. 3/4). Both of them shows reduced cell viability to the cells at 15 M, whereas at 10 M no signs of impairment in cell viability was found (FIG. 3/4). On this basis, combination treatment was designed in which sub-effective doses of the inhibitor compound A (10 M) and calcipotriol (10 M) were combined. Combination of compound A and calcipotriol were also compared with already established combination of betamethasone and calcipotriol. Following 24 hours of treatment, 10 M of calcipotriol and 50 M of betamethasone shows 45% reduction of cell viability which increased to nearly 70% when same concentration of calcipotriol is given with compound A 10 M (FIG. 5). This observed trend of synergistic effects on cell viability indicates better beneficial effects calcipotriol combination with compound A than betamethasone dipropionate on skin disorders.

EXAMPLE 5

[0152] 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:

[0153] 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:

[0154] 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:

[0155] 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.

[0156] In the same way, Calcipotriol 8 M and Betamethasone 30 M does not have any effect on cell viability. Nevertheless, addition of 7 M 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.

[0157] These results show that Compound A can 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.

EXAMPLE 6

Methods

[0158] Blood from healthy, consenting blood donors was collected at St. Olays Hospital HF, Trondheim, Norway.

Isolation and Cell Culture of Peripheral Blood Mononuclear Cells.

[0159] From each donor, 26 ml heparin tubes with blood were used. Peripheral blood mononuclear cells (PBMC) were isolated using 50 mL SepMate tubes (STEMCELL Technology, UK) and Lymphoprep (Fresenius Kabi, N) density gradient according to the manufacturers instructions.

[0160] PBMC were resuspended in RPMI-1640 cell culture medium complemented with 5% heat-inactivated fetal bovine serum (FBS), 0.03% glutamine, 0.01 mg/mL gentamicin and 3.4 ppm -mercaptoethanol (all reagents from Sigma-Aldrich). 110.sup.6 PBMC were seeded per well in 24-well plates and pre-treated for 2 hrs with Compound A and calcipotriol prior to the addition of Lipopolysaccaride (LPS, 10 ng/mL, total volume pr. well 1000 pt). Cells were incubated for 72 hrs (5% CO2, 37 C.) before the cells were centrifuged (10 min, 2000 rpm, 4 C.) to clear the supernant of cells. The supernatant were aliquoted and stored at 80 C. until analysis.

PGE2 Analysis

[0161] PGE2 enzyme immunoassay analysis of PBMC supernatants was performed according to the kit protocol (Cayman Chemicals, #514010, USA). Samples were diluted 1:100 except for the untreated control PBMC supernatant that was assayed undiluted. The samples were hybridized overnight (18 h, 4 C.). The plate was read at OD 420 nm using a Cytation 5 imaging reader from BioTek and data acquired with the corresponding software Gen5 2.09. A four-parameter logistic model was fit to the absorbance data to determine PGE2 levels using the Cayman PGE2 protocol. PGE2 levels for all treatments are shown relative to the untreated PBMC supernatant.

Results

[0162] As described previously, in vitro cellular experiments using HaCaT keratinocytes have shown significant synergistic effects on reducing keratinocyte viability when sub-optimal doses of Compound A and calcipotriol are combined. However, the viability assays performed to date cannot always reveal if the combination treatment may also yield synergistic anti-inflammatory effects, locally or systemically. Therefore, it was of interest to evaluate the effects of the Compound A and calciptriol combination in another physiologically and clinically relevant model system with an anti-inflammatory read-out.

[0163] Accordingly, we isolated peripheral blood mononuclear cells, treated them with various doses and combinations of Compound A and calcipotriol and finally added LPS as a trigger to mount a broad inflammatory response. The cell supernatants collected following 72 hrs of LPS stimulation were next analyzed with respect to levels of PGE2, a key proinflammatory mediator close to the cPLA2 target.

[0164] LPS clearly induced PGE2 production, confirming that cPLA2 and COX2 are activated by LPS (FIG. 7). PGE2 levels in untreated PBMC were in the 120-200 pg/mL range. LPS induced levels to varying degree in different donors to 10-47 000 pg/mL, i.e. 80-200 fold induction, which indicates considerable individual variation.

[0165] In addition to PGE2, LPS also induced a more general proinflammatory PBMC profile, including production of the cytokines TNF, IL-1 and IL6 (FIG. 8). PBMC cytokine production was dose-dependently reduced by Compound A, showing that the cPLA2c enzyme is regulating signaling leading to LPS-induced cytokine production.

[0166] In response to Compound A alone, a clear dose-dependent reduction in PGE2 levels were found, with an estimated IC50 of 10 M. Compound A in doses 0.3 M and 1 M were found to be sub-optimal with little or no inhibition and were chosen for subsequent combo-experiments. In response to high doses of calcipotriol alone (<0.5 M), PGE2 levels increased, indicating that calcipotriol may act as a stressor above a given threshold dose. The suboptimal calcipotriol dose 0.5 M was chosen for inclusion in combo-experiments with Compound A.

[0167] When suboptimal doses of Compound A and calcipotriol were combined, the reduction of PGE2 was better than for each inhibitor alone; Compound A at 1 M reduced PGE2 by 18%, Compound A at 0.3 M showed no inhibition, and calcipotriol at 0.5 M increased PGE2 by 18%. In contrast, the combination of Compound A (1 M) with calcipotriol (0.5 M) reduced PGE2 by 51%. The combination of Compound A (0.3 M) with calcipotriol (0.5 M) also reduced PGE2 levels by 42%, indicating synergistic effects of the combination compared to each inhibitor alone.

[0168] The synergy found with the Compound A and calcipotriol combination on PGE2 production in PBMC challenged with LPS, indicates that in addition to the synergistic effects on keratinocyte viability, beneficial systemic, clinically relevant, anti-inflammatory effects may be achieved as well.

[0169] The combination of Compound A and calcipotriol represents a novel, non-steroidal treatment for psoriasis and related diseases that offers improved efficacy at low doses and less adverse effects compared to existing therapeutic modalities.