COMPOSITIONS COMPRISING ELECTROHYDRODYNAMICALLY OBTAINED FIBRES FOR ADMINISTRATION OF SPECIFIC DOSES OF AN ACTIVE SUBSTANCE TO SKIN OR MUCOSA

Abstract

The present invention relates to electrospun fibers comprising i) a hydrophilic polymer that is soluble in a first solvent, ii) a bioadhesive substance that is slightly soluble in said first solvent, iii) optionally, a drug substance.

Claims

1.-37. (canceled)

38. Electrospun fibers comprising (i) a hydrophilic polymer that is soluble in a first solvent, wherein the hydrophilic polymer comprises one or more selected from polyvinylpyrrolidone (PVP), ethylcellulose, hydroxypropylcellulose, acrylates, polymers of acrylic/methacrylic esters, and mixtures thereof; (ii) a bioadhesive substance that has a solubility in said first solvent of 0.5 g/100 ml or less at 25 C., wherein the bioadhesive substance comprises one or more selected from dextrans, polyethylene oxides (PEOs), alginate, tragacanth, carrageenan, pectin, guar, xanthan, gellan, methylcellulose, hydroxypropylmethylcellulose (HPMC), polyvinylalcohol (PVA), polymers of acrylic acids (PAAs), chitosan, lectins, thiolated polymers, nonionic water-soluble polyethylene oxide resins (polyox WSR), and PAA polyethylene glycol copolymers (PAA-co-PEG), and mixtures thereof; and (iii) optionally, a drug substance.

39. Electrospun fibers according to claim 38 comprising the optional drug substance.

40. Electrospun fibers according to claim 38, wherein the hydrophilic polymer has a solubility in said first solvent of 3 g/100 ml or more at 25 C.

41. Electrospun fibers according to claim 38, wherein the bioadhesive substance is at the most very slightly soluble in said first solvent at 25 C.

42. Electrospun fibers according to claim 38, wherein at least 90% w/w of the bioadhesive substance is present in undissolved form.

43. Electrospun fibers according to claim 38, wherein at least 95% w/w of the bioadhesive substance is present in undissolved form.

44. Electrospun fibers according to claim 38, wherein said first solvent is ethanol or an ethanol-water mixture.

45. Electrospun fibers according to claim 44, wherein said first solvent is an ethanol-water mixture containing 10% v/v water or less.

46. Electrospun fibers according to claim 44, wherein said first solvent is an ethanol-water mixture containing 5% v/v water.

47. Electrospun fibers according to claim 38, wherein the hydrophilic polymer comprises PVP, polymers of acrylic/methacrylic esters, or mixtures thereof.

48. Electrospun fibers according to claim 38, wherein the bioadhesive substance comprises dextran having an average molecular weight of from 400,000 Da to 2,000,000 Da.

49. Electrospun fibers according to claim 38, wherein the bioadhesive substance comprises dextran having an average molecular weight of about 2,000,000 Da.

50. Electrospun fibers according to claim 38, wherein the bioadhesive substance comprises polyethylene oxide having an average molecular weight of from 100,000 Da to 4,000,000 Da.

51. Electrospun fibers according to claim 38, wherein the bioadhesive substance comprises polyethylene oxide having an average molecular weight of 2,000,000 Da.

52. Electrospun fibers according to claim 38, wherein the weight ratio between the bioadhesive substance and the hydrophilic polymer in the fibers is in a range of from 0.1 to 10.

53. Electrospun fibers according to claim 38, wherein the drug substance is selected from drug substances indicated for treatment of a disease of the skin or mucosa.

54. Electrospun fibers according to claim 38, wherein the drug substance is selected from drug substances indicated for treatment of a disease in the oral cavity.

55. Electrospun fibers according to claim 54, wherein the drug substance is selected from drug substances indicated for local treatment of a disease in the oral cavity.

56. Electrospun fibers according to claim 38, wherein the water content of the fibers is at the most about 5% w/w.

57. Electrospun fibers according to claim 38, further comprising a coating provided on an outer surface of the electrospun fibers.

58. Electrospun fibers according to claim 57, wherein the coating is water-impermeable.

59. Electrospun fibers according to claim 58, wherein the coating comprises one or more of carbothane, polycapronelactone, and polyethylene-co-vinyl acetate.

60. A composition comprising electrospun fibers according to claim 38.

61. A composition according to claim 60, wherein the concentration of the electrospun fibers in the composition is from 70 to 100% w/w.

62. A composition according to claim 60 in the form of a layered composition.

63. A pharmaceutical composition comprising electrospun fibers according to claim 39.

64. A kit comprising (i) a composition according to claim 60, and (ii) an applicator for applying the composition in an oral cavity.

65. A method for preparing electrospun fibers according to claim 39, comprising: (i) dissolving the hydrophilic polymer in the first solvent to obtain a solution; (ii) suspending the bioadhesive substance in the solution to obtain a dispersion; (iii) adding the drug substance to the dispersion to obtain a mixture; and (iv) electrospinning the mixture; wherein at least 90% w/w of the bioadhesive substance is present in undissolved form in the electrospun fibers.

66. A method for preparing electrospun fibers according to claim 39, comprising: (i) dissolving the hydrophilic polymer in a first portion of the first solvent to obtain a solution; (ii) dissolving or suspending the drug substance in the first solution to obtain a mixture; (iii) suspending the bioadhesive substance in a second portion of the first solvent to obtain a dispersion, (iv) dual-electrospinning the mixture obtained and the dispersion, wherein at least 90% w/w of the bioadhesive substance is present in undissolved form in the electrospun fibers.

Description

LEGENDS TO FIGURES

[0160] FIG. 1 shows electron microscopic pictures of electrospun fibres according to the invention

[0161] FIG. 2 shows a test apparatus suitable for bioadhesiveness testing

[0162] FIG. 3 shows an apparatus for testing buccal penetration

[0163] FIG. 4 shows various embodiments of fibres or compositions of the invention

[0164] FIG. 5 shows electron microscopy micrographs of a sample of electrospun polyvinylpyrrolidone (PVP). A 10 wt % PVP (Kollidon 90F) solution was prepared by dissolving the appropriate amount of PVP in ethanol and stirring for a minimum time of 3 hours. A volume of the solution (2 mL) was then loaded into a syringe and placed on a syringe pump, pushing the solution through a metallic needle (20 gauge) while a 14 kV electrical current was applied to the needle. This resulted in the formation of a jet of fibres travelling from the tip of the needle to a collecting plate located at a distance of 17 cm. The syringe pump was set at a flow rate of 10 mL/hour.

[0165] The images show that the material was composed of a mesh of fibres deposited on a random fashion. The fibres generally exhibited a smooth surface and no apparent defects, were cylindrical in shape, and had a diameter under 2 m.

[0166] FIG. 6 shows a scanning electron microscopy micrograph of a sample of electrospun PVP with dextran particles located on the surface of the fibres.

[0167] Electrospinning solutions were prepared by first mixing the appropriate amount of PVP and dextran powders, and then adding ethanol to complete the desired mass. A suspension of dextran particles in dissolved PVP was formed after stirring for a minimum time of 3 hours, which was then used for electrospinning under the conditions described in FIG. 5. Two molecular weights (i.e. 500,000 and 2,000,000) and various amounts of dextrans (i.e. up to 15 wt %) were used in the solutions.

[0168] The material was composed of random fibres exhibiting a smooth surface and no apparent defects. The dextran particles were generally significantly larger than the fibres and appeared to attach to their surface, although it is possible that smaller particles were also embedded within the PVP fibres.

[0169] Additionally, the optical microscopy image of an electrospun PVP sample containing dextrans shows that the dextran particles were present on the surface of the material.

[0170] FIG. 7 shows an optical microscopy image of a sample of electrospun PVP containing alcian blue dye.

[0171] A 10 wt % solution of PVP was prepared by dissolving the appropriate amount of PVP in a 1% w/v solution of alcian blue 8GX in ethanol. The mixture was stirred for a minimum time of 3 hours, and then was electrospun under the conditions described in FIG. 5.

[0172] It was observed that the surface of the material exhibited a homogeneous blue coloration, demonstrating the potential of electrospun PVP to encapsulate ethanol-soluble substances (i.e. dyes, drugs) within the fibres and to deliver them after dissolution of PVP.

[0173] FIG. 8 (left image) illustrates the quick dissolution of a sample of electrospun PVP in artificial saliva. The measured dissolution time was less than 1 second for a sample of dimensions 2 cm0.5 cm, and was observed to be similar in the case of samples dissolved in other water-based media. This quick dissolution allows for the rapid release of any drug encapsulated within the electrospun fibres.

[0174] After dissolution, electrospun PVP formed a gel with bioadhesive properties, as observed in FIG. 8 (right image). In this case, several samples of a dual layer system made of electrospun PVP and electrospun poly(caprolactone) (PCL) were placed on pig cheek mucosa sprayed with artificial saliva. The PVP layer quickly formed a gel after contact with the mucosa, while the PCL layer remained undamaged as the material is not water-soluble. Additionally, the PCL layer was able to remain on place for a length of time due to the bioadhesiveness of the PVP gel.

[0175] FIG. 9 shows scanning electron microscopy micrographs of a dual layer system made of electrospun PVP and electrospun PCL. These samples were processed using a thermal treatment intended to create an attachment between the two layers. A 10 wt % solution of PVP in ethanol was prepared and electrospun as previously described. Also, a 10 wt % solution of PCL (80,000 average Mw) in a blend of dichloro-methane and dimethylformamide (i.e. DCM/DMF, 90%/10% vol %) was prepared and electrospun on top of the PVP layer. Afterwards, samples were cut from the mats, placed between glass slides, and exposed to a temperature of 65 C. for 15 minutes in an electric furnace. Finally, all the samples were allowed to cool down at room temperature

[0176] The images show that the thermal treatment resulted in the melting of the electrospun PCL layer and the subsequent formation of a non-porous and dense film attached to the PVP layer. The PVP layer was unaffected by the thermal treatment due to the melting temperature of PVP being much higher than the melting temperature of PCL (PCL, 60 C.; PVP, >180 C.).

[0177] FIG. 10. The images show pictures of a sample of electro spun fibres after electrospinning of a PVP gel with suspended imiquimod.

[0178] FIG. 11 shows data from tensile testing experiments.

[0179] Patches are clamped into the claws of a Bose electrophorus 3100 and the arms separated at 0.02 mm/sec. The stress and strain are measured electronically as the patch distends (see graph). From the graph the tensile strength, 5 elongation and Youngs modulus (a measure of stress & stain) of the patch is measured.

[0180] FIG. 12. The buccal mucosa isolated from pig cheeks is firmly adhered to a petri-dish using cyanoacrelaye glue. Mucosal patches (PVP with increasing % dextran) of equal dimensions are then applied for 5 seconds with approx equal force and then sub-merged in PBS and then rotated at different speeds using a mechanical stirrer. The time for the patch to be dislodged from the mucosa is measured in minutes.

[0181] FIG. 13. A PCL/PVP (+dextrans) is applied to the surface of pig mucosa with constant force for 30 min. The mucosa with patch is then snap frozen in liquid nitrogen and then stored at 80 C in optimum cutting temperature mountant. 8 uM sections were then cut using a cryostat and the sections stained with haemotoxylin & eosin before being mounted on slides. The figure clearly shows that the patch is tightly adhered to but does not penetrate into the mucosal epithelium.

[0182] FIGS. 14. A and B show electrospun fibres, where Eudragit L100-55 is the fibre-forming hydrophilic polymer and sodium alginate is a bioadhesive substance, that is present in undissolved form and attached to the fibres. In C and D sodium carboxymethylcellulose has been used as bioadhesive material.

[0183] FIG. 15. Fibres formed with Eudragit 100-55. In A and B chitosan is a bioadhesive substance and in C and D polyvinylalcohol has been used. The bioadhesive material is present in undissolved form attached to the fibres.

[0184] FIG. 16. Results of bioadhesive tests as described in Example 23.

MATERIALS

[0185] The following materials are used in the experiments reported in the Examples below.

Polyvidone 90.000 (Kollidone 90K) is obtained from BASF, Germany
Klucel LF is obtained from Hercules Incorporated, US
Eudragit E, is obtained from Evonik Industires

Eudragit RS, Evonik Industires

[0186] Dermacryl 79, is obtained from AkzoNobel
Tributyl citrate, Ethanol, Sodium acetate, Hydrochloric acid & Bethamethasone dipropionate and clobetasol propionate are obtained from Sigma-Aldrich
Dextran, Molecular weight 500.000, 750.000, 1.000.000 are obtained from Pharmacosmos Denmark
Polyethylene oxide 400.000, 2.000.000, 4.000.000 are obtained from The Dow Chemical Company
Medium chain glyceride, Henry Lamotte Oils GmbH
Imiquimod and clobetasol propionate are obtained from APIChem Technology Co., Ltd.
Carbothane is obtained from Lubrizol Corporation US

Methods

[0187] Analysis of Bethamethasone dipropionate or clobetasol propionate by HPLC:
Column: Sunfire C18; 3.5 m or 5 m; 1504.6 mm ID or equivalent
Mobile Phase: Acetonitrile/0.01 M (NH.sub.4).sub.2HP0.sub.4 pH 6.4, 70:30 (v/v).
Flow rate: 0.8 ml/min

Detection Wavelength: 240 nm

Analysis of Imiquimod by HPLC:

[0188] Column: Phenomenex C.sub.18 column or equivalent
Mobile phase: 40:60 Acetonitril to water containing 1% trifluoroacetic acid
Flow rate: 1 ml/min
Detection wavelength: 242 nm

EXAMPLES

Example 1

Preparation of Alcoholic Gel Ready for ElectrospinningDextran as Bioadhesive Substance

[0189]

TABLE-US-00003 Fibre-forming Bioadhesive substance hydrophilic Dextran.sup.1 Dextran.sup.1 Dextran.sup.1 Solvent polymer 500.000 750.000 2.000.000 Ethanol Polyvidone x x x x x Kollindon 90F10% Klucel LF x X x 5%(HPC) Eudragit E x X X 15% Eudragit RS x x X x X 15% Dermacryl 79 x x X 10%
1) The content of the different dextrans varied between 2.5, 5.0 and 7.5% by weight of the gel or between 25 to 75% by weight of the fibre-forming hydrophilic polymer in those cases where PVP or Dermacryl was used. Experiments have shown that it is possible to use at least up 20% w/w of dextran. The weight ratios between the bioadhesive substance and the hydrophilic polymer are 0.1 to 1.5, namely 0.1, 0.16, 0.25, 0.33, 0.5, 0.75, 1, 1.5.

[0190] To prepare the gels, dextrans were suspended in the ethanol by stirring and ultra sound followed by slowly addition of the fibre-forming hydrophilic polymer while slowly stirring. The resulting suspension was stirred overnight to complete the dissolution of the fibre-forming hydrophilic polymer.

Example 2

Preparation of Alcoholic Gel Ready for ElectrospinningPolyethylene Oxide as Bioadhesive Substance

[0191]

TABLE-US-00004 Bioadhesive substance Fibre-forming Polyethylene Polyethylene Polyethylene hydrophilic oxide - oxide - oxide - Solvent polymer 400.000 2.000.000 4.000.000 Ethanol Polyvidone - X x x X x Kollidon 90F010% Klucel LF 5% X X x Eudragit E X X X 15% Eudragit RS X x X X X 15% Dermacryl 79 X x x 10%

[0192] The content of the different dextrans varied between 2.5, 5.0 and 7.5% by weight of the gel or between 25 to 75% by weight of the fibre-forming hydrophilic polymer in those cases where PVP or Dermacryl was used. Experiments have shown that it is possible to use at least up 20% w/w of dextran. The weight ratios between the bioadhesive substance and the hydrophilic polymer are 0.1 to 1.5, namely 0.1, 0.16, 0.25, 0.33, 0.5, 0.75, 1, 1.5.

[0193] To prepare the gels Polyethylene oxide was suspended in ethanol by stirring and ultra sound followed by slowly addition of the fibre-forming hydrophilic polymer while slowly stirring. The resulting suspension was stirred overnight to complete the dissolution of the fibre-forming hydrophilic polymer.

Example 3

Preparation of Alcoholic Gel Containing the Drug Substance Imiquimod and Ready for Electrospinning

[0194] Two different methods were used:

[0195] 1.5 g of imiquimod was suspended by stirring in 20 g ethanol to which 80 g of a 10% PVP 90K in ethanol was added and stirred slowly for 2 hours.

[0196] 2.5 g imiquimod was suspended by stirring in 20 g 0.1M acetate buffer pH 4.0 for 2 hours, whereby imiquimod partly dissolves. Then 80 g of a 10% PVP 90.000 in ethanol was added and stirred slowly for 2 hours.

[0197] After dissolution of the fibre-forming hydrophilic polymer, imiquimod and the bioadhesive substance were added to obtain a suspension. The suspension was then electrospun as described herein.

[0198] The following bioadhesive substances have been used in both methods:

Dextran 500,000 Da

Dextran 750,000 Da

Dextran 2,000,000 Da

[0199] Polyethylene oxide 400,000 Da
Polyethylene oxide 2,000,000 Da
Polyethylene oxide 4,000,000 Da

[0200] The bioadhesive substances were added in proportion to the fibre-forming hydrophilic polymer so that the weight ratio between the bioadhesive substance and the fibre-forming hydrophilic polymer was in the range of from 0.1-5. Specific weight ratios obtained were: 0.2, 0.25, 0.3, 0.4, 0.6, 0.7, 0.75, 0.8, 1, 1.2, 1.25, 1.3, 1.5, 1.6, 1.7, 2, 2.4, 2.7, 3, and 4.

Example 4

Preparation of Fibres Containing the Drug Substance Imiquimod

[0201] Two different methods were used:

[0202] 1.5 g of imiquimod was suspended by stirring in 20 g ethanol to which 80 g of a fibre-forming hydrophilic polymer in ethanol was added and stirred slowly for 2 hours.

[0203] 2.5 g imiquimod was suspended by stirring in 20 g 0.1 M acetate buffer pH 4.0 for 2 hours, whereby imiquimod partly dissolves. Then 80 g of a fibre-forming hydrophilic polymer in ethanol was added and stirred slowly for 2 hours.

[0204] The following fibre-forming hydrophilic polymers were used in both methods:

Eudragit E as a 15% solution in ethanol
Eudragit RS as a 15% solution in ethanol
Dermacryl 79 as a 10% solution in ethanol

[0205] The following bioadhesive substances have been used in both methods:

Dextran 500,000 Da

Dextran 750,000 Da

Dextran 2,000,000 Da

[0206] Polyethylene oxide 400,000 Da
Polyethylene oxide 2,000,000 Da
Polyethylene oxide 4,000,000 Da

[0207] The bioadhesive substances were added in proportion to the fibre-forming hydrophilic polymer so that the weight ratio between the bioadhesive substance and the fibre-forming hydrophilic polymer was in the range of from 0.1-2. Specific weight ratios obtained were: 0.2, 0.25, 0.3, 0.4, 0.6, 0.7, 0.75, 0.8, 1, 1.2, 1.25, 1.3, 1.5, 1.6, 1.7, and 2.

[0208] After dissolution of the fibre-forming hydrophilic polymer, imiquimod and the bioadhesive substance were added to obtain a suspension. The suspension was then electrospun as described herein.

Example 5

Preparation of Two-Layered Composition Comprising Fibres Containing Imiquimod Layered on a Hydrophobic Backing Layer

[0209] The fibres described in Example 3 and 4 were prepared, but spun on a hydrophobic layer of containing poly(caprolactone) to obtain a two-layered composition.

Example 6

Preparation of Alcoholic Gel Containing the Drug Substance Betamethasone Di-Proprionate or Clobetasol Propionate and Dextran as Bioadhesive Substance and Ready for Electrospinning

[0210]

TABLE-US-00005 Composition Ingredients (mg) I II III IV Polyvidone - 100 100 100 100 Kollidon 90F Dextran 75 75 75 75 750.000 Tributyl citrate 0 50 100 0 Medium chain 0 0 0 75 glyceride Betamethasone 5 5 5 5 dipropionate (BDP) or clobetasol propionate Ethanol 1000 1000 1000 1000

[0211] BDP or clobetasol propionate, tributyl citrate and/or medium chain triglyceride were dissolved in ethanol. Then dextran with a molecular weight of approximately 750.000 was added by stirring and ultra sound, and finally Polyvidone 90.000 was added during slowly stirring.

[0212] The resulting suspension was stirred overnight to complete the dissolution of the fibre-forming hydrophilic polymer. The suspension was then electrospun as described herein.

Example 7

Preparation of Alcoholic Gel Containing the Drug Substance Betamethasone Di-Proprionate or Clobetasol Propionate and Polyethylene Oxide as Bioadhesive Substance and Ready for Electrospinning

[0213]

TABLE-US-00006 Composition Ingredients (mg) I II III IV V Eudragit RS 150 150 150 150 150 Polyoxyethylene 75 75 75 75 75 750,000 Tributyl citrate 0 50 100 0 75 Medium chain 0 0 0 75 75 glyceride Betamethasone 5 5 5 5 5 dipropionate (BDP) or clobetasol propionate Ethanol 1000 1000 1000 1000 1000

[0214] To prepare an alcoholic gel ready for spinning BDP or clobetasol propionate, Tributyl citrate and/or Medium chain glyceride were dissolved in ethanol. Then polyethylene oxide with a molecular weight of approximately 750.000 was added by stirring and ultra sound, and finally Eudragit RS was added during slowly stirring.

[0215] The resulting suspension gel was stirred overnight to complete the dissolution of the fibre-forming hydrophilic polymer. The suspension was electrospun as described herein.

Example 8

Preparation of Alcoholic Gel Containing the Drug Substance Imiquimod and Dextran as Bioadhesive Substance and Ready for Electrospinning

[0216]

TABLE-US-00007 Ingredients Composition mg I II III Iv V VI Imiquimod 25 25 25 25 25 25 Acetate buffer 200 200 200 200 200 200 0.22M pH 4.65 Acetic acid, A few A few A few A few A few A few glacial drop to drop to drop to drop to drop to drop to dissolve dissolve dissolve dissolve dissolve dissolve imiquimod imiquimod imiquimod imiquimod imiquimod imiquimod Denature 1000 1000 1000 1000 1000 1000 Ethanol Luvitec 90K 100 100 100 100 0 0 (Polyvinylpyrrolidone (PVP)) Eudragit RS 0 0 0 0 450 450 100 Dextran T750 75 75 75 75 75 75 Tributyl citrate 0 50 100 50 0 150 97% Captex 300 0 0 0 50 0 0 (Medium chain glyceride)

[0217] Imiquimod is suspended in acetate buffer to obtain a suspension, wherein imiquimod is partly dissolved. Tributyl citrate and/or medium chain triglyceride were dissolved in ethanol. The imiquimod suspension obtained is added. Then dextran with a molecular weight of approximately 750.000 was added by stirring and ultra sound, and finally Eudragit RS was added during slowly stirring.

[0218] The resulting suspension was stirred overnight to complete the dissolution of the fibre-forming hydrophilic polymer. The suspension was then electrospun.

[0219] The gels given in the above example were fabricated to sheets using an electrospun manufacturing process with the following settings:

Distance from tip to collector: 25 cm
Electric field at tip: 20 kV
Electric field at collector: +6 kV
Tip geometry: 18 gauge
Flow rate: 10 ml/h
Temperature: room temperature

Humidity: 60%

Example 9

Preparation of a Two-Layered Composition Containing a Drug-Containing Layer and a Backing Layer

[0220]

TABLE-US-00008 Formulation Ingredients mg I - electrospin II - coating III - coating Polyvidone - Kollisone 100 100 100 90F Dextran 750.000 75 75 75 BDP or clobetasol 5 5 5 propionate Ethanol 1,000 1,000 1,000 Lubrizol - Carbothane 0 + +

[0221] Four compositions were made two of which were without any coating and the II-coating was sprayed on the electrospun fibres, whereas the III coating was made spun on top of the electrospun fibres.

Example 10

In Vivo Adhesion Testing of Compositions

[0222] The electrospun fibres exemplified in the examples herein were tested for bioadhesion by placing 1 cm1 cm sheet on the middle of the tongue. The subject tested the fibres was asked to evaluate the bioadhesiveness on a scale from 0 to 5, where 5 is strong bioadherence and 0 is no bioadherence.

Example 11

Electron Microscopic Analysis

[0223] Electron microscope pictures from the two different compositions given in Example 3 are shown in FIG. 1. From the figure it is seen that the size of the drug particles are much smaller in the fibres, where the drug substance is suspended in acetate buffer, i.e. confirming that part of the drug substance is dissolved in the acetate buffer before spinning.

Example 12

In Vitro Adhesion Testing of Electrospun Fibres

[0224] The bioadhesive forces of the electrospun fibres were determined by means of a bioadhesive measuring device shown in FIG. 2. Buccal mucosa was cut into strips/pieces and washed with tyroide solution. At time of testing a section of buccal mucosa (c) was secured keeping the mucosal side out, on the upper glass vial (B) using rubber band and aluminium cap. The diameter of each exposed mucosal membrane was 1 cm. The vial with the sheep buccal mucosa (C) was stored at 37 C. for 10 min. Then one vial with section of buccal mucosa (C) and another vial were fixed on height adjustable pan (E). To a lower vial electrospun fibres (D) were placed with the help of bilayered adhesive tape, adhesive side facing downward. The height of the lower vial was adjusted so that the electrospun fibres could adhere to the sheep buccal mucosa on the upper vial. A constant force was applied on the upper vial for 2 min, after which it was removed and the upper vial was then connected to the balance. Then the weight on right side pan was slowly added in an increment of 0.5 g till the two vials just separated from each other. The total weight (g) required to detach two vials was taken as a measure of bioadhesive strength. From this bioadhesive strength, the force of adhesive was calculated.

[0225] Other suitable methods may also be used such as the in vitro and in vivo methods described by Kockish et al. in Journal of Controlled Release, 77 (2001) 1-6, which is incorporated by reference in its entirety.

Example 13

In Vitro Relase Testing of Compositions

[0226] The purpose of the study is to explore the effect of polymer and bioadhesive polymer, plasticizer and oily release-enhancing agent on the in vitro release of betamethasone dipropionate, BDP, or clobetasol propionate from compositions described herein

Membrane:

[0227] Dow Corning 7-4107 Silicone Elastomer Membrane, 751 m.

Diffusion Cell System:

[0228] Modified dialysis cells.

[0229] Receptor compartment: 3.75 ml. The actual volume of each cell is registered by weighing of the assembled cell before and after filling of the receptor compartment. Diameter: 1.55 cm, corresponding to an available diffusion area of 1.89 cm.sup.2.

[0230] Sheets of silicone membrane are cut to size (circles, 0=22 mm). The membrane is placed between the two compartments of the dialysis cells with the glossy side facing the donor compartment.

[0231] The electron spun patch formulation is applied directly onto the membrane by pressing the actuator.

[0232] The receptor compartment is filled with preheated and degassed receptor medium (the actual volume of each cell is registered by weighing) and possible air bubbles removed.

[0233] The sampling arm is sealed with a plastic bung and parafilm to prevent evaporation of the receptor medium. Uniform mixing of the receptor phase is obtained with a magnetic bar placed in the receptor compartment. The diffusion cells are placed in a heating cabinet set at 37 C. to maintain a temperature of 32 C. at the membrane surface. The stirring bed is set.

Receptor Medium:

[0234] 10% w/w methyl-13-cyclodextrin in 0.05M acetate buffer pH 4.0. The receptor medium is degassed in an ultrasound water bath for minimum 20 minutes prior to the start of the experiment and before 24 h and 48 h sampling. It was ensured that sink conditions were present at all times during the study period; i.e. that the concentration of the drug compounds in the recipient phase was below 10% of the solubility of the drug substances in the medium.

Exposure and Sampling Times:

[0235] Samples of 1500 l (the actual volume is weighed and registered) are withdrawn from each cell at regular time intervals. After each sampling the receptor compartment is re-filled (the exact same volume as withdrawn) with preheated fresh receptor medium. The withdrawn samples are stored in brown sealed HPLC vials at 2-8 C. and protected from light until quantification by HPLC analysis at the end of the experiment. Sampling time points: 0, 1, 6, 24, 30, 48, 54, 72 h.

Study Design:

[0236] Each formulation is tested in 3 replicates (n=3).

Example 14

In Vitro Skin Penetration Studies

[0237] To investigate the skin penetration and permeation of imiquimod from compositions according to example 3 and 6 a skin diffusion experiment was conducted. Full thickness skin from pig ears was used in the study. The skin was cleaned and kept frozen at 18 C. before use. On the day prior to the experiment the skin was placed in a refrigerator (53 C.) for slow defrosting.

[0238] Static Franz-type diffusion cells with an available diffusion area of 3.14 cm.sup.2 and receptor volumes ranging from 8.6 to 11.1 ml were used in substantially the manner described by T. J. Franz, The finite dose technique as a valid in vitro model for the study of percutaneous absorption in man, in Current Problems in Dermatology, 1978, J. W. H. Mall (Ed.), Karger, Basel, pp. 58-68. The specific volume was measured and registered for each cell. A magnetic bar was placed in the receptor compartment of each cell. After mounting the skin, physiological saline (35 C.) was filled into each receptor chamber for hydration of the skin. The cells were placed in a thermally controlled water bath which was placed on a magnetic stirrer set at 300 rpm. The circulating water in the water baths was kept at 351 C. resulting in a temperature of about 32 C. on the skin surface. After 30 min the saline was replaced by the receptor medium, consisting of 1 part acetate buffer (100 mM, pH 4,0) and 1 part saline.

[0239] The in vitro skin permeation of each test composition containing imiquimod was tested in 3 replicates (i.e. n=6). Each test composition was applied on the skin membrane at 0 hours using a pipette. The skin penetration experiment was allowed to proceed for 24 hours. Samples were then collected from the receptor compartments for up to 72 hours.

[0240] The concentration of imiquimod in the samples was determined by HPLC.

Example 15

In Vitro Penetration in Buccal Tissue Culture

[0241] The apparatus used is shown in FIG. 3.

[0242] A bethamethasone dipropionate or clobetasol propionate containing spun sheet formulation is applied directly onto the membrane by pressing the actuator. The cells were kept at 37 C in a heating cabinet. The receptor compartment is filled with preheated receptor medium. The actual volume of each cell is registered by weighing. The receptor medium consists of 10 w/w methyl-13 cyclodekstrin in 0.05M acetate buffer pH 4.0. At different time intervals for up to 48 hours samples of receiver fluid is removed and replaced by fresh preheated receptor medium. Withdrawn samples are stored in brown sealed HPLC vials at 2-8.sup.0donor medium and protected from light until quantification by HPLC analysis at the end of the experiment. Each experiment was run in triplicate.

Example 16

In Vitro Skin Irritation Studies in Human Cell Culture

[0243] In vitro skin irritation studies in human cell culture was tested in accordance to OECD's Test Guidelines OECD Guidelines for the testing of chemicalsIn Vitro Skin Irritation: Reconstructed Human Epidermis Test Method. 439, adopted 26 Jul. 2013.

Example 17

Determination of Solubility of Bioadhesive Substances

[0244] The solubility of the bioadhesive substances was determined using a method recommended by the European Pharmacopoeia 5.0 (Section 5.11, p. 565).

[0245] The European Pharmacopoeia uses the following terms to define the solubility of a substance in a particular solvent (Section 1.4, p. 7):

TABLE-US-00009 Approximate volume of solvent Descriptive term in mL per g of solute Very soluble Less than 1 Freely soluble From 1 To 10 Soluble From 10 To 20 Sparingly soluble From 30 To 100 Slightly soluble From 100 To 1000 Very slightly soluble From 1000 To 10000 Practically insoluble More than 10000

[0246] The experimental method used to determine the solubility of dextrans and polyethylene oxide is described in the following:

Dissolving Procedure:

[0247] Shake tube (1 min) and place in a constant temperature device at a temperature of 250.5 C. for 15 min. If the substance is not completely dissolved, repeat the shaking (1 min) and place the tube in the constant temperature device for 15 min.

Method:

[0248] 1) Weigh 100 mg of finely powdered substance in a stoppered tube (16 mm in internal diameter and 160 mm long), add 0.1 ml of the solvent and proceed as described under Dissolving Procedure. If the substance is completely dissolved, it is very soluble. [0249] 2) If the substance is not completely dissolved, add 0.9 ml of the solvent and proceed as described under Dissolving Procedure. If the substance is completely dissolved, it is freely soluble. [0250] 3) If the substance is not completely dissolved, add 2.0 ml of the solvent and proceed as described under Dissolving Procedure. If the substance is completely dissolved, it is soluble. [0251] 4) If the substance is not completely dissolved, add 7.0 ml of the solvent and proceed as described under Dissolving Procedure. If the substance is completely dissolved, it is sparingly soluble. [0252] 5) If the substance is not completely dissolved, weigh 10 mg of finely powdered substance in a stoppered tube, add 10.0 ml of the solvent and proceed as described under Dissolving Procedure. If the substance is completely dissolved, it is slightly soluble. [0253] 6) If the substance is not completely dissolved, weigh 1 mg of finely powdered substance in a stoppered tube, add 10.0 ml of the solvent and proceed as described under Dissolving Procedure. If the substance is completely dissolved, it is very slightly soluble.

Materials

Substances:

[0254] 1) DEX20: Dextran with Mw 2,000,000 (Pharmacosmos) [0255] 2) PEO20: Polyethylene oxide with Mw 2,000,000 (Sigma Aldrich)

Solvent:

[0256] 1) 3 vol % distilled water in ethanol

Results

[0257]

TABLE-US-00010 Step DEX20 PEO20 1 Not dissolved Not dissolved 2 Not dissolved Not dissolved 3 Not dissolved Not dissolved 4 Not dissolved Not dissolved 5 Not dissolved Not dissolved 6 Not dissolved Not dissolved

Discussion and Conclusions

[0258] Both bioadhesive substances (i.e. dextran and polyethylene oxide) had not completely dissolved after the last step of the method recommended by the European Pharmacopoeia, in which 1 mg of substance is added to 10 ml of the solvent. [0259] This means that more than 10,000 ml of solvent are needed to dissolve 1 g of both substances. [0260] Therefore, using the terminology defined in the European Pharmacopoeia, the bioadhesive substances used in the fabrication of a composition of the invention (i.e. dextran and polyethylene oxide) may be described as practically insoluble in 3 vol % distilled water in ethanol.

Example 18

[0261] Determination of Maximum Amount of Water Added to Ethanol that Results in the Successful Formation of Fibres

[0262] The maximum amount of water that can be added to the solvent system was determined by preparing a series of solutions of polyvinylpyrrolidone (PVP) and/or Eudragit RS100 in blends of distilled water and ethanol, which were then electrospun to confirm the formation of fibres.

Composition of Solutions

[0263] PVP=10 wt % [0264] Eudragit RS100=0 wt % and 5 wt % [0265] Solvent=blend of distilled water and ethanol at various proportions

Electrospinning Conditions

[0266] 15 gauge needle [0267] Voltage=16 kV [0268] Distance=19 cm [0269] Flow rate=5 ml/h
Results [0270] Solutions prepared with up to 50 vol % water were easily processed, generating fibres and materials of good quality. [0271] Solutions prepared with 60 vol % water could generate fibres after modification of the electrospinning conditions. Resulting materials were of unsatisfactory quality. [0272] Solutions made of only PVP with 75 vol % and 100 vol % distilled water could generate fibres after modification of electrospinning conditions. Resulting materials were of unsatisfactory quality. [0273] Solution made of PVP and Eudragit RS100 with 75 vol % and 100 vol % distilled water could not be processed as Eudragit RS100 did not dissolve.

Results

[0274] Electrospun PVP and Eudragit RS100 also appear to show increased solubility and reduced material integrity when exposed to water as the water content in the solvent system increases.

CONCLUSIONS

[0275] Up to 50 vol % distilled water can be added to ethanol and produce good electrospun fibres made of PVP and/or Eudragit RS100. [0276] In practice, the concentration of water in the solvent system used is a balance between i) ensuring a good solubility of the fibre-forming hydrophilic polymer and a poor solubility of the bioadhesive substance, and ii) the properties of the bioadhesive substance upon contact with water; the bioadhesive substance should only to a minor extent affect the viscosity of the solvent system as a highly viscous solvent system makes it difficult to electrospin the fibres. [0277] Increased water content (>20 vol %) in solvent system affects behaviour of PVP+Eudragit RS100 electrospun fibres when exposed to water.

Example 19

[0278] Demonstration that Electrospun Fibres May Also be Produced with Additional Polymers

[0279] Various hydrophilic polymers could be suitable for fibre formation: polyvinylpyrrolidone, polyvinyl alcohol, ethyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, acrylates and acrylic copolymers.

[0280] A brief literature review of the field was performed in order to learn the potential solution and processing conditions that may facilitate the production of electrospun fibres.

[0281] Then, the possibility of electrospinning polymers other than PVP and Eudragit was investigated using solutions of the following polymers in various solvent systems: [0282] 1) Poly(vinyl alcohol), 99+% hydrolyzed, Mw 146,000-186,000 [0283] 2) Sodium carboxymethyl cellulose, Mw 250,000 [0284] 3) Hydroxypropyl cellulose, Mw 100,000 [0285] 4) Ethyl cellulose, ethoxyl content 48%, 10 cps

[0286] Initially, the solvents selected were ethanol and distilled water. According to FAO (Food and Agriculture Organization of the United Nations), the solubility of these polymers in ethanol and water is as follows:

TABLE-US-00011 Polymers Solubility in ethanol Solubility in water Poly(vinyl alcohol) Sparingly soluble Soluble Ethyl cellulose Soluble if ethyl cellulose Practically insoluble contains 46-48% or more of ethoxyl groups Hydroxypropyl Forms smooth and clear Forms smooth cellulose solution at <38 C. and clear solution at <38 C. Carboxymethyl Not soluble Yields viscous cellulose colloidal solution

[0287] Thus, based on this information PVA and CMC are not freely soluble in ethanol.

Poly(Vinyl Alcohol) (PVA)

[0288] PVA dissolved in distilled water at (70-90) C. under continuous stirring until formation of clear solution. [0289] Concentration=6 wt % [0290] Electrospun fibres formed when using 20 kV and 1.25 ml/h. [0291] Irregular formation of fibresCurrently not adequate for use as a fibre-forming hydrophilic polymer in the fabrication of a composition according to the invention.

Ethyl Cellulose (EC)

[0292] EC dissolved well in ethanol and tetrahydrofuran. [0293] Concentration=10-15 wt % [0294] Not possible to electrospin under a wide range of processing conditions, but proper adjustment of process parameters may enable processing of fibres [0295] However, could produce electrospun fibres and whole mats by blending with PVP (i.e. 10 wt % PVP and 5 wt % EC) [0296] Resulting material exhibited reduced solubility in water, similar to electrospun PVP and RS100.

Hydroxypropyl Cellulose (HPC)

[0297] HPC dissolved well in ethanol and tetrahydrofuran. [0298] Concentration=10-15 wt % [0299] Not possible to electrospin under a wide range of processing conditions, but proper adjustment of process parameters may enable processing of fibres [0300] However, could produce electrospun fibres and whole mats by blending with PVP (i.e. 10 wt % PVP and 5 wt % HPC) [0301] Addition of HPC to PVP did not reduce solubility of electrospun fibres.

Carboxymethyl Cellulose (CMC)

[0302] CMC dissolved well in distilled water. [0303] Concentration=1-3 wt % [0304] Not possible to electrospin under a wide range of processing conditions. [0305] Results partially improved after blending with polyethylene oxide (CMC:PEO 1:2) and addition of 25 vol % ethanol to distilled water, although fibre formation was not observed.

Conclusions

[0306] PVA can be electrospun although current results are not adequate for fabrication of a composition according to the invention. [0307] EC and HPC can be electrospun if blended with PVP. [0308] CMC cannot be currently electrospun. [0309] Results for EC, HPC and CMC differ with what is reported in the literature. [0310] Probably possible with further adjustments of the process parameters.

Example 20

Demonstration of the Spinnability of Various Eudragit Compositions

[0311] Various Eudragit compositions were mentioned in the patent, and it was considered important to find out which compositions can be used successfully to produce electrospun fibres.

[0312] The following compositions were identified as interesting to be investigated:

TABLE-US-00012 Eudragit Chemical Composition E100 Basic Butylated Methacrylate Copolymer L100 Methacrylic Acid - Methyl Methacrylate Copolymer (1:1) S100 Methacrylic Acid - Methyl Methacrylate Copolymer (1:2) L100-55 Methacrylic Acid - Ethyl Acrylate Copolymer (1:1) Type A RL100 Ammonio Methacrylate Copolymer, Type A RS100 Ammonio Methacrylate Copolymer, Type B Plastoid B Neutral copolymer based on butyl methacrylate and methyl methacrylate

Eudragit RS100

[0313] Ammonio Methacrylate Copolymer, Type B. [0314] Dissolved in 3 vol % distilled water in ethanol. [0315] Possible to electrospin. [0316] Good fibre formation when blended with PVP.

Eudragit L100-55

[0317] Methacrylic AcidEthyl Acrylate Copolymer (1:1) Type A. [0318] Dissolved in ethanol. [0319] Possible to electrospin forming materials of good quality. [0320] If a blend of two or more fibre-forming hydrophilic polymers are used for fibre formation, then the polymers used should be able to blend in the solvent system used and they should be dissolved.

Example 21

Molecular Weight of Bioadhesive Substances Used

[0321] The aim of this example is to demonstrate that the bioadhesive substances suggested for use in a composition of the invention can be employed within the molecular weight ranges stated.

[0322] One requirement is that the bioadhesive substance must not be freely soluble in the solvent system used, the solubility should be sparingly soluble or less. This sets a limitation with respect to molecular weight as eg dextran and PEO with low molecular weight do not fulfil the solubility criteria.

Our Experimental Work Had Demonstrated:

[0323] Bioadhesive strength of a polymer tends to increase as the molecular weight increases. [0324] This is related to the critical molecular length necessary to produce an inter-penetrating layer and entanglements with the surface of the soft tissues. [0325] In the case of polyethylene oxide, which has a highly linear configuration, adhesive strengths increases up to molecular weights of 4,000,000. [0326] In the case of dextrans, which present a more coiled configuration, are reported to display similar bioadhesive strengths at both low and high molecular weights due to shielding of the functional dextran groups.

[0327] For polyethylene oxide, an experimental study of the bioadhesive properties of the electrospun composition was performed using PEO with molecular weights of 400,000 and 2,000,000. [0328] Although there were no significant differences between both compositions, the patches with polyethylene oxide of 2,000,000 presented results with smaller variability and greater average adhesion times.

[0329] For dextrans, the experimental study was performed on electrospun materials containing dextrans with molecular weights of 500,000 and 2,000,000. [0330] Similarly, although there were no significant differences between both compositions, the patches with dextrans of 2,000,000 presented results with greater average adhesion times.

[0331] In conclusion, the high molecular weights substances were selected in the bioadhesion study as they exhibited more clearly defined results than those of lower molecular weight.

Example 22

[0332] Determination of Maximum Amount of Bioadhesive Substance that can be Added to the Electrospinning Solution

[0333] The maximum amount of bioadhesive substances that can be added to the electrospun materials was determined by preparing a series of solutions of polyvinylpyrrolidone (PVP) in ethanol with increasing amounts of bioadhesive substance, which were then electrospun to confirm the formation of fibres.

Composition of Solutions

[0334] PVP=10 wt % [0335] Bioadhesive substances=polyethylene oxide, Mw 2,000,000 (PEO20) and dextrans, Mw 2,000,000 (DEX20) [0336] Solvent=ethanol

Electrospinning Conditions

[0337] 15 gauge needle [0338] Voltage=16 kV [0339] Distance=19 cm [0340] Flow rate=5 ml/h
Results [0341] Solutions with up to 30 wt % DEX20 and 20 wt % PEO20 were easily processed, generating fibres and materials of good quality. [0342] Solutions with 40 wt % DEX20 and 30 wt % PEO20 could be electrospun but the resulting materials were of unsatisfactory quality due to the high viscosity of the solution. [0343] Preparations with 50 wt % DEX20 and 40 wt % PEO20 could not be processed. Their viscosity was too high to be electrospun, and their appearance was more paste-like than solution-like.

Example 23

[0344] Demonstration that Electrospun Fibres May Also be Produced Using Additional Bioadhesive Substances

[0345] Experiments with a range of bioadhesive substances other than dextran and polyethylene oxide have been performed.

[0346] A brief literature review of the field was performed in order to identify potential bioadhesive substances that may be added to the electrospun fibres. Then, the following hydrophilic substances were proposed:

TABLE-US-00013 Bioadhesive Solubility in ethanol Solubility in water Sodium alginate Not soluble Dissolves slowly, forming a viscous solution Sodium carboxymethyl Not soluble Forms viscous colloidal cellulose solution Chitosan Not soluble Not soluble unless pH <6 or deacetylated Poly(vinyl alcohol) Sparingly soluble Soluble

[0347] The substances used were: [0348] 1) Alginic acid sodium salt from brown algae, medium viscosity [0349] 2) Sodium carboxymethyl cellulose, Mw 250,000 [0350] 3) Chitosan, medium molecular weight [0351] 4) Poly(vinyl alcohol), 99+% hydrolyzed, Mw 146,000-186,000

[0352] Particle size of the substances as supplied was too large to be added to the electrospun fibres. Therefore, all substances were milled and sieved to produce powders with particle size<150 m.

[0353] Electrospinning solutions were then prepared and processed under the following conditions: [0354] 20 wt % Eudragit L100-55 in ethanol+10 wt % bioadhesive substance [0355] 15 gauge needle [0356] Voltage=16 kV [0357] Distance=19 cm [0358] Flow rate=2.5 ml/h [0359] The results showed that other bioadhesive substances are suitable for use in the present context. [0360] Poly(vinyl alcohol) and chitosan particles were visible after drying of the samples while the other substances were not apparent. This suggests that poly(vinyl alcohol) and chitosan may be the substances with the least bioadhesive potential due to slow dissolution in water at room temperature.

Example 24

Fibre Formation of PVP Using Ethanol as Solvent

[0361] Experiments were conducted to investigate whether fibre-formation of PVP is dependent on the concentration of PVP in ethanol. The following results were obtained:

1) 2.5 wt % PVPNo formation of fibre. Electrospraying (i.e. formation of particles rather than fibres) was observed instead, even after reducing the flow rate to 2.5 mL/h and 1 mL/h.
2) 5 wt % PVPFibre formation was observed. Good formation of membrane made of individual fibres.
3) 7.5 wt % PVPFibre formation was observed. Good formation of membrane made of individual fibres.
4) 10 wt % PVPFibre formation was observed. Good formation of membrane made of individual fibres.
5) 12.5 wt % PVPFibre formation was observed. Good formation of membrane made of individual fibres.
6) 15 wt % PVPFibre formation was observed. Good formation of membrane made of individual fibres.
7) 20 wt % PVPFibre formation was observed. Membrane made of individual fibres could be fabricated after reducing flow rate to 2.5 mL/h and increasing the distance to collector to 23 cm.
8) 25 wt % PVPFibre formation was observed. A membrane could be fabricated after reducing the flow rate to 1 mL/h and increasing the distance to collector to 23 cm. However, the resulting membrane was of less good quality than the fibres obtained in 1)-7).

[0362] All solutions were prepared in ethanol.

[0363] The electrospinning conditions were as follows, except when otherwise indicated: [0364] Voltage=15 kV [0365] Flow rate=5 mL/h [0366] Distance to collector=19 cm

[0367] The diameter of the fibres was observed to increase as the concentration of PVP increased. In the case of 20 wt % and 25 wt % PVP this resulted in slower solvent evaporation and the fusion of the fibres after deposition, forming a film. In these cases the distance to the collector was increased to 23 cm in order to obtain membranes made of individual fibres. Additionally, the area of fibre deposition on the collector decreased as the concentration increased.

[0368] The viscosity of 20 wt % and 25 wt % PVP was significantly greater than in the other solutions, probably causing the issues with fibre formation mentioned above. In the specific case of 25 wt % it was difficult to eliminate air bubbles from the solution prior to electrospinning due to its viscous nature. Fibres could be generated from 20 wt % and 25 wt %.

[0369] These results suggested that the optimal range of PVP concentrations for the fabrication of fibres according to the invention may be between 5 wt % and 20 wt %, with concentrations around 10 wt % producing very good results.