DESC:Field of the Invention
The present invention describes a combination of clobetasol and hyaluronate with an average molecular weight of between 40 and 60 kDa for the treatment of Psoriasis. The formulation is preferably in a topical form. The topical pharmaceutical composition comprises, a therapeutically effective amount of 0.01 to 0.05% clobetasol by weight and 0.01 % to 0.5% sodium hyaluronate by weight with an average molecular weight of 40 and 60 kDa along with at least one suitable pharmaceutically acceptable excipient. The present application relates to use of the topical pharmaceutical composition for the treatment or prevention of a suitable disease or condition, such as Psoriasis. The invention also describes the preparation of such compositions.

Background of the Invention
Psoriasis is a lifelong immune-mediated inflammatory skin disease, associated with morbidities such as psoriatic arthropathy, psychological, cardiovascular and hepatic diseases. In 2014, the World Health Organization recognized psoriasis as a serious non-communicable disease and highlighted the distress related to misdiagnosis, inadequate treatment and stigmatization of this disease.1 The Global Burden of Disease Study estimated that psoriasis accounted for 5.6 million all-age disability-adjusted life-years (DALYs) in 2016; at least three-fold that of inflammatory bowel disease.2The severity and course of psoriasis can vary greatly depending on the individual, but in general this condition recurs throughout the life of the individual causing significant psychological and social distress, and significantly impacting on quality of life. About 15 percent of people with psoriasis have joint inflammation that produces arthritis symptoms. This condition is called psoriatic arthritis. The exact mechanism which triggers the abnormal cell proliferation is not known, though researchers believe psoriasis occurs when faulty signals in the immune system cause skin cells to grow too rapidly. There are many treatments available but because of its chronic recurrent nature psoriasis is a challenge to treat.
Topical treatments performed by applying agents to the skin that slow down or normalize the excessive cell reproduction and reduce inflammation are usually the first line of therapy for psoriasis and other skin disorders. Topical corticosteroids are the most prescribed treatment for mild to moderate psoriasis and may also complement other psoriasis treatments for moderate to severe psoriasis. However, if corticosteroids are used long-term on large areas of skin or mucous membranes, they can be absorbed into the body. This increases the risk of local side effects such as skin thinning, and of systemic side effects, such as adrenal suppression, where the adrenal glands become unable to regulate hormones being released in the body; and tachyphylaxis, where the body develops immunity to a certain treatment regimen. Moreover, the need for long-term application may be a daunting task for many patients. Poor adherence to treatment over time, due to an undesirable vehicle, may result in poor treatment outcomes.
Hyaluronic acid (HA) is a glycosaminoglycan distributed widely throughout connective, epithelial, and neural tissues. HA acts as a lubricating agent in the synovial joint fluid, thus protecting the articular cartilage3. It also plays beneficial roles in inflammation, tissue injury and repair, wound healing and immunosuppression4. HA is a major component of skin, in the dermis HA regulates water balance, osmotic pressure, ion flow and sieve function. HA is demonstrated to be collagen booster as it induces collagen synthesis in in-vitro (cultured human fibroblast) and in-vivo in clinic5.
WO2012053007 disclosed topical pharmaceutical compositions for the treatment of psoriasis. However, this patent application does not provide any biological data of said composition.
WO2009010488 disclosed anti-inflammatory dermatological composition comprising corticosteroids and hyaluronate fragments with an average molecular weight of between 20 and 500 kDa and uses thereof. However, this patent application disclosed broader range of hyaluronate fragment.
The current therapies for psoriasis have lot of side effects, longer duration of treatment and these therapies are not able to cure psoriasis completely. It has now surprisingly been found that the topical formulation of clobetasol in combination with hyaluronic acid with an average molecular weight of between 40 and 60 kDa provides a particularly beneficial effect on psoriasis with no observed adverse effects. Such combination is therefore particularly useful for the treatment of Psoriasis.
The present invention provides a topical pharmaceutical composition comprising clobetasol in combination with a sodium hyaluronate for the treatment of psoriasis.

Embodiments of the invention
In an embodiment of the present invention is provided a topical pharmaceutical composition comprising 0.01 to 0.05 % clobetasol by weight and 0.01 to 0.5 % hyaluronic acid by weight with an average molecular weight of between 40 and 60 kDa for the treatment of psoriasis.
In another embodiment is provided a topical pharmaceutical composition comprising 0.01 % to 0.05 % clobetasol by weight and 0.01% to 0.5 % hyaluronic acid by weight with an average molecular weight of between 40 and 60 kDa for the treatment of humans and other mammals in need thereof.
In a preferred embodiment is provided a topical pharmaceutical composition comprising 0.01 % to 0.05 % clobetasol propionate by weight and 0.01 to 0.5 % sodium hyaluronate by weight with an average molecular weight of between 40 and 60 kDa along with one or more suitable pharmaceutical excipients.
In a preferred embodiment is provided a topical pharmaceutical composition comprising 0.01 % to 0.05 % clobetasol propionate by weight and 0.01 to 0.5 % hyaluronate by weight with an average molecular weight of between 40 and 60 kDa for the treatment of Psoriasis.

Description of the figures

Figure 1: Effect of topical preparation of clobetasol alone or in combination with sodium hyaluronate with molecular weight of 8.2 kDa and 40 kDa in imiquimod-induced psoriasis model in mice (Change in Ear thickness (mm)
Figure 2: Effect of topical preparation of clobetasol alone or in combination with sodium hyaluronate with molecular weight of 40 kDa, 27 kDa and 130 kDa in imiquimod-induced psoriasis model in mice (PASI Score)
Figure 3: Effect of topical preparation of clobetasol alone or in combination with sodium hyaluronate with molecular weight of 40 kDa, 27 kDa and 130 kDa in imiquimod-induced psoriasis model in mice (Change in ear weight)

Detailed Description of the Invention
Term ‘treatment’ used anywhere in the specification means prevention or reduction or delay in progression of clinical symptoms of disease or disorder.
Term ‘therapeutically effective amount’ used anywhere in the specification means amount of compound/ drug/ composition to treat disease but low enough to avoid any serious side effects.
Term ‘pharmaceutically acceptable’ used anywhere in specification means it allowed for human use as well as veterinary use. Term ‘subject’ used anywhere in the specification describe human or animal possessing clinical symptoms of disease or disorder.
In an embodiment of the present invention is provided a topical pharmaceutical composition comprising 0.01 to 0.05 % clobetasol by weight and 0.01 to 0.5 % hyaluronic acid by weight with an average molecular weight of between 40 and 60 kDa for the treatment of psoriasis.
In a preferred embodiment of the present invention is provided a topical pharmaceutical composition comprising 0.01 to 0.05 % clobetasol propionate by weight and 0.01 to 0.5 % sodium hyaluronate by weight with an average molecular weight of between 40 and 60 kDa for the treatment of psoriasis.
In another preferred embodiment is provided a topical pharmaceutical composition comprising 0.01 % to 0.05 % clobetasol propionate by weight and 0.01 to 0.5 % sodium hyaluronate by weight with an average molecular weight of between 40 and 60 kDa along with one or more suitable pharmaceutical excipients.
In a further preferred embodiment is provided a topical pharmaceutical composition comprising 0.01 % to 0.05 % clobetasol propionate by weight and 0.01 to 0.5 % sodium hyaluronate by weight with an average molecular weight of 40 kDa along with one or more suitable pharmaceutical excipients.
The compound clobetasol propionate can be prepared by the general processes and examples disclosed in prior art.
Sodium hyaluronate with different fragments was obtained from Sahndong focusfreda biotech co. ltd.
In the treatment of the present invention, the active medicaments are preferably administered in topical pharmaceutical composition form.
The compositions are preferably in topical form in an amount appropriate for the relevant daily dosage.
As used herein the term `pharmaceutically acceptable` use embraces both human and veterinary use.
The compositions are prepared and formulated according to conventional methods, such as those disclosed in standard reference texts and are well within the scope of a skilled person. Thus the composition can be in the form of individual components delivered as a kit or may comprise of suitable co-formulation of the two ingredients using suitable excipients. A skilled person is aware of the alternate ways to deliver a topical composition.
No adverse toxicological effects were seen for the topical compositions or methods of the invention in the above mentioned strength. Further the topical composition of the present invention was found suitable for the treatment of psoriasis.
The present invention discloses a topical formulation comprising 0.01 % to 0.05 % clobetasol propionate by weight and 0.01 to 0.5 % sodium hyaluronate by weight with an average molecular weight of between 40 and 60 kDa. Topical formulation includes gel, ointment, creams, solution, lotion, spray, shampoo and foams preferably is lotion and process for the preparation thereof. The topical formulations of this invention are suitable for the treatment of psoriasis.
In a further another embodiment is provided the use of different formulations such as gel, ointment, creams, solution and foams comprising 0.01 % to 0.05 % clobetasol propionate by weight and 0.01 to 0.5 % sodium hyaluronate by weight with an average molecular weight of between 40 and 60 kDa.
In an embodiment is provided a topical formulation comprising clobetasol propionate, salicylic acid, hyaluronic acid, thickening agents, pH adjusting agent, stabilizer, preservatives or anti-oxidant agent, vehicle and pharmaceutically acceptable carriers.

Thickening agents used may be selected from the group of hydrophilic polymers such as carbomers, carbomer polymer, carbomer derivative, hydroxyethyl cellulose, anionic polymers or mixture thereof.
Preservatives or antioxidant agents are selected from suitable acids such as citric acid, benzoic acid, esters, alcohols such as bronopol, chlorobactenes, monothioglycerol, phenols such as cresol, Butylated hydroxylanisol, Butylated hydroxyltoluene, and quaternary ammonium compounds.
pH adjusting agent are selected from which can be used include one or more of organic or inorganic acids and bases including sodium hydroxide, potassium hydroxide, ammonium hydroxide, phosphate buffers, citric acid, acetic acid, fumaric acid, hydrochloric acid, malic acid, nitric acid, phosphoric acid, propionic acid, sulfuric acid, tartaric acid or mixture thereof.
stabilizers are selected from one or more of ionic polysorbate surfactant, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80,
vehicle is selected from water, solvent and mixture thereof.
The compositions are prepared and formulated according to conventional methods, such as those disclosed in standard reference texts and are well within the scope of a skilled person.
The invention is further exemplified by the following non-limiting examples, which are illustrative representing the preferred modes of carrying out the invention. The invention’s scope is not limited to these specific embodiments only but should be read in conjunction with what is disclosed anywhere else in the specification together with those information and knowledge which are within the general understanding of a person skilled in the art.

Example -1: Composition for combination of clobetasol propionate and sodium hyaluronate (40 kDa)
Ingredient 40 K Da Sodium Hyaluronate
%w/w
Clobetasol Propionate 0.05
Salicylic Acid 6.00
Sodium Hyaluronate 0.10
Polysorbate 80 3.00
Glycerin 5.00
Absolute Alcohol 5.00
soybean Oil 9.00
Butylated hydroxytoluene (BHT) 0.01
Hydroxyethyl cellulose (HEC) 0.75
Sodium Hydroxide 1.75
Purified water 69.34
Total 100.00

Manufacturing process:
Clobetasol propionate and BHT were mixed with alcohol, salicylic acid, sodium hydroxide, water, polysorbate 80, glycerine and soyabean oil. The resulting blend was homogenized to reduce the droplet size to D90 particle size of less than 300 nm by high pressure homogenization to get the nano emulsion. The aqueous dispersion of HEC and Sodium hyaluronate was mixed with nano emulsion to get lotion.

Example – 2 : Composition for combination of clobetasol propionate and sodium hyaluronate (8.2 kDa)
Ingredient 8.2 kDa Sodium Hyaluronate
%w/w
Clobetasol Propionate 0.05
Salicylic Acid 6.00
Sodium Hyaluronate 0.10
Polysorbate 80 3.00
Glycerin 5.00
Absolute Alcohol 5.00
soybean Oil 9.00
Butylated hydroxytoluene (BHT) 0.01
Hydroxyethyl cellulose (HEC) 0.75
Sodium Hydroxide 1.75
Purified water 69.34
Total 100.00

Example – 3 : Composition for combination of clobetasol propionate and sodium hyaluronate (27.5 kDa)
Ingredients 27.5 K Da Sodium Hyaluronate
% w/w
Clobetasol Propionate 0.05
Absolute Alcohol 5.00
Polysorbate 80 3.00
Glycerin 5.00
Butylated hydroxytoluene 0.01
Soybean Oil 9.00
Sodium Hydroxide 1.75
Salicyclic Acid 6.00
Purified Water 34.67
Purified Water 34.67
Sodium hyaluronate 0.10
Hydroxyethylcellulose 0.75
100.00

Example – 4 : Composition for combination of clobetasol propionate and sodium hyaluronate (130 kDa)
Ingredients 130 K Da Sodium Hyaluronate
% w/w
Clobetasol Propionate 0.05
Absolute Alcohol 5.00
Polysorbate 80 3.00
Glycerin 5.00
Butylated hydroxytoluene 0.01
Soybean Oil 9.00
Sodium Hydroxide 1.75
Salicyclic Acid 6.00
Purified Water 34.67
Purified Water 34.67
Sodium hyaluronate 0.10
Hydroxyethylcellulose 0.75
100.00

Biological studies:
Experiment 1: Evaluation of effect of topical preparation of clobetasol alone or in combination with sodium hyaluronate with molecular weight of 8.2 kDa and 40 kDa in imiquimod-induced psoriasis model in mice
Study design and treatment
Male SD rats of 6-8-week-old were randomly assigned into different groups of 10-12 each. Each of the study animal received topical application of 50µL of respective treatments on to the right ear as per the following groups in Table 1:
Table 1:
Sr. No. Groups Treatment
1 Placebo Treatment applied topically (50 µL/rat) onto the right ear once daily from day-0 to 13
2 Clobetasol 0.05% + Sodium hyaluronate (40 kDa) 0.1%
3 Clobetasol 0.05% + Sodium hyaluronate (8.2 kDa) 0.1%
4 Clobetasol 0.05%

Ear thickness was measured on day-0 (before treatment initiation) and on the day of study termination i.e. day-14 using a digital caliper. Change in ear thickness was of each animal was calculated by subtracting thickness of day-14 from day-0.

Results:
Fourteen day treatment with all the formulations with clobetasol exhibited significant (p<0.05) reduction in ear thickness as compared to the placebo group (Table-2, Figure-1). Clobetasol alone treatment showed the highest reduction in ear thickness (-0.09±0.01) versus day-0 (Table-2). Treatment with combination of clobetasol and sodium hyaluronate (40 kDa) showed lowest reducion in ear thickness (-0.06±0.01) versus day-0 (Table-2) which was statistically significant as compared to combination of clobetasol with sodium hyaluronate (8.2 kDa) and clobetasol alone group (Figure-1).
Table 2:
Treatment Change in Ear thickness (mm) vs Day-0 (MEAN±SEM)
Placebo 0.00 ± 0.00
Clobetasol 0.05% + Sodium hyaluronate (40 kDa) 0.1% -0.06 ± 0.01
Clobetasol 0.05% + Sodium hyaluronate (8.2 kDa) 0.1% -0.08 ± 0.00
Clobetasol 0.05% -0.09 ± 0.01

Conclusion:
Combination of sodium hyaluronate of 40 kDa showed a significant protection in clobetasol induced reduction in skin thickness. This novel combination will be highly beneficial in mitigating adverse event like skin thinning associated with long-term clobetasol treatment.

Experiment 2: Evaluation of effect of topical preparation of clobetasol in combination with sodium hyaluronate (40 kDa) in imiquimod-induced psoriasis model in mice

Study design and treatment
Male C57 mice of 6-8-week-old were randomly assigned into different groups of six each. Each of the study animal received topical application of 65µL of 5% imiquimod cream (Imiquad cream, Glenmark Pharmaceuticals Ltd.) on to the shaved back and ear for 5-days. Then after 6h of imiquimod applications animals of different groups received 100µL of the respective treatments as per the following groups in Table 3:

Table 3:
Sr. No. Groups Psoriasis inducer Treatment
1 Placebo 5% imiquimod cream applied topically (65 µL/mouse) once daily from day-1 to 5 Treatment applied topically (100 µL/mouse) once daily from day-1 to 5
2 Clobetasol 0.05% + Sodium hyaluronate (40 kDa) 0.1%
3 Clobetasol 0.05%

Skin thickness was measured on day-0 and on the day of study termination i.e. day-6 using a digital caliper. Clinical score for erythema and scaling was performed on day-6. PASI (Psoriasis Area and Severity Index) score was evaluated by combining individual scores of skin thickness, erythema and scaling scores. Then the animals were sacrificed and skin punch sample of each animal was collected using a 6 mm biopsy punch and weight of the tissue was recorded.

Results:
Treatment with combination of clobetasol and sodium hyaluronate (40 kDa) showed 75.5% whereas, clobetasol alone had 66% suppression in PASI score as compared to the placebo treated group (Table 4). Similarly, combination showed 75% reduction in skin punch weight while, clobetasol alone treatment exhibited only 55% reduction in skin punch weight when compared with placebo group (Table 5).

Table 4:
Treatment PASI score
(MEAN±SEM) % inhibition vs Placebo
Placebo 8.83 ± 0.48
Clobetasol 0.05% + Sodium hyaluronate (40 kDa) 0.1% 2.17 ± 0.38 75.5
Clobetasol (0.05%) 3.00 ± 0.32 66.0

Table 5:
Treatment Change in skin punch weight (mg) % inhibition vs Placebo
Placebo 3.33 ± 0.68
Clobetasol 0.05% + Sodium hyaluronate (40 kDa) 0.1% 0.83 ± 0.22 75.0
Clobetasol (0.05%) 1.50 ± 0.17 55.0

Conclusion:
The results of the present studies indicate that treatment with combination of clobetasol and sodium hyaluronate (40 kDa) demonstrated superior efficacy as compared to clobetasol alone in ameliorating imiquimod-induced psoriasis in mice. This novel combination will be advantageous in terms of efficacy as well as skin-related adverse events over clobetasol alone.

Experiment 3: Evaluation of effect of topical preparation of clobetasol in combination with sodium hyaluronate in imiquimod-induced psoriasis model in mice

Biological studies:
Protocol for pharmacological studies data:
Study design and treatment
Male C57 mice of 6-8-week-old were randomly assigned into different groups of six each. Each of the study animal received topical application of 65µL of 5% imiquimod cream (Imiquad cream, Glenmark Pharmaceuticals Ltd.) on to the shaved back and ear for 5-days. Then after 6h of imiquimod applications animals of different groups received 100µL of the respective treatments as per the following groups in Table:

Table 6:
Sr. No. Groups Psoriasis inducer Treatment
1 Placebo 5% imiquimod cream applied topically (65 µL/mouse) once daily from day-1 to 5 Treatment applied topically (100 µL/mouse) once daily from day-1 to 5
2 Clobetasol 0.05% + Sodium hyaluronate (40 kDa) 0.1%
3 Clobetasol 0.05% + Sodium hyaluronate (27 kDa) 0.1%
4 Clobetasol 0.05% + Sodium hyaluronate (130 kDa) 0.1%
5 Clobetasol 0.05%

Skin thickness was measured on day-0 and on the day of study termination i.e. day-6 using a digital caliper. Clinical score for erythema and scaling was performed on day-6. PASI (Psoriasis Area and Severity Index) score was evaluated by combining individual scores of skin thickness, erythema and scaling scores. Then the animals were sacrificed, and skin punch sample of each animal was collected using a 6 mm biopsy punch and weight of the tissue was recorded.

Results:
Treatment with combination of 40 kDa sodium hyaluronate along with clobetasol showed 72% whereas, 27 and 130 kDa sodium HA showed 63 & 57% suppression in PASI score as compared to the placebo treated group (Table 7, Fig. 2). Clobetasol alone had 66% suppression in PASI score as compared to the placebo treated group (Table 7, Fig. 2). Similarly, combination of 40, 27 & 130 kDa of sodium hyaluronate with clobetasol demonstrated 89, 74 & 76% suppression in skin punch weight respectively as compared to the placebo group (Table 8, Fig. 3). Whereas clobetasol alone treatment exhibited 79% reduction in skin punch weight (Table 8, Fig. 3). These data indicate that combination of 40 kDa sodium hyaluronate showed superior efficacy as compared to alone clobetasol.

Table 7:
Treatment PASI score
(MEAN±SEM) % inhibition vs Placebo
Placebo 7.77 ± 0.39
Clobetasol 0.05% + Sodium hyaluronate (40 kDa) 0.1% 2.17 ± 0.31 72.1
Clobetasol 0.05% + Sodium hyaluronate (27 kDa) 0.1% 2.86 ± 0.40 63.2
Clobetasol 0.05% + Sodium hyaluronate (130 kDa) 0.1% 3.29 ± 0.29 57.7
Clobetasol (0.05%) 2.62 ± 0.40 66.3

Table 8:
Treatment Change in skin punch weight (mg) % inhibition vs Placebo
Placebo 3.60 ± 0.34
Clobetasol 0.05% + Sodium hyaluronate (40 kDa) 0.1% 0.83 ± 0.22 89.3
Clobetasol 0.05% + Sodium hyaluronate (27 kDa) 0.1% 1.97 ± 0.20 74.6
Clobetasol 0.05% + Sodium hyaluronate (130 kDa) 0.1% 1.83 ± 0.20 76.5
Clobetasol (0.05%) 1.60 ± 0.18 79.5

Conclusion:
The results of the present studies indicate that treatment with combination of clobetasol and 40 kDa sodium hyaluronate demonstrated superior efficacy as compared to clobetasol alone in ameliorating imiquimod-induced psoriasis in mice. This novel combination will be advantageous in terms of efficacy as well as skin-related adverse events over clobetasol alone.

References:
1. World Health Organization. Global report on psoriasis. World Health Organization, 2016.
2. A systematic analysis for the Global Burden of Disease Study 2016. Lancet 2017; 390:1260–344.
3. Tamer TM. Hyaluronan and synovial joint: function, distribution and healing. Interdiscip Toxicol. 2013;6(3):111-125.
4. Fallacara A, Baldini E, Manfredini S, Vertuani S. Hyaluronic Acid in the Third Millennium. Polymers (Basel). 2018;10(7):701.
5. Wang F, Garza LA, Kang S, et al. In vivo stimulation of de novo collagen production caused by cross-linked hyaluronic acid dermal filler injections in photodamaged human skin. Arch Dermatol. 2007;143(2):155-163.
,CLAIMS:We claim:

1. A topical pharmaceutical composition comprising 0.01 to 0.05 % clobetasol by weight and 0.01 to 0.5 % hyaluronic acid by weight with an average molecular weight of between 40 and 60 kDa for the treatment of psoriasis.

2. The topical pharmaceutical composition as claimed in claim 1 comprising 0.01 to 0.05 % clobetasol propionate by weight and 0.01 to 0.5 % sodium hyaluronate by weight with an average molecular weight of between 40 and 60 kDa for the treatment of psoriasis.

3. The topical pharmaceutical composition as claimed in any one of claims can be in the form of a gel, ointment, creams, solution, lotion, spray, shampoo and foams.

4. The topical pharmaceutical composition as claimed in claim 3 is lotion.

5. The topical pharmaceutical composition comprising 0.01 % to 0.05 % clobetasol propionate by weight and 0.01 to 0.5 % sodium hyaluronate by weight with an average molecular weight of between 40 and 60 kDa along with one or more suitable pharmaceutical excipients.

6. The topical pharmaceutical composition as claimed in any one of claims 1-4, wherein pharmaceutical acceptable excipients is selected from salicylic acid, thickening agents, pH adjusting agent, stabilizer, preservatives or antioxidant agent, vehicle or combination thereof.

7. The topical pharmaceutical composition as claimed in claims 1 to 5 for use as an active therapeutic substance.

8. The topical pharmaceutical composition as claimed in claim 1 for use in the treatment of Psoriasis.

9. The topical composition of claim 6, wherein the excipients are those described in the specification.