Betulin-containing birch bark extracts and their formulation

Abstract

To provide wound-healing formulations containing solid birch bark extracts that may be applied to the skin without worsening the patient's condition.SOLUTION: A solid birch bark extract comprises at least about 70 wt.% of betulin, and one or more triterpenes selected from the group consisting of betulinic acid, oleanolic acid, erythrodiol, and lupeol.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 613,646, filed January 4, 2018, the entire contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] The present disclosure relates to pharmaceutical preparations derived from extracts of birch bark. [Background technology]

[0003] The triterpenes found in birch bark extract are known to have wound healing properties.The method of extracting these triterpenes from birch bark is reported in U.S. Patent No. 7,482,383.These methods provide a solid birch bark extract that can be used in pharmaceutical preparations.For example, an emulsion containing such an extract is described in U.S. Patent No. 7,482,383, and an oleogel containing such an extract is described in U.S. Patent No. 9,352,041, U.S. Patent No. 8,828,444, and U.S. Patent No. 8,536,380.

[0004] For clinical use in wound healing, the oleogel must be applied by touching the area of ​​skin requiring treatment. Contact application is disadvantageous in the treatment of certain skin conditions (e.g., epidermolysis bullosa) because the simple act of applying the oleogel can lead to a worsening of the skin condition. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Provisional Application No. 62 / 613,646 [Patent Document 2] U.S. Patent No. 7,482,383 [Patent Document 3] U.S. Patent No. 7,482,383 [Patent Document 4] U.S. Patent No. 9,352,041 [Patent Document 5] U.S. Patent No. 8,828,444 [Patent Document 6] U.S. Patent No. 8,536,380 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a need for a wound healing preparation containing solid birch bark extract that can be applied to the skin without worsening the patient's condition. [Means for solving the problem]

[0007] The present disclosure provides clinically advantageous wound healing formulations (including oleogels, emulsions and foams) with improved rheological properties that overcome the disadvantages of known solid birch bark-containing emulsions and oleogels.

[0008] The present disclosure provides a solid birch bark extract containing at least about 70% by weight of betulin and one or more triterpenes selected from the group consisting of betulinic acid, oleanolic acid, erythrodiol, and lupeol. The solid birch bark extract described herein can be formulated into an oleogel with clinically advantageous rheological properties.

[0009] The present disclosure also provides methods for making a solid birch bark extract dispersible in a non-polar solvent, comprising the steps of: (a) contacting birch bark with a suitable solvent to form an extract solution containing betulin and at least one triterpene; (b) separating the birch bark from the extract solution; (c) cooling the extract solution to crystallize a portion of the betulin and triterpene from the solution; (d) separating the crystallized betulin and triterpene; and (e) drying the separated crystallized betulin and triterpene to form a solid birch bark extract, providing a clinically advantageous oleogel. The present disclosure also provides solid birch bark extracts prepared according to these methods.

[0010] The present disclosure provides a clinically advantageous oleogel comprising about 1% to about 20% by weight of solid birch bark extract particles dispersed in about 80% to about 99% of one or more non-polar liquids, the solid birch bark extract being dispersed in a suitable non-polar liquid to form the oleogel. Also provided are pads and sterile wound dressings comprising a therapeutically effective amount of the oleogel.

[0011] The present disclosure provides emulsions comprising the oleogels of the present disclosure, and further provides foams comprising the emulsions of the present disclosure.

[0012] The present disclosure also provides a method of treating a wound in a patient by topically administering to at least a portion of the wound an effective amount of an oleogel, emulsion, or foam of the present disclosure.

[0013] The present disclosure also provides a method of treating epidermolysis bullosa in a patient in need thereof comprising topically administering to the area of ​​epidermolysis bullosa an effective amount of an oleogel, emulsion, or foam of the present disclosure.

[0014] The present disclosure provides solid birch bark extracts, oleogel, sterile wound dressings, preparation processes, emulsions, foams, pressurized containers, methods of treating wounds, and methods of treating epidermolysis bullosa, according to the following numbered embodiments: 1. A solid birch bark extract comprising at least about 70% by weight of betulin and one or more triterpenes selected from the group consisting of betulinic acid, oleanolic acid, erythrodiol and lupeol. 2. A solid birch extract, when chromatographed according to the following HPLC method, has an HPLC chromatogram substantially identical to that in Figure 1; [Table 1] 2. The solid birch bark extract of embodiment 1, wherein betulinic acid has a relative retention time of about 0.75-0.90, oleanolic acid has a relative retention time of about 0.84-0.97, betulin has a relative retention time of 1.00, erythrodiol has a relative retention time of about 1.25-1.40, and lupeol has a relative retention time of about 3.50-4.15. 3. (a) contacting birch bark with a pharmaceutically acceptable solvent, thereby forming an extract solution containing betulin and one or more triterpenes; (b) separating the birch bark from the extraction solution; (c) cooling the extract solution, whereby a portion of the betulin and one or more triterpenes crystallize from the cooled extract solution; and (d) separating the crystallized betulin and one or more triterpenes from the cooled extract solution; and (e) drying the separated crystallized betulin and one or more triterpenes to provide a solid birch bark extract; 3. The solid birch bark extract of embodiment 1 or 2, wherein after drying, about 1% to about 20% by weight of the dried solid birch bark extract of step (e) dispersed in the refined sunflower oil forms an oleogel. 4. One or more of steps (a), (c), and (e) are subject to the following conditions: (i) the contacting in step (a) is carried out at a temperature of about 60°C to about 130°C; (ii) the cooling in step (c) is carried out at a temperature of about −20° C. to about 35° C.; (iii) The solid birch bark extract of embodiment 3, wherein said drying in step (e) is carried out at a temperature of about 75° C. to about 95° C. at a pressure of less than about 70 mbar. 5. Two or more of steps (a), (c), and (e) are subject to the following conditions: (i) the contacting in step (a) is carried out at a temperature of from about 60°C to about 130°C; (ii) the cooling in step (c) is carried out at a temperature of about −20° C. to about 35° C.; (iii) The solid birch bark extract of embodiment 3, wherein said drying in step (e) is carried out at a temperature of about 75° C. to about 95° C. at a pressure of less than about 70 mbar. 6. Steps (a), (c), and (e) are subject to the following conditions: (i) the contacting in step (a) is carried out at a temperature of from about 60°C to about 130°C; (ii) the cooling in step (c) is carried out at a temperature of about −20° C. to about 35° C.; (iii) The solid birch bark extract of embodiment 3, wherein said drying in step (e) is carried out at a temperature of about 75° C. to about 95° C. at a pressure of less than about 70 mbar. 7. The solid birch bark extract of any one of embodiments 3-6, wherein the pharmaceutically acceptable solvent is a hydrocarbon or an alcohol, or a combination thereof. 8. The solid birch bark extract of any one of embodiments 3-7, wherein the pharmaceutically acceptable solvent is a hydrocarbon selected from the group consisting of n-pentane, n-hexane, or n-heptane. 9. The solid birch bark extract of embodiment 8, wherein the pharmaceutically acceptable solvent is n-heptane and the contacting is carried out at a temperature of about 60°C to about 130°C for about 8 to 12 minutes. 10. The solid birch bark extract of any one of embodiments 3-9, wherein in step (c), the cooled extraction solution is at least about 2-fold supersaturated. 11. The solid birch bark extract of embodiment 10, wherein the cooled extraction solution is about 5 times supersaturated. 12. The solid birch bark extract of any one of embodiments 3-11, wherein the amount of residual extraction solvent is about 0.5% by weight or less. 13. An oleogel comprising about 1% to about 20% by weight of particles of the solid birch bark extract of any one of embodiments 1 to 12, having an average particle size of less than about 50 μm, dispersed in about 80% to about 99% by weight of one or more non-polar liquids. 14. The oleogel of embodiment 13, wherein the dispersed solid birch bark extract particles are the only oleogel-forming agent in the oleogel. 15. The oleogel of embodiment 13 or 14, comprising about 10% by weight of solid birch bark extract particles. 16. The oleogel of any one of embodiments 13 to 15, wherein the non-polar liquid comprises at least one triglyceride. 17. The oleogel of any one of embodiments 13-16, wherein the non-polar liquid comprises at least one C7 or higher hydrocarbon. 18. The oleogel of embodiment 16, wherein the non-polar liquid comprises one or more vegetable oils. 19. The oleogel of embodiment 18, wherein the non-polar liquid comprises sunflower oil. 20. The oleogel of any one of embodiments 13-19, wherein the non-polar liquid has a peroxide value of less than about 10. 21. The oleogel of embodiment 20, wherein the peroxide value is about 3 or less. 22. The oleogel of any one of embodiments 13-21, wherein the oleogel is substantially free of solid birch bark extract particles having a size greater than about 50 μm. 23. The oleogel of any one of embodiments 13 to 22, wherein the oleogel is sterile. 24. The oleogel of embodiment 23, wherein the oleogel is sterilized by ionizing radiation at a dose of less than about 20 kGy. 25. The oleogel of embodiment 24, wherein the oleogel is sterilized by ionizing radiation at a dose ranging from about 11 to about 20 kGy. 26. The oleogel of any one of embodiments 13 to 25, wherein separation of non-polar liquid from the oleogel after centrifugation at 2750 g for 30 minutes at 25° C. is less than about 10%. 27. The oleogel according to any one of embodiments 13 to 26, wherein the viscosity of the oleogel at 200 s is in the range of about 0.5 to about 4.0 Pa.s and the thixotropy value of the oleogel is in the range of about 200 to about 1200 Pa.s when measured according to the rotational viscometer method described in Ph.Eur.2.2.10 using a cone and plate viscometer. 28. The oleogel of any one of embodiments 13 to 27, wherein the consistency of the oleogel is in the range of about 300 to 3000 mN as measured by use of a texture analyzer. 29. The oleogel of any one of embodiments 13 to 28, further comprising a bactericide. 30. The oleogel of embodiment 29, wherein the disinfectant is selected from the group consisting of ethanol, n-propanol, and isopropanol. 31. The oleogel of any one of embodiments 13 to 28, further comprising a lipophilic antibiotic. 32. A sterile wound dressing comprising: (a) a pad; (b) a therapeutically active layer disposed on at least one surface of the pad, the therapeutically active layer comprising the oleogel of any of embodiments 13-31. 33. A sterile wound dressing as described in embodiment 32, wherein the pad is an absorbent pad. 34. (a) contacting birch bark with a pharmaceutically acceptable solvent, thereby forming an extract solution containing betulin and one or more triterpenes; (b) separating the birch bark from the extraction solution; (c) cooling the extract solution, whereby a portion of the betulin and one or more triterpenes crystallize from the cooled extract solution; and (d) separating the crystallized betulin and one or more triterpenes from the cooled extract solution; and (e) drying the separated crystallized betulin and one or more triterpenes to provide a solid birch bark extract; 3. The method for preparing a solid birch bark extract according to embodiment 1 or 2, wherein after drying, about 1% to about 20% by weight of the dried solid birch bark extract of step (e) dispersed in the refined sunflower oil forms an oleogel. 35. One or more of steps (a), (c), and (e) may be subject to the following conditions: (i) the contacting in step (a) is carried out at a temperature of from about 60°C to about 130°C; (ii) the cooling in step (c) is carried out at a temperature of about −20° C. to about 35° C.; (iii) The method of embodiment 34, wherein said drying in step (e) is carried out at a pressure of less than about 70 mbar and a temperature of about 75°C to about 95°C. 36. Two or more steps (a), (c), and (e) are subject to the following conditions: (i) the contacting in step (a) is carried out at a temperature of from about 60°C to about 130°C; (ii) the cooling in step (c) is carried out at a temperature of about −20° C. to about 35° C.; (iii) The solid birch bark extract of embodiment 34, wherein said drying in step (e) is carried out at a temperature of about 75°C to about 95°C at a pressure of less than about 70 mbar. 37. Steps (a), (c), and (e) are subject to the following conditions: (i) the contacting in step (a) is carried out at a temperature of from about 60°C to about 130°C; (ii) the cooling in step (c) is carried out at a temperature of about −20° C. to about 35° C.; (iii) The solid birch bark extract of embodiment 34, wherein said drying in step (e) is carried out at a temperature of about 75°C to about 95°C at a pressure of less than about 70 mbar. 38. The method of any one of embodiments 34-37, wherein the pharmaceutically acceptable solvent is a hydrocarbon selected from the group consisting of n-pentane, n-hexane, or n-heptane. 39. The method of any one of embodiments 34-38, wherein the pharmaceutically acceptable solvent is n-heptane and the contacting is carried out at a temperature of about 115°C to about 130°C for about 8 to 12 minutes. 40. The method of any one of embodiments 34-39, wherein in step (c), the cooled extraction solution is at least about 2-fold supersaturated. 41. The method of embodiment 40, wherein the cooled extraction solution is about 5 times supersaturated. 42. The method of any one of embodiments 34 to 41, wherein the amount of residual extraction solvent is about 0.5% by weight or less. 43. An emulsion comprising the solid birch bark extract of any one of embodiments 1 to 12. 44. An emulsion comprising an oleogel according to any one of embodiments 13 to 31. 45. An emulsion described in embodiment 43 or 44, wherein the emulsion is a water-in-oil emulsion. 46. ​​The emulsion of embodiment 45, which is substantially free of emulsifiers. 47. An emulsion according to any one of embodiments 43 to 46, consisting essentially of solid birch bark extract or oleogel, oil, and water. 48. An emulsion according to any one of embodiments 44 to 46, consisting of an oleogel, oil, and water. 49. The emulsion of any one of embodiments 43 to 46, further comprising a bactericide. 50. An emulsion described in embodiment 49, wherein the disinfectant is selected from the group consisting of ethanol, n-propanol, and isopropanol. 51. An emulsion described in any one of embodiments 43 to 46, further comprising a lipophilic antibiotic. 52. A foam comprising the emulsion of any one of embodiments 43 to 51. 53. The foam of embodiment 52, further comprising a disinfectant. 54. The foam of embodiment 53, wherein the disinfectant is selected from the group consisting of ethanol, n-propanol, and isopropanol. 55. The foam described in embodiment 52, further comprising a lipophilic antibiotic. 56. A foam according to any one of embodiments 52-55, wherein the interfacial tension of the emulsion is greater than about 4 mN / m. 57. The foam of any one of embodiments 52-56, wherein the foam index is greater than about 2. 58. The foam of embodiment 52, wherein the oleogel consists of about 5% to about 10% by weight of solid birch bark extract, and the emulsion is a water-in-oil emulsion consisting of oleogel and about 20% to about 30% by weight of water. 59. A foam according to embodiment 58, wherein the oleogel consists of about 7% by weight of solid birch bark extract and the amount of water in the emulsion is about 25% by weight. 60. The foam of any one of embodiments 52-56, further comprising an emulsifier selected from the group consisting of phosphatidylcholine, polyglyceryl-3-methylglucose distearate, and combinations thereof. 61. A pressurized container filled with a mixture comprising the emulsion of any one of embodiments 43 to 51 and a pharmaceutically acceptable propellant, wherein the emulsion forms a foam upon decanting at least a portion of the mixture from the container. 62. A method of treating a wound in a patient, comprising topically administering to at least a portion of the wound an oleogel according to any one of embodiments 13 to 31. 63. An oleogel according to any one of embodiments 13 to 31 for use in a method for treating a wound. 64. The method or oleogel for use of embodiment 63, wherein the wound is selected from the group consisting of burns, surgical skin lesions, superficial body injuries, chronic wounds, pressure ulcers, diabetic foot ulcers, chronic venous ulcers, arterial insufficiency ulcers, wounds resulting from aesthetic skin treatments, wounds resulting from ablative laser skin treatments, wounds resulting from chemical peels, wounds resulting from skin abrasion, wounds resulting from adverse drug reactions, wounds resulting from toxic epidermal necrolysis, wounds resulting from Lyell's syndrome, wounds resulting from Stevens-Johnson syndrome, and wounds resulting from radiation dermatitis. 65. A method of treating a wound in a patient, comprising topically administering to at least a portion of the wound an emulsion described in any one of embodiments 43 to 51. 66. An emulsion described in any one of embodiments 43 to 51 for use in a method for treating a wound. 67. The method or emulsion for use of embodiment 66, wherein the wound is selected from the group consisting of burns, surgical skin lesions, superficial body injuries, chronic wounds, pressure ulcers, diabetic foot ulcers, chronic venous ulcers, arterial insufficiency ulcers, wounds resulting from aesthetic skin treatments, wounds resulting from ablative laser skin treatments, wounds resulting from chemical peels, wounds resulting from skin abrasion, wounds resulting from side effects of medications, wounds resulting from toxic epidermal necrolysis, wounds resulting from Lyell's syndrome, wounds resulting from Stevens-Johnson syndrome, and wounds resulting from radiation dermatitis. 68. A method of treating a wound in a patient, comprising topically administering to at least a portion of the wound a foam described in any one of embodiments 52-60. 69. A foam according to any one of embodiments 52 to 60 for use in a method for treating a wound. 70. The method or foam for use of embodiment 69, wherein the wound is selected from the group consisting of burns, surgical skin lesions, superficial body injuries, chronic wounds, pressure ulcers, diabetic foot ulcers, chronic venous ulcers, arterial insufficiency ulcers, wounds resulting from aesthetic skin treatments, wounds resulting from ablative laser skin treatments, wounds resulting from chemical peels, wounds resulting from skin abrasion, wounds resulting from side effects of medications, wounds resulting from toxic epidermal necrolysis, wounds resulting from Lyell's syndrome, wounds resulting from Stevens-Johnson syndrome, and wounds resulting from radiation dermatitis. 71. A method of treating epidermolysis bullosa in a patient in need thereof, comprising topically administering to the area of ​​epidermolysis bullosa of the patient an oleogel according to any one of embodiments 13-31. 72. An oleogel according to any one of embodiments 13 to 31 for use in a method for treating epidermolysis bullosa. 73. A method of treating epidermolysis bullosa in a patient in need thereof, comprising topically administering to the patient's area of ​​epidermolysis bullosa an emulsion of any one of embodiments 43-51. 74. An emulsion described in any one of embodiments 43 to 51 for use in a method for treating epidermolysis bullosa. 75. A method of treating epidermolysis bullosa in a patient in need thereof, comprising topically administering to the area of ​​epidermolysis bullosa of the patient a foam of any one of embodiments 52-60. 76. A foam according to any one of embodiments 52 to 60 for use in a method for treating epidermolysis bullosa. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 shows HPLC chromatograms obtained from solid birch bark extract, monitoring at 210 nm (top) and 320 nm (bottom). The x-axis of Figure 1 is retention time (minutes) and the y-axis is peak intensity (mAU). [Figure 2] FIG. 2 illustrates the sequential extraction of birch bark to provide an extract solution containing betulin and one or more triterpenes. DETAILED DESCRIPTION OF THE INVENTION

[0016] definition Although the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the subject matter of the present disclosure.

[0017] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Preferred methods, devices, and materials are described, but any methods and materials similar or equivalent to those described herein can be used to practice or test this disclosure.All references cited herein (including U.S. Patent Nos. 9,352,041, 8,828,444, 8,536,380, and 7,482,383) are incorporated by reference in their entirety for all purposes.

[0019] Following long-standing patent law convention, the terms "a," "an," and "the" when used in this application, including the claims, refer to "one or more." Thus, for example, reference to a "carrier" includes one or more carriers, mixtures of two or more carriers, etc.

[0020] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and the like used in the specification and claims should be understood as modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present application. In general, when used herein in reference to a measurable value, such as weight, time, dose, and the like, the term "about" is meant to encompass variations of ±15% or ±10% from the particular amount in some instances, ±5% in other instances, ±1% in other instances, and ±0.1% in still other instances, where such variations are appropriate for practicing the disclosed methods.

[0021] "Administering" includes any mode of administration, such as oral, subcutaneous, sublingual, transmucosal, parenteral, intravenous, intraarterial, buccal, sublingual, topical, vaginal, rectal, ophthalmic, otic, nasal, inhalation, and transdermal. "Administering" can also include prescribing or filling a prescription for a dosage form containing a particular compound. "Administering" can also include providing instructions for practicing a method involving a particular compound or a dosage form containing that compound.

[0022] As used herein, the verb "comprise" and its conjugations as used in the specification and claims are used in their open-ended sense, meaning that the items following the word are included but not excluding items not specifically mentioned.

[0023] The term "birch bark" means the bark layer of the white-barked birch tree. Preferred embodiments include birch bark from Betula pendula Roth and Betula pubescens Ehrh, as well as hybrids of both species.

[0024] The term "relative retention time" (or "RRT") refers to the ratio of the retention time of an unknown peak to the retention time of the betulin peak, and is calculated according to the following equation: RRT=RT unknown / RT betulin During the ceremony, RT unkown is the retention time of the unknown peak, and RT betulin is the retention time of the betulin peak (210 nm), measured by the following HPLC method. [Table 2]

[0025] The term "substantially identical," as used herein, refers to an analytical spectrum, such as an HPLC chromatogram or NMR spectrum, that closely resembles a reference spectrum in both peak positions and their intensities. For example, an HPLC chromatogram is "substantially identical" to a reference chromatogram if the peak positions (relative retention times) in the HPLC chromatogram vary by no more than ±5% from the peak positions (relative retention times) in the reference chromatogram. In some embodiments, the relative peak intensities of the HPLC chromatogram may vary by no more than ±10% from the peak intensities in the reference chromatogram.

[0026] "Therapeutically effective amount" means the amount of an active substance that, when administered to a subject for treating a disease, disorder, or other undesirable medical condition, is sufficient to have a beneficial effect on the disease, disorder, or condition. The therapeutically effective amount varies depending on the chemical identity and formulation of the active substance, the disease or condition and its severity, and the age, weight, and other relevant characteristics of the patient being treated. Determining the therapeutically effective amount of a given active substance is within the skill of the art and typically requires only routine experimentation.

[0027] The term "substantially free" means that the indicated component is absent or present in only trace amounts. In one embodiment, "substantially free" means less than about 10% (e.g., less than about 10% by weight). In other embodiments, "substantially free" means less than about 5% (e.g., less than about 5% by weight), less than about 2% (e.g., less than about 2% by weight), or less than about 1% (e.g., less than about 1% by weight), or about 0% (e.g., about 0% by weight). For example, an emulsion that is substantially free of emulsifiers does not contain a substantial amount of emulsifier (e.g., contains less than about 10%, less than about 5%, less than about 2%, or less than about 1% by weight of emulsifier, or about 0% by weight of emulsifier). Similarly, an oleogel that is substantially free of solid birch bark extract particles having a size greater than about 50 μm does not contain a substantial amount of solid birch bark extract particles having a size greater than about 50 μm (e.g., contains less than about 10% by weight, less than about 5%, less than about 2%, or less than about 1% by weight of solid birch bark extract particles having a size greater than about 50 μm, or contains 0% by weight of solid birch bark extract particles having a size greater than about 50 μm).

[0028] Solid birch bark extract The present disclosure provides a solid birch bark extract that can be formulated into a clinically beneficial oleogel. Without being bound by any theory, it is believed that the chemical composition and particle morphology of the solid birch bark extract produced according to the methods of the present disclosure are responsible for these benefits.

[0029] The solid birch bark extracts of the present disclosure can be characterized based on their chemical composition. In some embodiments, the solid birch bark extracts of the present disclosure contain lupane and oleanane triterpenes. In particular, the birch bark extract can contain the lupane triterpenes betulin, lupeol, and betulinic acid, and the oleanane triterpenes erythrodiol and oleanolic acid.

[0030] The presence of specific triterpenes in birch bark extracts can be determined using relative retention times obtained from HPLC chromatography. The following HPLC method is used to determine the chemical composition of the solid birch bark extracts of the present disclosure. [Table 3]

[0031] The Phenomenex Kinetix C18 column is an example of an HPLC column using a C-18 modified silica stationary phase on a core-shell silica solid support (2.6 μm HILIC 100 Å) with the column dimensions described above.

[0032] When the solid birch bark extract is subjected to the above-mentioned HPLC chromatography method, betulinic acid has a relative retention time of about 0.75-0.90, oleanolic acid has a relative retention time of about 0.84-0.97, betulin has a relative retention time of 1.00, erythrodiol has a relative retention time of about 1.25-1.40, and lupeol has a relative retention time of about 3.50-4.15.

[0033] In certain embodiments, the solid birch bark extract has an HPLC chromatogram substantially identical to FIG.

[0034] In some embodiments, the solid birch bark extract comprises at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% by weight of betulin and one or more triterpenes, in some embodiments, the one or more triterpenes are selected from the group consisting of betulinic acid, oleanolic acid, erythrodiol, and lupeol.

[0035] In some embodiments, the solid birch bark extract comprises at least one of the following substances: 3-β-caffeoylbetulin, acetate salt of the methyl ester of betulinic acid, acetyloleanolic acid, allobetulin, betulinic aldehyde, betulonic acid, betulonic aldehyde, lupane-3β,20,28-triol, lupane-3β,20-diol (monodinol), oleanolic aldehyde, sitosterol, ursolic acid, or β-amyrin.

[0036] The solid birch bark extract of the present disclosure can be characterized by the particle size of the solid birch bark extract.In some embodiments, the average particle size of the solid birch bark extract particles is less than about 100 μm, less than about 90 μm, less than about 80 μm, less than about 70 μm, less than about 60 μm, less than about 50 μm, less than about 40 μm, less than about 30 μm or less than about 25 μm.

[0037] In other embodiments, the solid birch bark extract of the present disclosure is substantially free of solid birch bark extract particles having a particle size greater than about 30 μm, greater than about 40 μm, greater than about 50 μm, greater than about 60 μm, greater than about 70 μm, greater than about 80 μm, greater than about 90 μm, or greater than about 100 μm.

[0038] In a preferred embodiment, the solid birch bark extract is derived from Betula pendula Roth and Betula pubescens Ehrh, as well as hybrids of both species.

[0039] Method for producing solid birch bark extract The present disclosure provides a method for preparing a solid birch bark extract that can be formulated into a clinically beneficial oleogel. Generally, the method includes obtaining birch trees, stripping and processing bark from the birch trees, contacting the processed birch bark with a suitable solvent to provide an extract solution containing betulin and one or more triterpenes, and isolating and drying the birch bark extract containing betulin and one or more triterpenes from the extract solution. In some embodiments, the isolated birch bark extract is in a solid form.

[0040] In some embodiments, the method includes: (a) contacting birch bark with a pharmaceutically acceptable solvent to form an extract solution containing betulin and one or more triterpenes; (b) separating the birch bark from the extract solution; (c) cooling the extract solution, whereby a portion of the betulin and one or more triterpenes crystallizes from the cooled extract solution; (d) separating the crystallized betulin and one or more triterpenes from the cooled extract solution; and (e) drying the separated crystallized betulin and one or more triterpenes to provide a solid birch bark extract.

[0041] In some embodiments, the birch bark used in step (a) is first treated to improve the extraction efficiency of betulin and one or more triterpenes from the birch bark. For example, experiments have shown that the yield of dry extract from birch bark is highest when the extracted birch bark particles are small. In certain embodiments, before step (a), the particle size of the birch bark is reduced using a mill or other suitable device until the obtained birch bark passes through a sieve with a mesh size of about 1.25 mm.

[0042] In some embodiments, the birch bark is contacted with the pharmaceutically acceptable solvent at a temperature of about 50° C. to about 200° C. In other embodiments, the temperature is about 60° C. to about 100° C., about 60° C. to about 110° C., about 60° C. to about 120° C., about 60° C. to about 130° C., about 70° C. to about 130° C., about 80° C. to about 130° C., about 90° C. to about 130° C., or about 100° C. to about 150° C. In certain further embodiments, the temperature is about 115° C. to 130° C.

[0043] In some embodiments, the birch bark is contacted with the pharmaceutically acceptable solvent at a pressure of about 2-10 bar, hi one embodiment, the pressure is about 4.5 bar.

[0044] Pharmaceutically acceptable solvents are known to those skilled in the art and include hydrocarbons, alcohols, ketones, ether esters, sulfoxides, etc. Examples of pharmaceutically acceptable solvents include, but are not limited to, 1-butanol, 1-pentanol, 1-propanol, 2-butanol, 2-methyl-1-propanol, 2-propanol, 3-methyl-1-butanol, acetic acid, acetone, anisole, butyl acetate, dimethyl sulfoxide, ethanol, ethyl acetate, ethyl ether, ethyl formate, formic acid, heptane, hexane, isobutyl acetate, isopropyl acetate, methyl acetate, methyl ethyl ketone, methyl isobutyl ketone, pentane, propyl acetate, tert-butyl methyl ether, diisopropyl ether, methyl tert-butyl ether, methyl isopropyl ketone, and methyl tetrahydrofuran.

[0045] In some embodiments, two or more pharmaceutically acceptable solvents are mixed to provide a pharmaceutically acceptable solvent.

[0046] In some embodiments, the birch bark is contacted with n-heptane at a temperature of about 60° C. to 130° C. for 8 to 12 minutes.

[0047] In some embodiments, (a) contacting birch bark with a pharmaceutically acceptable solvent to form an extract solution containing betulin and one or more triterpenes, and (b) separating the birch bark from the extract solution are carried out using a continuous extraction method (shown in FIG. 2).

[0048] In one embodiment of the continuous extraction method, birch bark is mixed with n-heptane in a ratio of about 1:14 to 1:16 (w / v). The mixture is continuously extracted using n-heptane as the extraction solvent at a temperature of about 115-130°C and a pressure of about 4.5 bar (nitrogen). At the bottom of the precipitation vessel, the extracted cork is washed with cold n-heptane, and at the top of the precipitation vessel, the extract (hot solution of triterpenes in n-heptane) is filtered (10 μm filter) and then passed through a second filter (1 μm filter) to the crystallization step.

[0049] In some embodiments, the cooling in step (c) is carried out at a temperature of about -20°C to about 35°C, about -15°C to about 35°C, -10°C to about 35°C, about -5°C to about 35°C, or about 0°C to about 35°C. In other embodiments, in step (c), the cooled extraction solution is at least about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, or about 6-fold supersaturated. In preferred embodiments, in step (c), the cooled extraction solution is about 2-fold or about 5-fold supersaturated.

[0050] In some embodiments, drying step (e) is carried out under vacuum at a temperature of at least 50°C, at least 60°C, at least 70°C, at least 80°C, or at least 85°C. In certain embodiments, drying step (e) is carried out at a temperature of about 65°C to about 75°C, about 70°C to about 80°C, about 75°C to about 85°C, or about 85°C to about 95°C.

[0051] In some embodiments, drying step (e) is carried out under vacuum at a pressure of less than about 90 mbar, less than about 80 mbar, less than about 70 mbar, less than about 60 mbar, or less than about 50 mbar. In certain embodiments, drying step (e) reduces the amount of pharmaceutically acceptable solvent in the dried birch bark extract to less than about 0.5% (w / w).

[0052] The present invention also provides a solid birch bark extract prepared according to the above-described method.

[0053] Compositions containing solid birch bark extract The present disclosure provides clinically advantageous wound-healing formulations containing solid birch bark extract. The compositions include oleogels, emulsions, foams, and oleogel-impregnated sterile wound dressings. The compositions are useful as topical wound-healing agents.

[0054] Gels are finely dispersed systems containing a liquid and a solid phase. The solid phase forms a coherent three-dimensional framework, and the two phases are interpenetrating. Oleogels are hydrophobic gels based on non-polar liquids (e.g., oils, waxes, or paraffins) to which gel-forming agents are added to achieve desired physical properties.

[0055] The present disclosure provides oleogels comprising a non-polar liquid and an oleogel-forming agent. Suitable non-polar liquids for use in the oleogels of the present disclosure include, for example, vegetable, animal, or synthetic oils, waxes, and paraffins. In some embodiments, the non-polar liquid is a vegetable oil selected from the group consisting of castor oil, peanut oil, jojoba oil, sunflower oil, olive oil, avocado oil, and almond oil. In a preferred embodiment, the non-polar liquid is sunflower oil.

[0056] In some embodiments, the non-polar liquid comprises at least one triglyceride. In certain embodiments, the at least one triglyceride is miglyol. In other embodiments, the non-polar liquid comprises at least one C7 or higher hydrocarbon. In certain embodiments, the at least one C7 or higher hydrocarbon is paraffin.

[0057] In some embodiments, the non-polar liquid used in the oleogel has a peroxide value of less than about 15, less than about 10, less than about 5, less than about 4, less than about 3, or less than about 2. In certain embodiments, the non-polar liquid has a peroxide value of about 3 or less. The term "peroxide value," as used herein, means that the peroxide value is determined in accordance with Ph.Eur. 2.5.5.

[0058] The present disclosure provides methods of making an oleogel. In some embodiments, after the solid birch bark extract is dried, about 1% to about 20% by weight of the dried solid birch bark extract is dispersed in a non-polar liquid to form an oleogel. In certain embodiments, the non-polar liquid is sunflower oil.

[0059] In certain embodiments, the oleogel is sterilized. The oleogel may be sterilized by any suitable method known to those skilled in the art, for example, using ionizing radiation, such as electron beam (EB), X-ray, or gamma. In some embodiments, the oleogel is sterilized by ionizing (e.g., gamma) radiation at a dose of less than about 40 kGy, less than about 35 kGy, less than about 30 kGy, less than about 25 kGy, less than about 20 kGy, or less than about 15 kGy. In certain embodiments, the oleogel is sterilized by ionizing (e.g., gamma) radiation at a dose ranging from about 5 to about 25 kGy, about 9 to about 25 kGy, about 10 to about 25 kGy, about 11 to about 25 kGy, about 12 to about 25 kGy, or about 11 to about 20 kGy.

[0060] In some embodiments, the oleogel comprises from about 1% to about 30% by weight of solid birch bark extract dispersed in from about 70% to about 99% by weight of one or more non-polar liquids, the oleogel containing at least one oleogel-forming agent in addition to the solid birch bark extract particles. In some embodiments, the oleogel comprises from about 1% to about 20% by weight of solid birch bark extract dispersed in from about 80% to about 99% by weight of one or more non-polar liquids, the oleogel containing at least one oleogel-forming agent in addition to the solid birch bark extract particles.

[0061] In other embodiments, the oleogel comprises from about 1% to about 30% by weight of solid birch bark extract particles dispersed in from about 70% to about 99% by weight of one or more non-polar liquids, wherein the dispersed solid birch bark extract particles are the only oleogel-forming agent in the oleogel. In some embodiments, the oleogel comprises from about 1% to about 20% by weight of solid birch bark extract dispersed in from about 80% to about 99% by weight of one or more non-polar liquids, wherein the oleogel contains at least one oleogel-forming agent in addition to the solid birch bark extract particles.

[0062] In certain embodiments, the oleogel comprises about 5% by weight of solid birch bark extract particles dispersed in about 95% by weight of one or more non-polar liquids, about 10% by weight of solid birch bark extract particles dispersed in about 90% by weight of one or more non-polar liquids, about 15% by weight of solid birch bark extract particles dispersed in about 85% by weight of one or more non-polar liquids, or about 20% by weight of solid birch bark extract particles dispersed in about 80% by weight of one or more non-polar liquids.

[0063] In the foregoing embodiments, the amount of solid birch bark extract particles (e.g., about 1% to about 20% by weight) includes up to about 0.5% by weight of solid birch bark extract particles dissolved in the non-polar liquid.

[0064] Stressing an oleogel by centrifugation provides information regarding the tendency of the oleogel to separate (or segregate) non-polar liquid. In some embodiments, oleogels of the present disclosure exhibit less than about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, about 0.5%, or about 0.1% separation of non-polar liquid from the oleogel after centrifugation at 2750 g (e.g., 4,400 rpm) at 25° C. for about 30 minutes.

[0065] The oleogels of the present disclosure may be characterized by their viscosity, in certain embodiments, the viscosity of the oleogels at 200 s is in the range of about 0.5 to about 4.0 Pa.s, and the thixotropy value of the oleogels is in the range of about 200 to about 1200 Pa.s, measured according to the rotational viscometer method described in Ph.Eur.2.2.10 using a cone and plate viscometer.

[0066] The oleogel of the present disclosure may also be characterized by its consistency value, as determined using a texture analyzer. Texture analysis is primarily concerned with measuring the mechanical properties of the product, calculated from the results of a two-cycle texture profile analysis test. The texture analyzer performs this test by applying a controlled force to the product and recording its response in the form of force, deformation, and time.

[0067] In this disclosure, consistency is the force [mN] required to penetrate 1 cm into a sample (e.g., an oleogel) measured using a material testing device (texture analyzer) equipped with a cylindrical piercing object (0.5 in = 1.27 cm). The penetration rate is 0.4 mm / sec.

[0068] The oleogels of the present disclosure may be distinguished from simple thickened mixtures of solid birch bark extract based on their consistency values. Specifically, the oleogels of the present disclosure may be characterized as having a consistency value of less than about 3000 mN and at least about 250 mN, as measured by a texture analyzer. In some embodiments, the oleogels have a consistency value of about 300-2000 mN, as measured by a texture analyzer.

[0069] The present disclosure also provides methods for preparing an oleogel, in some embodiments, the methods comprising dispersing solid birch bark extract in a suitable non-polar liquid.

[0070] In some embodiments, a sterile wound dressing is provided, comprising a pad comprising an oleogel of the present disclosure and a therapeutically active layer. In certain embodiments, the pad is an absorbent pad. In other embodiments, the pad is any solid material suitable for covering a wound, including cotton gauze. In some embodiments, the therapeutically active layer of oleogel is applied to one or more surfaces of the pad (e.g., the surface of the pad intended to directly contact the patient's skin or wound). In other embodiments, the pad is immersed in oleogel such that the oleogel is disposed on at least a portion of the exterior surface of the pad and, optionally, on at least a portion of the interior of the pad.

[0071] In some embodiments, the pad may comprise a material suitable for covering a wound that dissolves upon use to release the oleogel. Such materials may include absorbent materials including collagen, alginates, etc.

[0072] The term emulsion relates to a heterogeneous system of two immiscible or only limitedly miscible liquids, commonly referred to as phases, in which one of the two liquids is dispersed in the other in the form of minute droplets.

[0073] In some embodiments, an emulsion is provided comprising the solid birch bark extract of the present disclosure. Other embodiments provide an emulsion comprising the oleogel of the present disclosure.

[0074] In some embodiments, the emulsions of the present disclosure include an emulsifier. In certain embodiments, the emulsifier is (hydropropyl)methylcellulose. In certain other embodiments, the emulsions are substantially free of emulsifiers.

[0075] Foams may be superior to oleogels because to treat skin wounds, application of oleogels requires contact, whereas foams can be applied to wounds with little or no contact. Foams are typically based on emulsions in which a propellant is mixed with the dispersed lipid phase of the emulsion.

[0076] The present disclosure provides a foam comprising a solid birch bark extract-containing emulsion, as described above.

[0077] In certain embodiments, the foam comprises an oleogel comprised of about 5% to about 10% by weight of solid birch bark extract, and the emulsion is a water-in-oil emulsion comprised of oleogel and about 20% to about 30% by weight of water.

[0078] In certain other embodiments, the foam comprises an oleogel consisting of about 7% by weight of birch bark extract solids, and the emulsion is a water-in-oil emulsion consisting of oleogel and about 25% by weight of water.

[0079] In certain embodiments, the foam of the present disclosure further comprises an emulsifier. Emulsifiers are known in the art as substances that stabilize emulsions and include surfactants. Emulsifiers useful in the present invention are those that are acceptable for medical use, particularly for contact with skin or wounds. In some embodiments, suitable emulsifiers include emulsifying wax, cetearyl alcohol, polysorbate 20, ceteareth 20, and the like. In certain further embodiments, the emulsifier is selected from the group consisting of phosphatidylcholine, polyglyceryl-3-methylglucose distearate, and combinations thereof.

[0080] In certain embodiments, the oleogels, emulsions, and foams of the present disclosure further comprise a disinfectant. In certain further embodiments, the disinfectant is selected from the group consisting of ethanol, propan-1-ol (n-propanol), and propan-2-ol (isopropanol). In other embodiments, the oleogels, emulsions, and foams of the present disclosure further comprise an antibiotic, particularly a lipophilic antibiotic such as a fluoroquinolone, a macrolide, tigecycline, lincosamide, rifampin, linezolid, tetracycline, and chloramphenicol.

[0081] In certain embodiments, the foams of the present disclosure have certain physical properties. In some embodiments, the foam index is greater than about 2. In other embodiments, the emulsion used in the foam exhibits an interfacial tension greater than about 4 nM / m using methods known in the art. The term "foam index," as used herein, is the ratio of the foamed density and the unfoamed density of a particular material. For example, the foam index of a particular foam is the ratio of the density of the foamed material to the density of the material before foaming.

[0082] The present disclosure also provides a pressurized container filled with an emulsion of the present invention and a pharmaceutically acceptable propellant, whereby the emulsion forms a foam upon decanting at least a portion of the mixture from the container.

[0083] How to use solid birch bark extract The present disclosure also provides a method of treating a wound in a patient by topically administering to at least a portion of the wound an effective amount of an oleogel, emulsion, or foam of the present disclosure.

[0084] In certain embodiments, the wound to be treated is selected from the group consisting of burns (e.g., mild to severe burns), surgical skin lesions, superficial body injuries, chronic wounds (e.g., pressure ulcers, diabetic foot ulcers, chronic venous ulcers, arterial insufficiency ulcers), aesthetic skin treatments (e.g., ablative laser skin treatments, chemical peels, dermabrasion), wounds caused by side effects of medications (e.g., toxic epidermal necrolysis, Lyell's syndrome, Stevens-Johnson syndrome, radiation dermatitis, chemotherapy dermatitis), rare skin diseases (e.g., epidermolysis bullosa, pemphigus vulgaris, pemphigoid, bullous pemphigoid, pemphigus foliaceus, pyoderma gangrenosum), and combinations thereof.

[0085] The present disclosure also provides a method of treating epidermolysis bullosa in a patient in need thereof, comprising topically administering to the area of ​​epidermolysis bullosa an effective amount of an oleogel, emulsion, or foam of the present disclosure.

[0086] Incorporation by Reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not and should not be construed as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country throughout the world.

Claims

[Claim 1] The invention described in the specification.

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