Topical composition

Abstract

The present invention relates to a composition for topical application comprising a polyaphron dispersion, the polyaphron dispersion comprising a continuous aqueous phase and at least one discontinuous oil phase, wherein the polyaphron dispersion comprises calcipotriol, betamethasone dipropionate, alpha-tocopherol and butylated hydroxyanisole, and wherein the composition has a pH of 7.75±0.5.

Claims

1. A composition for topical application comprising a polyaphron dispersion, the polyaphron dispersion comprising a continuous aqueous phase and at least one discontinuous oil phase, wherein the composition comprises, by weight of the composition, from 0.001 to 0.01 wt % calcipotriol, betamethasone dipropionate, from 0.001 to 0.005 wt % alpha-tocopherol, and from 0.05 to 0.5 wt % butylated hydroxyanisole, and wherein the composition has a pH of 7.75±0.5.

2. A composition according to claim 1, wherein the composition has a pH of 7.75±0.25.

3. A composition according to claim 1 wherein the composition comprises, by weight of the composition: from 0.02 to 0.1 wt % betamethasone.

4. A composition according to claim 1 wherein: the at least 60 wt % of the calcipotriol is in at least one of said discontinuous oil phase(s); and/or the at least 60 wt % of the betamethasone dipropionate is at least 60 wt % of the betamethasone dipropionate is in at least one of said discontinuous oil phase(s); and/or the at least 60 wt % of the alpha-tocopherol is in at least one of said discontinuous oil phase(s); and/or the at least 60 wt % of the butylated hydroxyanisole is in at least one of said discontinuous oil phase(s).

5. A composition according to claim 1, wherein at least one of said discontinuous oil phase(s) comprises medium chain triglycerides and isopropyl myristate, wherein the isopropyl myristate and medium chain triglycerides are present in a weight ratio of from 3:1 to 12:1.

6. A composition according to claim 1, wherein the polyaphron dispersion further comprises a discontinuous phase comprising a non-solvent oil.

7. A composition according to claim 1, wherein said discontinuous oil phase(s) comprises a first discontinuous phase, a second discontinuous phase and, optionally a third discontinuous phase comprising mineral oil, at least 60 wt % of the calcipotriol is in the first discontinuous phase, at least 60 wt % of the betamethasone dipropionate is in the second discontinuous phase, and at least 60 wt % of the the alpha-tocopherol and the butylated hydroxyanisole are in the first discontinuous phase, or predominantly in the first and second discontinuous phases collectively.

8. A composition according to claim 7, wherein the first discontinuous phase and the second discontinuous phase each comprise the same pharmaceutically acceptable oil, wherein the pharmaceutically acceptable oil is a blend of caprilic capric triglycerides (CCT) and isopropyl myristate, and wherein the isopropyl myristate and caprilic capric triglycerides are present in a weight ratio of from 3:1 to 12:1.

9. A composition according to claim 1, wherein the continuous aqueous phase comprises at least 4 wt % isopropanol by weight of the composition.

10. A composition according to claim 1, wherein the composition is chemically stable for at least 6 months at 25° C.±2° C., as measured at 60% RH±5%; and/or wherein the composition is chemically stable for at least 12 months at 5° C.±3° C., as measured at 60% RH±5%.

11. A composition according to claim 6, wherein the non-solvent oil comprises mineral oil.

Description

(1) The present invention will now be described in relation to the following non-limiting figures:

(2) FIG. 1 depicts the chemical structure of calcipotriol.

(3) FIG. 2 depicts the chemical structure of betamethasone dipropionate.

(4) FIG. 3 shows the variation in the amounts of the principal degradation products of calcipotriol (24-epi calcipotriol, lighter filled circles) and betamethasone dipropionate (betamethasone 21-propionate, darker filled circles) with pH in polyaphron compositions containing the two actives, as measured after storage for 9 months at 5° C. The y-axis represents the level of a degradation product, as determined from an HPLC chromatogram and including related peaks, as a percentage of the total active pharmaceutical ingredient from which it is derived.

(5) FIG. 4 shows the chemical stability of calcipotriol in polyaphron compositions as determined by the purity method described herein for different combinations of antioxidants. The error bars indicate standard deviation.

(6) FIG. 5 shows the cumulative amount of calcipotriol diffused through human skin over 72 hours from the three formulations tested in Example 9. The x-axis represents time in hours and the y-axis represents the mean cumulative amount of calcipotriol diffused through the skin in ng/cm.sup.2. The darkest filled circles represent MC01-53, the intermediate filled circles represent MC01-42, and the lightest filled circles represent MC01-54. The error bars indicate standard error of the mean.

(7) FIG. 6 shows the cumulative amount of betamethasone dipropionate diffused through human skin over 72 hours from the three formulations tested in Example 9. The x-axis represents time in hours and the y-axis represents the mean cumulative amount of betamethasone dipropionate diffused through the skin in ng/cm.sup.2. The darkest filled circles represent MC01-53, the intermediate filled circles represent MC01-42, and the lightest filled circles represent MC01-54. The error bars indicate standard deviation.

(8) FIG. 7 shows the cumulative amount of calcipotriol diffused through human skin over 72 hours from the polyaphron dispersion-containing formulations tested in Example 10. The x-axis represents time in hours and the y-axis represents the mean cumulative amount of calcipotriol diffused through the skin in ng/cm.sup.2. The small dark triangles represent 7% CCT, the larger lighter triangles represent IPM only, and the squares represent 13% CCT. The error bars indicate standard deviation.

(9) FIG. 8 shows the cumulative amount of betamethasone dipropionate diffused through human skin over 72 hours from the polyaphron dispersion-containing formulations tested in Example 10. The x-axis represents time in hours and the y-axis represents the mean cumulative amount of betamethasone dipropionate diffused through the skin in ng/cm.sup.2. The small dark triangles represent 7% CCT, the larger lighter triangles represent IPM only, and the squares represent 13% CCT. The error bars indicate standard deviation.

(10) FIG. 9 shows the cumulative amount of calcipotriol diffused through human skin over 72 hours from a formulation in accordance with the invention (MC01-17, triangles) compared with commercially available Dovobet® ointment (diamonds). The x-axis represents time in hours and the y-axis represents the mean cumulative amount of calcipotriol diffused through the skin in ng/cm.sup.2. The error bars indicate standard deviation.

(11) FIG. 10 shows the cumulative amount of betamethasone dipropionate diffused through human skin over 72 hours from a formulation in accordance with the invention (MC01-17, triangles) compared with commercially available Dovobet® ointment (diamonds). The x-axis represents time in hours and the y-axis represents the mean cumulative amount of betamethasone dipropionate diffused through the skin in ng/cm.sup.2. The error bars indicate standard deviation.

(12) The present invention will now be described in relation to the following non-limiting examples.

EXAMPLE 1

(13) A composition in accordance with the present invention was prepared by combining the following components.

(14) TABLE-US-00001 Component Ingredient % wt/wt % wt/wt A (Calcipotriol Oil Calcipotriol base 0.005 28.12466 containing phase Isopropyl myristate 16.668 polyaphron) Capric/caprylic triglycerides 5.556 Laureth-4 0.25 Butylated hydroxyanisole 0.05 α-tocopherol 0.0006625 Aqueous Poloxamer 407 0.28 phase Isopropyl alcohol 1.00 Water 4.315 B (Betamethasone Oil Betamethasone dipropionate 0.0644 28.12506 dipropionate phase Isopropyl myristate 16.625 containing Capric/caprylic triglycerides 5.54 polyaphron) Laureth-4 0.25 Butylated hydroxyanisole 0.05 α-tocopherol 0.0006625 Poloxamer 407 0.28 Aqueous Isopropyl alcohol 1.00 phase Water 4.315 C (Mineral oil Oil Mineral oil 26.70 33.75 polyaphron) phase Laureth-4 0.30 Aqueous Cremophor RH40 0.337 phase Isopropyl alcohol 2.025 Water 4.388 D (Buffer phase) Disodium phosphate heptahydrate 0.0291 1.00 Mono sodium phosphate 0.0044 monohydrate Water 0.9665 E (Gel phase) Carbomer (Ultrez 10) 0.20 7.00 Water 6.80 F Water 0.30 0.30 G (pH and adjust) Triethanolamine q.s. q.s. (50% wt/wt aqueous solution) Water q.s. 100.00

(15) The polyaphron sub-component A was made by firstly mixing the components of the oil phase together at 40° C. until fully dissolved and then allowed to cool. The aqueous phase components were also mixed with suitable stirring until fully dissolved. The oil phase was then slowly added to the aqueous phase with continuous moderate stirring. After the full oil addition the dispersion was then mixed for a further 30 minutes. The polyaphron sub-components B & C were made using the same method. The buffer phase D was mixed in a separate vessel. In another suitable vessel the gel phase (E) was made by adding the carbomer to the water with vigorous mixing until fully dispersed and hydrated.

(16) The final formulation was then made by mixing the polyaphron sub components (A, B & C) together in a vessel using moderate mixing. The gel phase (E) was then added to this and mixed followed by the addition of the Buffer phase (D) and the additional isopropanol (F). The formulation was then adjusted to pH7.75 using the required amount of triethanolamine 50% wt aqueous solution before being adjusted to 100% wt by the addition of water. The formula was made on a 1 kg scale.

EXAMPLE 2

(17) A further composition in accordance with the present invention was prepared by combining the following components:

(18) TABLE-US-00002 Component Ingredient % wt/wt % wt/wt A (Calcipotriol Oil Calcipotriol base 0.005 28.12466 containing phase Isopropyl myristate 20.204 polyaphron) Capric/caprylic triglycerides 2.02 Laureth-4 0.25 Butylated hydroxyanisole 0.05 α-tocopherol 0.0006625 Aqueous Poloxamer 407 0.28 phase Isopropyl alcohol 2.00 Water 3.315 B (Betamethasone Oil Betamethasone dipropionate 0.0644 28.12506 dipropionate phase Isopropyl myristate 20.15 containing Capric/caprylic triglycerides 2.015 polyaphron) Laureth-4 0.25 Butylated hydroxyanisole 0.05 α-tocopherol 0.0006625 Aqueous Poloxamer 407 0.28 phase Isopropyl alcohol 2.00 Water 3.315 C (Mineral oil Oil Mineral oil 26.70 33.75 polyaphron) phase Laureth-4 0.30 Aqueous Cremophor RH40 0.337 phase Isopropyl alcohol 2.50 Water 3.913 D (Buffer phase) Disodium phosphate heptahydrate 0.0291 1.00 Mono sodium phosphate 0.0044 monohydrate Water 0.9665 E (Gel phase) Carbomer (Ultrez 10) 0.20 7.00 Water 6.80 F Isopropyl alcohol 0.30 0.30 G (pH and adjust) Triethanolamine q.s. q.s. (50% wt/wt aqueous solution) Water q.s. 100.00

(19) The components were combined analogously to Example 1.

EXAMPLE 3

(20) A composition in accordance with the present invention was prepared by combining the following components:

(21) TABLE-US-00003 Component Ingredient % wt/wt % wt/wt A (Calcipotriol Oil Calcipotriol base 0.005 28.12466 containing phase Isopropyl myristate 20.514 polyaphron) Capric/caprylic triglycerides 1.71 Laureth-4 0.25 Butylated hydroxyanisole 0.05 α-tocopherol 0.0006625 Aqueous Poloxamer 407 0.28 phase Isopropyl alcohol 2.00 Water 3.315 B (Betamethasone Oil Betamethasone dipropionate 0.0644 28.12506 dipropionate phase Isopropyl myristate 20.46 containing Capric/caprylic triglycerides 1.705 polyaphron) Laureth-4 0.25 Butylated hydroxyanisole 0.05 α-tocopherol 0.0006625 Aqueous Poloxamer 407 0.28 phase Isopropyl alcohol 2.00 Water 3.315 C (Mineral oil Oil Mineral oil 13.35 16.875 polyaphron) phase Laureth-4 0.15 Aqueous Cremophor RH40 0.1685 phase Isopropyl alcohol 1.25 Water 1.9565 D (Buffer phase) Disodium phosphate heptahydrate 0.0291 1.00 Mono sodium phosphate 0.0044 monohydrate Water 0.9665 E (Gel phase) Carbomer (Ultrez 10) 0.20 7.00 Water 6.80 F Water 17.00 18.25 Isopropyl alcohol 1.25 G (pH and adjust) Triethanolamine q.s. q.s. (50% wt/wt aqueous solution) Water q.s. 100.00

(22) The components were combined analogously to Example 1.

EXAMPLE 4

(23) A composition in accordance with the present invention was prepared by combining the following components:

(24) TABLE-US-00004 Component Ingredient % wt/wt % wt/wt A (Calcipotriol Oil Calcipotriol base 0.005 28.12466 containing phase Isopropyl myristate 18.849 polyaphron) Capric/caprylic triglycerides 3.375 Laureth-4 0.25 Butylated hydroxyanisole 0.05 α-tocopherol 0.0006625 Aqueous Poloxamer 407 0.28 phase Isopropyl alcohol 1.70 Water 3.615 B (Betamethasone Oil Betamethasone dipropionate 0.0644 28.12506 dipropionate phase Isopropyl myristate 18.79 containing Capric/caprylic triglycerides 3.375 polyaphron) Laureth-4 0.25 Butylated hydroxyanisole 0.05 α-tocopherol 0.0006625 Aqueous Poloxamer 407 0.28 phase Isopropyl alcohol 1.70 Water 3.615 C (Mineral oil Oil Dimethicone (Dow corning Q7-9120 26.70 33.75 polyaphron) phase Silicone Fluid, 350 cst) Laureth-4 0.30 Aqueous Cremophor RH40 0.337 phase Isopropyl alcohol 2.025 Water 4.388 D (Buffer phase) Disodium phosphate heptahydrate 0.0291 1.00 Mono sodium phosphate 0.0044 monohydrate Water 0.9665 E (Gel phase) Carbomer (Ultrez 10) 0.20 7.00 Water 6.8 F Isopropyl alcohol 0.30 0.30 G (pH and adjust) Triethanolamine q.s. q.s. (50% wt/wt aqueous solution) Water q.s. 100.00

(25) The components were combined analogously to Example 1.

EXAMPLE 5

(26) A composition in accordance with the present invention (MC01-42) was prepared by combining the following components:

(27) TABLE-US-00005 Component Ingredient % wt/wt % wt/wt A (Calcipotriol Oil Calcipotriol base 0.005 28.12466 containing phase Isopropyl myristate 18.849 polyaphron) Capric/caprylic triglycerides 3.375 Laureth-4 0.25 Butylated hydroxyanisole 0.05 α-tocopherol 0.0006625 Aqueous Poloxamer 407 0.28 phase Isopropyl alcohol 1.70 Water 3.615 B (Betamethasone Oil Betamethasone dipropionate 0.0644 28.12506 dipropionate phase Isopropyl myristate 18.79 containing Capric/caprylic triglycerides 3.375 polyaphron) Laureth-4 0.25 Butylated hydroxyanisole 0.05 α-tocopherol 0.0006625 Aqueous Poloxamer 407 0.28 phase Isopropyl alcohol 1.70 Water 3.615 C (Mineral oil Oil Mineral oil 26.70 33.75 polyaphron) phase Laureth-4 0.30 Aqueous Cremophor RH40 0.337 phase Isopropyl alcohol 2.025 Water 4.388 D (Buffer phase) Disodium phosphate heptahydrate 0.0291 1.00 Mono sodium phosphate 0.0044 monohydrate Water 0.9665 E (Gel phase) Carbomer (Ultrez 10) 0.20 7.00 Water 6.8 F Isopropyl alcohol 0.30 0.30 G (pH and adjust) Triethanolamine q.s. q.s. (50% wt/wt aqueous solution) Water q.s. 100.00

(28) The components were combined analogously to Example 1.

EXAMPLE 6

(29) A further composition in accordance with the present invention (MC01-17) was prepared by combining the following components:

(30) TABLE-US-00006 Component Ingredient % wt/wt % wt/wt A (Calcipotriol Oil Calcipotriol base 0.00447 28.125 containing phase Isopropyl myristate 18.82 polyaphron) Capric/caprylic triglycerides 3.375 Laureth-4 0.250 Butylated hydroxyanisole 0.050 α-tocopherol 0.001 Aqueous Calcipotriol 0.00053 phase Poloxamer 407 0.28 Isopropyl alcohol 1.70 Water 3.644 B (Betamethasone Oil Betamethasone dipropionate 0.0604 28.125 dipropionate phase Isopropyl myristate 18.82 containing Capric/caprylic triglycerides 3.375 polyaphron) Laureth-4 0.25 Butylated hydroxyanisole 0.05 α-tocopherol 0.001 Aqueous Betamethasone dipropionate 0.004 phase Poloxamer 407 0.28 Isopropyl alcohol 1.70 Water 3.5846 C (Mineral oil Oil Mineral oil 26.70 34.000 polyaphron) phase Laureth-4 0.30 Aqueous Cremophor RH40 0.34 phase Methyl parabens 0.025 Propyl parabens 0.025 Isopropyl alcohol 2.000 Water 4.610 D (Buffer phase) Disodium phosphate heptahydrate 0.0291 1.00 Mono sodium phosphate 0.0044 monohydrate Water 0.9665 E (Gel phase) Carbomer (Ultrez 10) 0.20 7.00 Isopropyl alcohol 0.30 Water 6.50 Triethanolamine q.s. to (50% wt/wt aqueous solution) pH 7 Water q.s. F (pH and adjust) Triethanolamine q.s. q.s. (50% wt/wt aqueous solution) Water q.s. 100.00

(31) The polyaphron sub-component A was made by firstly mixing the components of the oil phase together at 40° C. until fully dissolved and then allowed to cool. The aqueous phase components were also mixed with suitable stirring until fully dissolved. The oil phase was then slowly added to the aqueous phase with continuous moderate stirring. After the full oil addition the dispersion was then mixed for a further 30 minutes. The polyaphron sub-components B & C were made using the same method. The buffer phase D was mixed in a separate vessel. In another suitable vessel the gel phase E was made by adding the carbomer to the bulk water with vigorous mixing until fully dispersed and hydrated. The isopropanol was then added and sufficient triethanolamine (50% wt aqueous solution) was added to adjust to pH7.0. Water was then added to make the sub-component up to weight.

(32) The final formulation was then made by mixing the polyaphron sub components (A, B & C) into the gel phase (E) using moderate mixing. This was followed by the addition of the Buffer phase (D). The formulation was then adjusted to pH7.75 using the required amount of triethanolamine 50% wt aqueous solution before being adjusted to 100% wt by the addition of water. The formula was made on a 1 kg scale.

EXAMPLE 7

(33) Variants of MC01-17 were prepared by an analogous method to that of Example 6. The formulae were identical except that they different amounts of TEA were included in component F to achieve different formulation pH values. In particular, formulations having a pH of 7, 7.25, 7.5 7.75 and 8.0 were prepared.

(34) The samples were stored for 9 months at 5° C. in sealed amber glass jars with less than 5% headspace. The jars were sealed in air. The purity (% area) value is calculated from the HPLC trace as the ratio of the API peak to other API related peaks present in the analysis. The results are shown in FIG. 3.

(35) It can be seen from FIG. 3 that the level of the principal calcipotriol degradation product (24-epi calcipotriol) decreases with increasing pH whilst the principal BDP degradation product (betamethasone 21-propionate) increases with increased pH. The calcipotriol degradation was found to increase significantly at pH values below 7.25. The pH range of 7.75±0.5, and preferably 7.75±0.25, was therefore preferred.

EXAMPLE 8

(36) A selection of six formulations including a control were developed with varying solo antioxidants and combinations of them. The samples were stored for 6 months at 40° C. in sealed amber glass jars with less than 5% headspace. The jars were sealed but were not sparged with nitrogen prior to sealing.

(37) The antioxidant is split evenly between the two API oil phases. The antioxidants used were α-tocopherol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), disodium edetate (EDTA) and citric acid. The levels used for the BHA and BHT were 0.1%. The EDTA was used at 0.05% and the citric acid was used at 0.1%.

(38) TABLE-US-00007 Formulation MC01-39 BHA only MC01-40 α-tocopherol only MC01-41 BHT only MC01-42 BHA + α-tocopherol MC01-43 No antioxidant (control) MC01-46 BHT + α-tocopherol

(39) MC01-42 is described in Example 5. The remaining formulations in the table are identical to MC01-42 except for the choice of antioxidant and as indicated above.

(40) Samples were monitored and tested at intervals for pH and API purity.

(41) The calcipotriol purity was determined using the following method:

Chromatographic Conditions

(42) TABLE-US-00008 HPLC System Waters Photodiode Array Detector Waters Separation Module Waters Empower2 or Empower3 Data Processing Software Column Phenomenex Kinetex XB C18, 100 × 4.6 mm, 2.6 μm, 00D-4496-E0 Guard Column SecurityGuard Ultra Cartridges, XB-C18, AJ0-8768 Detection 266 nm Sample 25° C. Temperature Column 25° C. Temperature Flow Rate 1.0 mL/min Mobile Phase Mobile Phase A: Water Mobile Phase B: Acetonitrile Time (min) % A % B 0.0 90 10 25.0 10 90 30.0 10 90 30.1 90 10 35.0 90 10 Injection 30 μL Volume Run Time 35 min

Sample Preparation

(43) Acetonitrile is used as the sample diluent.

Procedure

(44) 1. Accurately weigh 0.5 g (±0.025 g) of sample into a 10 ml volumetric flask, minimizing sample on the neck of the flask. 2. Add approximately 5 mL sample diluent to the volumetric flask and vortex mix for 2 minutes. 3. Allow equilibration to room temperature. 4. Make the volumetric flask to volume with sample diluent, add a magnetic stirrer and stir for 2 hours. 5. Transfer the solution to a 20 mL volumetric flask, rinse with washings of heptane and make to volume. 6. Shake for 1 minute and stir for 5 minutes. 7. Transfer an aliquot of the lower layer to a 2 ml centrifuge tube and centrifuge for 10 minutes at 13000 rpm (16,060 g-force).

Standard Preparation

Prepare a Solution of Calcipotriol in Acetonitrile at a Concentration of 2.5 μg/mL

(45) 1. Prepare a Calcipotriol stock solution by accurately weight 25 mg Calcipotriol standard into a 250 mL volumetric flask, make to volume with diluent. 2. Prepare an intermediate solution by pipetting 5.0 mL of the stock solution into a 20 mL volumetric flask, making to volume with diluent. 3. Prepare the final standard solution by pipetting 1.0 mL Calcipotriol intermediate solution into a 10 mL volumetric flask, make to volume with diluent.

(46) A suitable equivalent preparation may also be used.

(47) Analysis

(48) Approximate Retention Times:

(49) TABLE-US-00009 Pre-Calcipotriol 15.9 min Trans Calcipotriol (Imp C) 16.1 min Calcipotriol 16.7 min 24-epi Calcipotriol (Imp D) 17.2 min

(50) Calculate the Percentage Peak Purity of Calcipotriol using the following equation:

(51) $\mathrm{Peak}\mathrm{Purity}\left(\%\right)=\frac{\mathrm{Calcipotriol}\mathrm{Peak}\mathrm{Area}+\mathrm{Pre}\text{-}\mathrm{Calcipotroil}\mathrm{Peak}\mathrm{Area}}{\begin{array}{c}\mathrm{Calcipotriol}\mathrm{Peak}\mathrm{Area}+\mathrm{Pre}\text{-}\mathrm{Calcipotroil}\mathrm{Peak}\mathrm{Area}+\\ \mathrm{Total}\mathrm{Calcipotriol}\mathrm{Related}\mathrm{Impurities}\mathrm{Peak}\mathrm{Area}\end{array}}$

(52) Note: For assay calculations, a response factor of 1.9 is applied to the Pre-Calcipotriol peak area.

(53) The BDP purity was determined using the following method:

Chromatographic Conditions

(54) TABLE-US-00010 HPLC System Waters Photodiode Array Detector Waters Separation Module Waters Empower2 or Empower3 Data Processing Software Column Waters Codecs UPLC C18, 150 × 2 mm, 1.6 μm Guard Column Vanguard pre-column, Acquity UPLC BEH C18, 2.1 × 5 mm, 1.7 μm (PN 186007096) Detection 240 nm Sample 25° C. (±2° C.) Temperature Column 40° C. (±2° C.) Temperature Flow Rate 0.28 mL/min Mobile Phase Mobile Phase A: Water Mobile Phase B: 40/60 Methanol/Acetonitrile Time (min) % A % B 0 85 15 6.0 50 50 15.0 58 42 16.0 30 70 23.0 29 71 27.0 10 90 32.0 10 90 32.1 85 15 45 85 15 Column rinse: Flush the column for 30 minutes in Acetonitrile/ Water 75/25% after use and store in the same solvent Weak wash Acetonitrile/Water 50/50% (needle wash) solvent Strong wash Acetonitrile solvent Seal wash Methanol/Water 10/90% solvent Injection 4 μL Volume Run Time 35 min

Mobile Phases

(55) Mobile Phase A: Rinse the bottle with Acetonitrile before adding water. It is recommended to use an amber bottle and change the water every 5 days, to reduce the growth of bacteria.

(56) Mobile Phase B: Mix Methanol and Acetonitirle in the ratio of 40/60% and sonicate for 10 minutes. Stable for 30 days when stored at ambient temperature.

Sample Preparation

(57) Acetonitrile is used as the sample diluent. Prepare in amber glassware, rinse with acetonitrile before use. Samples are stable for 4 days at ambient conditions.

(58) Procedure: 1. Accurately weigh 1.25 g of sample into a 25 ml volumetric flask, minimizing sample on the neck of the flask. 2. Add approximately 10 mL sample diluent and vortex mix for 1 minute. 3. Sonicate for 5 minutes. 4. Allow to cool to ambient temperature and make to volume with diluent. 5. Add a magnetic stirrer and stir for 2 hours. 6. Leave to stand for 15 minutes. 7. Centrifuge for 10 minutes at 13,000 rpm. Prepare enough vials to allow for filtering. 8. Transfer the solution to a syringe, filter through a 0.2 μm PTFE syringe filter, discarding the first 1 mL.

Standard Preparation

(59) Acetonitrile is used as the sample diluent. Prepare in amber glassware, rinse with acetonitrile before use. Samples are stable for 15 days stored at ambient temperature.

(60) Prepare duplicate solutions of BDP in Acetonitrile at a concentration of 32 μg/mL.

Example Procedure

(61) 1. Accurately weigh 25.0 mg (±2 mg) of BDP reference material into a 25 mL flask. Dissolve and dilute to volume with acetonitrile (100 μg/mL stock solution). 2. Pipette 0.8 ml of the stock solution into a 25 mL volumetric flask and dilute to volume with diluent (32 μg/mL working standard solution.) 3. Pipette 1.0 mL of the stock solution into a 100 mL volumetric flask and dilute to volume with diluent (0.32 μg/mL stock sensitivity solution). 4. Pipette 1.0 mL of the stock sensitivity solution into a 10 mL volumetric flask and dilute to volume with diluent (0.032 μg/mL working sensitivity solution, LOQ). 5. Pipette 1.0 mL of the stock sensitivity solution into a 20 mL volumetric flask and dilute to volume with diluent (0.016 μg/mL working sensitivity solution, LOD).

Analysis

(62) Relative Retention Times (RRT's) and Relative response Factors (RRF's):

(63) TABLE-US-00011 RRT RRF Betamethasone 17-propionate (Impurity B) 0.65 1.03 E isomer of Enol 21-aldehyde (Impurity E) 0.76 1.05 Betamethasone 21-propionate (Impurity C) 0.79 1.14 Betamethasone 21-Acetate 17-propionate (Impurity D): 0.95 0.98 BHA 1 (not reported) 0.63 BHA 2 (not reported) 0.65 Trans-Calcipotriene (not reported) 1.03 Calcipotriene (not reported) 1.05

(64) BDP retention time is approximately 18 minutes.

(65) Calculations:

(66) Inhibit integration between 0-5 minutes and from 25-35 minutes. Disregard peaks due to the placebo, BHA and Calcipotriene.

(67) Calculate the % w/w of the BDP and impurities using the following equation:

(68) $\mathrm{Assay}\left(\%\right)=\frac{\begin{array}{c}\mathrm{Sample}\mathrm{area}\times \mathrm{Standard}\mathrm{weight}\left(\mathrm{mg}\right)\times \\ \mathrm{Purity}\left(\mathrm{as}a\mathrm{decimal}\right)\times \mathrm{Dilution}\mathrm{Factor}\left(3.2\right)\end{array}}{\mathrm{Standard}\mathrm{area}\times \mathrm{Sample}\mathrm{weight}\left(\mathrm{mg}\right)\times \mathrm{RRF}}$

(69) The RRF's of any unknown peak is assumed to be 1.0.

(70) The results for betamethasone dipropionate are shown in the table overleaf.

(71) TABLE-US-00012 BDP Purity Formulation 6 months at 40° C. MC01-46 BHT + α-tocopherol 95.13 MC01-43 No antioxidant (control) 95.16 MC01-41 BHT only 95.14 MC01-42 BHA + α-tocopherol 95.04 MC01-40 α-tocopherol only 95.37 MC01-39 BHA only 95.81

(72) There appears to be no significant trend or differences with regards to the BDP purity that could not be accounted for by slight changes in pH.

(73) The results for calcipotriol are shown in the table below and in FIG. 4.

(74) TABLE-US-00013 Calcipotriol Purity Formulation 6 months at 40° C. MC01-42 BHA + α-tocopherol 95.17 MC01-46 BHT + α-tocopherol 93.87 MC01-39 BHA only 93.31 MC01-41 BHT only 92.81 MC01-43 No antioxidant (control) 87.2 MC01-40 α-tocopherol only 93.53

(75) BHA and alpha-tocopherol in combination were found to give significantly improved calcipotriol stability. In contrast, BHA alone, BHT alone, alpha-tocopherol alone and BHT and alpha-tocopherol in combination were found to have similar effects on calcipotriol stability.

EXAMPLE 9

(76) Diffusion experiments were conducted investigating the effect of isopropanol (IPA) level on the amount of calcipotriol and BDP diffused through human skin.

(77) Three formulations were investigated, as summarised in the table overleaf.

(78) TABLE-US-00014 FN ID IPA level MC01-42 5.7% MC01-53 0.5% MC01-54 .sup. 0%

(79) MC01-42 is described in Example 5. The remaining formulations in the table are identical to MC01-42 except for the IPA level as indicated above.

(80) Skin diffusion studies were conducted over 72 hours. For each of the three formulations nine cells were filled with receptor phase (70% phosphate buffer, 30% isopropyl alcohol) and loaded with 30 mg formulation. Receptor phase was collected at 16 h, 24 h, 40 h, 48 h, 64 h and 72 h. Prior to injection into the HPLC all samples and standards were filtered through a PTFE filter (13 mm diameter, 0.45 μm pore size, hydrophilic PTFE, Millipore, UK) to remove paper fibres and other particles from the sample.

(81) A Waters H-Class UPLC system was used for the HPLC analysis. The column used was a BEH C18 50×2.1 mm, 1.7 μm particle size (Waters, UK), fitted with a VanGuard pre-column filter (Waters, UK). The column was maintained at 40° C. throughout. Samples were held at ambient temperature during the analytical run. It was not recommended to chill the samples during the run because buffer salts will precipitate out of the receptor phase matrix.

(82) An isocratic HPLC method was used. Mobile phase of 35/45/20 v/v/v water/acetonitrile/methanol was pumped at 0.5 ml/min. A 10 μl injection volume was used throughout. Detection using a UV-vis photodiode array detector with chromatograms extracted at 240 nm (BDP) and 263 nm (calcipotriene). A run time of 3 min was used. Retention times of 0.9 min (BDP) and 1.3 min (calcipotriene) were observed.

(83) The cumulative amount of the two actives determined at each time-point are summarised in the tables below.

(84) Cumulative Amount Diffused, Calcipotriol Through Human Skin.

(85) TABLE-US-00015 MC01-42 MC01-53 MC01-54 Time (h) (ng/cm.sup.2) (ng/cm.sup.2) (ng/cm.sup.2) 16 17.5 ± 5.8 17.8 ± 1.6 32.3 ± 8.7 24 30.6 ± 5.9 24.6 ± 5.2 20.1 ± 8.2 40  90.2 ± 11.6  69.5 ± 12.3  57.7 ± 15.6 48 136.4 ± 17.1 112.1 ± 15.2  86.3 ± 18.4 64 320.2 ± 49.9 203.6 ± 30.0 149.1 ± 24.3 72 510.3 ± 96.8 377.2 ± 44.3 203.6 ± 32.0

(86) Cumulative Amount Diffused. BDP Through Human Skin.

(87) TABLE-US-00016 MC01-42 MC01-53 MC01-54 Time (h) (ng/cm.sup.2) (ng/cm.sup.2) (ng/cm.sup.2) 16 123 ± 74 47 ± 8 136 ± 84 24 253 ± 91 142 ± 20  203 ± 105 40  758 ± 163 368 ± 56  521 ± 215 48 1070 ± 202 667 ± 66  737 ± 227 64 1884 ± 333 1132 ± 142 1116 ± 270 72 2649 ± 393 1569 ± 241 1358 ± 289

(88) The results are shown graphically in FIGS. 5 and 6.

(89) For both betamethasone dipropionate and calcipotriol, cumulative diffusion at 72 hours was significantly higher when 5.7% isopropanol was included compared to 0.5% and 0%.

EXAMPLE 10

(90) Two variants of MC01-17 (see Example 6) were prepared: one in which a portion of the IPM from each of components A and B was replaced with CCT (to give a total CCT content of 13%), and one in which the CCT was replaced entirely with IPM. MC01-17 itself contained 7% CCT in total.

(91) The human skin diffusion of each of the three formulations was measured using the same method as Example 9. The mean cumulative flux of calcipotriol and BDP in ng/cm.sup.2 is shown at different time points in FIGS. 7 and 8 respectively.

(92) As can be seen from FIGS. 7 and 8, the CCT level of 7% shows significantly greater flux (ANOVA) for both calcipotriol (p=0.0015) and BDP (p=0.0162) over the neat IPM (0% CCT) or the 13% CCT. The result is surprising in that it could be thought that the additional IPM, a known permeation enhancer, would be more beneficial but it appears that in this case more is not necessarily better.

(93) A further experiment was carried out in which MC01-17 was compared with the commercially available Dovobet® ointment using the same skin diffusion tests described above. The mean cumulative flux of calcipotriol and BDP in ng/cm.sup.2 is shown at different time points in FIGS. 9 and 10 respectively. As can be seen from FIGS. 9 and 10, the skin penetration of both calcipotriol and BDP is surprisingly better for MC01-17 than Dovobet®.

(94) The foregoing detailed description has been provided by way of explanation and illustration, and is not intended to limit the scope of the appended claims. Many variations in the presently preferred embodiments illustrated herein will be apparent to one of ordinary skill in the art, and remain within the scope of the appended claims and their equivalents.