RU2797184C2 - Betulin-containing extracts from birch bark and composition based on them - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: invention relates to a sterile oleogel based on a solid extract from birch bark. Sterile oleogel contains from 1 to 20 wt.% particles of a solid extract from birch bark, characterized by an average particle size of less than 50 microns, dispersed in one or more non-polar liquids; where the solid extract of birch bark contains at least 70 wt.% betulin and one or more triterpenes selected from the group consisting of betulinic acid, oleanolic acid, erythrodiol and lupeol; where the oleogel is sterilized using ionizing radiation at doses ranging from 11 to 40 kGy.

EFFECT: above gel is sterile and effective for wound healing.

10 cl, 2 dwg, 3 tbl

Description

LINK TO RELATED APPLICATIONS

[1] This application claims benefit under U.S. Provisional Application No. 62/613646, filed January 4, 2018, the entire contents of which are hereby incorporated by reference in its entirety and for all purposes.

The field of technology to which the present invention relates

[2] The present invention relates to pharmaceutical compositions derived from birch bark extracts.

State of the art of the present invention

[3] Triterpenes contained in birch bark extracts are known to have wound healing properties. Methods for extracting these triterpenes from birch bark are described in US Pat. No. 7,482,383. Such methods provide solid extracts from birch bark that can be used in pharmaceutical formulations. For example, emulsions containing such extracts are described in US Pat. No. 7,482,383, and oleogels containing such extracts are described in US Pat. Nos. 9,352,041; 8828444 and 8536380.

[4] For clinical use in wound care, oleogel must be applied by touching the area of skin to be treated. Touch application is undesirable for the treatment of certain skin conditions (eg, epidermolysis bullosa) because the mere act of applying the oleogel may result in deterioration of the skin condition.

[5] Therefore, there is a need for wound healing formulations containing solid birch bark extracts that can be applied to the skin without aggravating the patient's condition.

[6] The present invention provides clinically advantageous wound healing formulations (including oleogels, emulsions and foams) with improved rheological properties that overcome the shortcomings of known emulsions and oleogels containing hard birch bark.

Brief description of the present invention

[7] The present invention provides a solid birch bark extract that contains 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 birch bark solid extracts described herein can be formulated into an oleogel which has clinically advantageous rheological properties.

[8] The present invention also provides a process for preparing a solid birch bark extract that can be dispersed in a non-polar solvent to form a clinically advantageous oleogel, which comprises the steps of (a) contacting the 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 crystallized betulin and triterpene; (e) drying the separated crystallized betulin and triterpene to obtain a solid birch bark extract. The present invention also provides solid extracts from birch bark obtained in accordance with such methods.

[9] The present invention provides a clinically advantageous oleogel comprising from about 1% to about 20% by weight of birch bark solid extract particles dispersed in from about 80% to about 99% of one or more non-polar liquids, wherein when solid extracts from birch bark are dispersed in a suitable non-polar liquid, an oleogel is formed. Also provided is a sterile wound dressing containing a liner and a therapeutically effective amount of oleogel.

[10] The present invention provides an emulsion containing the oleogel of the present invention, and further provides a foam containing the emulsion of the present invention.

[11] The present invention also provides methods for treating a wound in a patient by topically applying an effective amount of the oleogel, emulsion or foam of the present invention to at least a portion of the wound.

[12] The present invention also provides methods for treating epidermolysis bullosa in a patient in need thereof, comprising topically applying an effective amount of the oleogel, emulsion or foam of the present invention to the patient's epidermolysis bullosa site.

[13] The present invention provides a birch bark solid extract, an oleogel, a sterile wound dressing, a preparation method, an emulsion, a foam, a pressurized container, a method for treating a wound, and a method for treating epidermolysis bullosa according to the following numbered embodiments:

1. Solid extract from birch bark containing at least about 70 wt. % betulin and one or more triterpenes selected from the group consisting of betulinic acid, oleanolic acid, erythrodiol and lupeol.

2. The birch bark solid extract according to Embodiment 1, wherein the birch solid extract is characterized by an HPLC chromatogram that is substantially identical to that shown in Figure 1, obtained by chromatography according to the following HPLC method:

Time (min) Acetonitrile Water H ₃ PO ₄ (v/v) Flow rate (ml/min) 0 thirty 70 0.1% 0.6 5 thirty thirty 0.1% 0.6 15 98 2 0.1% 0.6 20 98 2 0.1% 0.6 21 thirty 70 0.1% 0.6 thirty thirty 70 0.1% 0.6

column: C18, 2.6 µm, 150 x 2.1 mm; detector: 210 and 320 nm;

where betulinic acid has a relative retention time of approximately 0.75-0.90, oleanolic acid has a relative retention time of approximately 0.84-0.97, betulin has a relative retention time of 1.00, erythrodiol has a relative retention time of a retention time of approximately 1.25-1.40 and lupeol has a relative retention time of approximately 3.50-4.15.

3. A solid birch bark extract according to embodiment 1 or 2 obtained by a method comprising:

(a) bringing the birch bark into contact 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, wherein part of the betulin and one or more triterpenes crystallize from the cooled extract solution;

(d) separating the crystallized betulin and one or more triterpenes from the cooled extract solution;

(e) drying the separated crystallized betulin and one or more triterpenes to obtain a solid birch bark extract;

where after drying, dispersion from about 1 wt. % to about 20 wt. % dried birch bark solid extract obtained in step (e) forms an oleogel in purified sunflower oil.

4. Solid birch bark extract according to embodiment 3, wherein one or more of steps (a), (c) and (e) are carried out under the following conditions:

(i) said bringing into contact in step (a) is carried out at a temperature of from about 60°C to about 130°C;

(ii) said cooling in step (c) is carried out at a temperature of from about -20°C to about 35°C;

(iii) said drying in step (e) is carried out at a temperature of from about 75°C to about 95°C at a pressure of less than about 70 mbar.

5. Solid birch bark extract according to embodiment 3, wherein two or more of steps (a), (c) and (e) are carried out under the following conditions:

(i) said bringing into contact in step (a) is carried out at a temperature of from about 60°C to about 130°C;

(ii) said cooling in step (c) is carried out at a temperature of from about -20°C to about 35°C;

(iii) said drying in step (e) is carried out at a temperature of from about 75°C to about 95°C at a pressure of less than about 70 mbar.

6. Solid birch bark extract according to embodiment 3, wherein steps (a), (c) and (e) are carried out under the following conditions:

(i) said bringing into contact in step (a) is carried out at a temperature of from about 60°C to about 130°C;

(ii) said cooling in step (c) is carried out at a temperature of from about -20°C to about 35°C;

(iii) said drying in step (e) is carried out at a temperature of from about 75°C to about 95°C at a pressure of less than about 70 mbar.

7. A solid birch bark extract according to any one of embodiments 3-6, wherein the pharmaceutically acceptable solvent is a hydrocarbon or an alcohol, or combinations thereof.

8. A solid birch bark extract according to any 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 said contacting is carried out at a temperature of from about 60°C to about 130°C for about 8-12 minutes.

10. A solid birch bark extract according to any one of embodiments 3-9, wherein in step (c) the chilled extraction solution is at least about 2 times supersaturated.

11. The solid birch bark extract of Embodiment 10 wherein the chilled extract solution is about 5 times supersaturated.

12. Solid birch bark extract according to any one of embodiments 3-11, wherein the amount of residual extractant is not more than about 0.5 wt. %.

13. Oleogel containing from about 1 wt. % to about 20 wt. % particles of a solid birch bark extract according to any one of embodiments 1-12, characterized by an average particle size of less than about 50 μm, dispersed in from about 80 wt. % to about 99 wt. % of one or more non-polar liquids.

14. The oleogel of Embodiment 13, wherein the dispersed particles of birch bark solid extract are the only oleogel forming agent in the oleogel.

15. Oleogel according to embodiment 13 or 14, containing approximately 10 wt. % particles of solid extract from birch bark.

16. An oleogel according to any one of embodiments 13-15, wherein the non-polar liquid contains at least one triglyceride.

17. An oleogel according to any one of embodiments 13-16, wherein the non-polar liquid contains at least one hydrocarbon with C7 or more carbon atoms.

18. The oleogel of Embodiment 16, wherein the non-polar liquid contains one or more vegetable oils.

19. The oleogel of Embodiment 18 wherein the non-polar liquid contains sunflower oil.

20. An oleogel according to 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 not greater than about 3.

22. An oleogel according to any one of embodiments 13-21, wherein the oleogel is substantially free of birch bark solid extract particles larger than about 50 microns.

23. An oleogel according to any one of embodiments 13-22, wherein the oleogel is sterile.

24. The oleogel of Embodiment 23 wherein the oleogel is sterilized with ionizing radiation at doses less than about 20 kGy.

25. The oleogel of Embodiment 24, wherein the oleogel is sterilized with ionizing radiation at doses ranging from about 11 kGy to about 20 kGy.

26. An oleogel according to any one of embodiments 13-25, wherein the non-polar liquid separation from the oleogel is less than about 10% after centrifugation at 25° C. for 30 minutes at 2750 g.

27. An oleogel according to any one of embodiments 13-26, wherein the viscosity at 200/s of the oleogel is in the range of about 0.5 to about 4.0 Pa s and the thixotropy index of the oleogel is in the range of about 200 to about 1200 Pa s , which is measured in accordance with the method of rotational viscometry described in monograph 2.2.10 of the European Pharmacopoeia, using a cone-plate viscometer.

28. An oleogel according to any one of embodiments 13-27, wherein the density of the oleogel is in the range of about 300 to 3000 mN as measured using a texture analyzer.

29. An oleogel according to any one of embodiments 13-28, further comprising a disinfectant.

30. The oleogel of Embodiment 29, wherein the disinfectant is selected from the group consisting of ethanol, n-propanol, and isopropanol.

31. An oleogel according to any one of embodiments 13-28 further comprising a lipophilic antibiotic.

32. Sterile wound dressing containing:

(a) a gasket and

(b) a therapeutically effective layer comprising an oleogel according to any one of embodiments 13-31 located on at least one surface of the pad.

33. The sterile wound dressing of Embodiment 32, wherein the pad is an absorbent pad.

34. A method for producing a solid extract from birch bark according to embodiment 1 or 2, comprising:

(a) bringing the birch bark into contact 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, wherein part of the betulin and one or more triterpenes crystallize from the cooled extract solution;

(d) separating the crystallized betulin and one or more triterpenes from the cooled extract solution;

(e) drying the separated crystallized betulin and one or more triterpenes to obtain a solid birch bark extract;

where after drying, dispersion from about 1 wt. % to about 20 wt. % dried birch bark solid extract obtained in step (e) forms an oleogel in purified sunflower oil.

35. The method according to embodiment 34, wherein one or more of steps (a), (c) and (e) is carried out under the following conditions:

(i) said bringing into contact in step (a) is carried out at a temperature of from about 60°C to about 130°C;

(ii) said cooling in step (c) is carried out at a temperature of from about -20°C to about 35°C;

(iii) said drying in step (e) is carried out at a temperature of from about 75° C. to about 95° C. at a pressure of less than about 70 mbar.

36. The method according to embodiment 34, wherein two or more of steps (a), (c) and (e) are carried out under the following conditions:

(i) said bringing into contact in step (a) is carried out at a temperature of from about 60°C to about 130°C;

(ii) said cooling in step (c) is carried out at a temperature of from about -20°C to about 35°C;

(iii) said drying in step (e) is carried out at a temperature of from about 75° C. to about 95° C. at a pressure of less than about 70 mbar.

37. The method according to embodiment 34, wherein steps (a), (c), and (e) are carried out under the following conditions:

(i) said bringing into contact in step (a) is carried out at a temperature of from about 60°C to about 130°C;

(ii) said cooling in step (c) is carried out at a temperature of from about -20°C to about 35°C;

(iii) said drying in step (e) is carried out at a temperature of from about 75° C. to about 95° C. at a pressure of less than about 70 mbar.

38. The method according to 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 according to any one of embodiments 34-38, wherein the pharmaceutically acceptable solvent is n-heptane, and said contacting is carried out at a temperature of from about 115°C to about 130°C for about 8-12 minutes.

40. The method according to any one of embodiments 34-39, wherein in step (c) the chilled extract solution is at least about 2 times supersaturated.

41. The method of embodiment 40 wherein the chilled extract solution is supersaturated by about 5 times.

42. The method according to any of embodiments 34-41, where the amount of residual extractant is not more than about 0.5 wt. %.

43. An emulsion containing a solid birch bark extract according to any one of Embodiments 1-12.

44. An emulsion containing an oleogel according to any one of embodiments 13-31.

45. An emulsion according to embodiment 43 or 44, wherein said emulsion is a water-in-oil emulsion.

46. The emulsion of Embodiment 45 which is substantially free of an emulsifier.

47. An emulsion according to any one of embodiments 43-46, consisting essentially of a solid extract of birch bark or oleogel, oil and water.

48. An emulsion according to any one of embodiments 44-46, consisting of oleogel, oil and water.

49. An emulsion according to any one of embodiments 43-46 further comprising a disinfectant.

50. The emulsion of Embodiment 49 wherein the disinfectant is selected from the group consisting of ethanol, n-propanol and isopropanol.

51. An emulsion according to any one of embodiments 43-46 further comprising a lipophilic antibiotic.

52. A foam containing an emulsion according to any one of embodiments 43-51.

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 of Embodiment 52 further comprising a lipophilic antibiotic.

56. Foam according to any one of embodiments 52-55, wherein the interfacial tension of the emulsion is greater than about 4 mN/m.

57. Foam according to any one of embodiments 52-56, wherein the foam index is greater than about 2.

58. Foam according to embodiment 52, where the oleogel consists of from about 5 wt. % to about 10 wt. % solid extract from birch bark, and the emulsion is a water-in-oil type emulsion consisting of an oleogel and from about 20 wt. % to about 30 wt. % water.

59. Foam according to embodiment 58, where the oleogel consists of approximately 7 wt. % solid extract from birch bark, and the amount of water in the emulsion is approximately 25 wt. %.

60. Foam according to 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 containing an emulsion according to any one of embodiments 43-51 and a pharmaceutically acceptable propellant, wherein the emulsion forms foam upon release of at least a portion of the mixture from the container.

62. A method of treating a wound in a patient, comprising topically applying an oleogel according to any one of embodiments 13-31 to at least a portion of the wound.

63. An oleogel according to any one of embodiments 13-31 for use in a method of treating a wound.

64. The method or oleogel for use according to embodiment 63, wherein the wound is selected from the group consisting of burns, surgical skin lesions, superficial lesions, chronic wounds, bedsores, ulcers due to diabetic foot syndrome, chronic trophic venous ulcers, ulcers due to insufficiency arterial circulation, wounds associated with aesthetic skin treatments, wounds associated with ablative laser skin treatments, wounds associated with types of chemical peels, wounds associated with dermabrasion, wounds caused by adverse drug reactions, wounds caused by toxic epidermal necrolysis, wounds caused by Lyell's syndrome, wounds caused by Stevens-Johnson syndrome, and wounds caused by radiation dermatitis.

65. A method of treating a wound in a patient, comprising topically applying an emulsion according to any one of embodiments 43-51 to at least a portion of the wound.

66. An emulsion according to any one of embodiments 43-51 for use in a method of treating a wound.

67. The method or emulsion for use according to embodiment 66, wherein the wound is selected from the group consisting of burns, surgical skin lesions, superficial lesions, chronic wounds, bedsores, diabetic foot ulcers, chronic trophic venous ulcers, insufficiency ulcers. arterial circulation, wounds associated with aesthetic skin treatments, wounds associated with ablative laser skin treatments, wounds associated with types of chemical peels, wounds associated with dermabrasion, wounds caused by adverse drug reactions, wounds caused by toxic epidermal necrolysis, wounds caused by Lyell's syndrome, wounds caused by Stevens-Johnson syndrome, and wounds caused by radiation dermatitis.

68. A method of treating a wound in a patient, comprising topically applying a foam according to any one of embodiments 52-60 to at least a portion of the wound.

69. Foam according to any one of embodiments 52-60 for use in a method of treating a wound.

70. The method or foam for use according to the embodiment of claim 69, where the wound is selected from the group consisting of burns, surgical skin lesions, superficial injuries, chronic wounds, bedsores, ulcers due to diabetic foot syndrome, chronic trophic venous ulcers, ulcers, arterial circulatory insufficiency, wounds associated with aesthetic skin treatments, wounds associated with ablative laser skin treatments, wounds associated with types of chemical peels, wounds associated with dermabrasion, wounds caused by adverse drug reactions, wounds caused by toxic epidermal necrolysis, wounds caused by Lyell's syndrome, wounds caused by Stevens-Johnson syndrome, and wounds caused by radiation dermatitis.

71. A method of treating epidermolysis bullosa in a patient in need thereof, comprising topically applying an oleogel to an epidermolysis bullosa site in the patient according to any one of embodiments 13-31.

72. An oleogel according to any one of embodiments 13-31 for use in a method for treating epidermolysis bullosa.

73. A method of treating epidermolysis bullosa in a patient in need thereof, comprising topical application to an epidermolysis bullosa site of the patient of an emulsion according to any one of embodiments 43-51.

74. An emulsion according to any one of embodiments 43-51 for use in a method for treating epidermolysis bullosa.

75. A method of treating epidermolysis bullosa in a patient in need thereof, comprising topically applying a foam to the patient's epidermolysis bullosa site according to any one of embodiments 52-60.

76. Foam according to any one of embodiments 52-60 for use in a method of treating epidermolysis bullosa.

Detailed description of graphic materials

[14] FIG. 1. HPLC chromatogram obtained from a birch bark solid extract is shown, with data recorded at 210 nm (upper figure) and 320 nm (lower figure). In FIG. 1, the x-axis represents the retention time (min) and the y-axis represents the maximum intensity (mFU - millirelative units).

[15] FIG. 2 illustrates the continuous extraction of birch bark to obtain an extract solution containing betulin and one or more triterpenes.

Detailed description of the present invention

Definitions

[16] Although it is believed that the following terms are well known to a person skilled in the art, the following definitions are provided to facilitate understanding of the disclosed subject matter.

[17] It should be understood that the terminology used herein is only intended to describe specific embodiments and is not intended to be limiting.

[18] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as generally understood by a person skilled in the art to which the present invention pertains. Preferred methods, devices, and materials are described, although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All references cited herein (including US Pat. Nos. 9,352,041, 8,828,444, 8,536,380, and 7,482,383) are incorporated by reference in their entirety and for all purposes.

[19] In accordance with historical convention on patent law, when used in this application, including in the claims, the terms in the form of a singular and plural means "one or more". Thus, for example, reference to "carrier" includes mixtures of one or more carriers, two or more carriers, and the like.

[20] Unless otherwise indicated, all numbers expressing amounts of ingredients, reaction conditions, etc. used in this specification and claims are to be understood as being modified in all instances by the term "approximately". Accordingly, unless otherwise indicated, the numerical parameters set forth in this description and the accompanying claims of the present invention are approximate values that may vary depending on the desired properties to be obtained by this application. As commonly used herein, the expression "approximately" when referring to a measurable value such as weight, time, dose, etc., is understood to cover in one example a variation of ±15% or ±10%, in another example ± 5%, in another example, ±1%, and in another example, ±0.1% of the indicated amount, since such deviations are suitable for the implementation of the disclosed method.

[21] "Use" includes any route of administration such as oral, subcutaneous, sublingual, transmucosal, parenteral, intravenous, intra-arterial, buccal, sublingual, topical, vaginal, rectal, ophthalmic, otic, nasal, inhalation, and transdermal. "Using" may also include prescribing or filling a prescription for a dosage form containing a particular compound. "Use" may also include the provision of instructions for carrying out a method involving the use of a particular compound or dosage form containing the compound.

[22] As used herein, the verb "comprise" as used herein and in the claims, and its conjugations, are used in their non-limiting sense to indicate that elements following the word are included, but elements that are not specifically excluded are not excluded. mentioned.

[23] The term "birch bark" means the bark of white birch trees. Preferred embodiments include birch bark obtained from Betula pendula Roth and Betula pubescens Ehrh, as well as hybrids of both species.

[24] The term "relative retention time" (or "RRT") means the ratio of the retention time for the peak of the unknown to the retention time for the peak of betulin, and it is calculated by the following equation:

RRT=RT _unknown /RT _betulin,

where RT _unknown is the retention time for the unknown peak and RT _betulin is the retention time for the betulin peak (210 nm) measured using the following HPLC method:

Time (min) Acetonitrile Water H ₃ PO ₄ (v/v) Flow rate (ml/min) 0 thirty 70 0.1% 0.6 5 thirty thirty 0.1% 0.6 15 98 2 0.1% 0.6 20 98 2 0.1% 0.6 21 thirty 70 0.1% 0.6 thirty thirty 70 0.1% 0.6

column: Phenomenex Kinetix C18, 2.6 µm, 150 x 2.1 mm.

[25] The term "substantially identical" as used herein means an analytical spectrum, such as HPLC chromatogram, NMR spectrum, etc., that is substantially similar to a reference spectrum in both peak arrangement and by their intensity. 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% of the peak intensities of the reference chromatogram.

[26] "Therapeutically effective amount" means the amount of an active substance that, when administered to a subject for the purpose of treating a disease, disorder, or other undesirable medical condition, is sufficient to achieve a beneficial effect on that disease, disorder, or condition. A therapeutically effective amount will vary depending on the chemical identity and formulation of the active substance, the disease or condition and its severity, as well as the age, weight and other relevant characteristics of the patient being treated. Determination of a therapeutically effective amount of a given active substance is well known to the person skilled in the art and usually requires simple experimentation.

[27] The term "essentially does not contain" means that the specified ingredient is absent or present only in minor quantities. In one embodiment, "substantially free" means less than about 10% (eg, less than about 10% by weight). In other embodiments, "substantially free" means less than about 5% (e.g., less than about 5 wt.%), less than about 2% (e.g., less than about 2 wt.%), or less than about 1% (e.g., less than about 1 wt %), or about 0% (eg, about 0 wt %). For example, an emulsion that is substantially free of an emulsifier does not contain any emulsifier in a significant amount (e.g., contains less than about 10 wt.%, less than about 5 wt.%, less than about 2 wt.%, or less than about 1 wt. % emulsifier or contains 0 wt % emulsifier). Similarly, an oleogel that is substantially free of birch bark solid extract particles greater than about 50 μm does not substantially contain any birch bark solid extract particles greater than about 50 μm (e.g., contains less than about 10 wt. less than about 5 wt %, less than about 2 wt %, or less than about 1 wt % birch bark solid extract particles greater than about 50 µm, or contains 0 wt % birch bark solid extract particles greater than approximately 50 µm).

Solid extracts from birch bark

[28] The present invention provides solid birch bark extracts that can be formulated into clinically advantageous oleogels. Without wishing to be bound by theory, these advantages are believed to be due to the chemical composition and particle morphology of the solid birch bark extracts obtained in accordance with the methods of the present invention.

[29] Solid extracts from birch bark of the present invention can be characterized based on their chemical composition. In some embodiments, the birch bark solid extracts of the present invention contain lupane and oleanan triterpenes. In particular, birch bark extracts may contain lupan triterpenes: betulin, lupeol and betulinic acid, and oleanan triterpenes: erythrodiol and oleanolic acid.

[30] The presence of a particular triterpene in a birch extract can be determined from relative retention times obtained by HPLC chromatography. The following HPLC method is used to determine the chemical composition of the birch bark solid extracts of the present invention:

Time (min) Acetonitrile Water H ₃ PO ₄ (v/v) Flow rate (ml/min) 0 thirty 70 0.1% 0.6 5 thirty thirty 0.1% 0.6 15 98 2 0.1% 0.6 20 98 2 0.1% 0.6 21 thirty 70 0.1% 0.6 thirty thirty 70 0.1% 0.6

column: Phenomenex Kinetix C18, 2.6 µm, 150 x 2.1 mm.

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

[32] When carrying out the above HPLC chromatography method with a birch bark solid extract, 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.

[33] In certain embodiments, the birch bark solid extract has an HPLC chromatogram substantially identical to that depicted in Figure 1.

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

[35] In some embodiments, the birch bark solid extract contains at least one of the following: 3-β-caffeoylbetulin, betulinic acid methyl ester acetate, acetyloleanolic acid, allobetulin, betulinaldehyde, betulonic acid, betulonic aldehyde, lupan- 3β,20,28-triol, lupan-3β,20-diol (monoginol), oleanolaldehyde, sitosterol, ursolic acid, or β-amirin.

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

[37] In other embodiments, the birch bark solid extract of the present invention is substantially free of birch bark solid extract particles with particle sizes greater than about 30 microns, greater than about 40 microns, greater than about 50 microns, greater than about 60 microns, greater than about 70 microns, greater than about 80 microns, greater than about 90 microns, or greater than about 100 microns.

[38] In preferred embodiments, birch bark solid extracts are obtained from Betula pendula Roth and Betula pubescens Ehrh, as well as hybrids of both species.

Methods for obtaining solid extracts from birch bark

[39] The present invention provides methods for preparing birch bark solid extracts that can be formulated into clinically advantageous oleogels. Typically, the methods include the steps of obtaining birch trees, removing the bark from said birches and processing it, bringing the treated birch bark into contact with a suitable solvent to obtain 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 the form of a solid.

[40] In some embodiments, the implementation of the method involves: (a) bringing birch bark into contact with a pharmaceutically acceptable solvent to obtain an extract solution containing betulin and one or more triterpenes; (b) separating the birch bark from the extract solution; (c) cooling the extract solution, wherein part of the betulin and one or more triterpenes crystallize 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 obtain a solid birch bark extract.

[41] 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, as a result of experiments, it was shown that the yield of dry extract from birch bark is maximum if the extractable particles of birch bark are small. In certain embodiments, the particle size of the birch bark is reduced prior to step (a) using a mill or other suitable equipment until the resulting birch bark passes through a sieve with a mesh size of approximately 1.25 mm.

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

[43] In some embodiments, the implementation of the birch bark is brought into contact with a pharmaceutically acceptable solvent at a pressure of from about 2 to 10 bar. In one embodiment, the pressure is about 4.5 bar.

[44] Pharmaceutically acceptable solvents are known to those skilled in the art and include hydrocarbons, alcohols, ketones, ethers, 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 methyltetrahydrofuran.

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

[46] In some embodiments, the implementation of the birch bark is brought into contact with n-heptane at a temperature of from about 60°C to about 130°C for 8-12 minutes.

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

[48] In one embodiment of the continuous extraction process, 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 extractant at a temperature of approximately 115-130° C. and at a pressure of approximately 4.5 bar (nitrogen). At the lower end of the settler, the bark layer to be extracted is washed with cold n-heptane, and at the upper end of the settler, the extract (hot solution of triterpenes in n-heptane) is filtered (through a 10 µm filter) and transferred through a second filter (through a 1 µm filter) to the crystallization stage .

[49] In some embodiments, the cooling in step (c) is carried out at a temperature of from about -20°C to about 35°C, from about -15°C to about 35°C, from -10°C to about 35°C. , from about -5°C to about 35°C or from 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 times supersaturated or about 5 times supersaturated.

[50] In some embodiments, the 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, the drying step (e) is carried out at a temperature of from about 65°C to about 75°C, from about 70°C to about 80°C, from about 75°C to about 85°C, or from about 85 °C to approximately 95°C.

[51] In some embodiments, the 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, the drying step (e) reduces the amount of pharmaceutically acceptable solvent in the dry birch bark extract to less than about 0.5% (w/w).

[52] The present invention also presents solid extracts from birch bark, obtained in accordance with the methods described above.

Compositions containing solid extracts from birch bark

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

[54] Gels are finely dispersed systems containing a liquid phase and a solid phase. The solid phase forms an integral three-dimensional framework, while these two phases penetrate each other. Oleogels are hydrophobic gels based on a non-polar liquid (eg oil, wax or paraffin) to which a gelling agent is added to achieve the desired physical properties.

[55] The present invention provides oleogels containing a non-polar liquid and an oleogel forming agent. Suitable non-polar liquids for use in the oleogels of the present invention 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.

[56] In some embodiments, the implementation of the non-polar liquid contains at least one triglyceride. In certain embodiments, at least one triglyceride is miglyol. In other embodiments, the implementation of the non-polar liquid contains at least one hydrocarbon with C7 or more carbon atoms. In certain embodiments, the implementation of at least one hydrocarbon with C7 or more carbon atoms is a paraffin.

[57] In some embodiments, the non-polar fluid 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 fluid is characterized a peroxide value of not more than about 3. The term "peroxide value" as used herein means that the peroxide value is determined in accordance with monograph 2.5.5. European Pharmacopoeia.

[58] The present invention provides methods for producing oleogels. In some embodiments, after drying the birch bark solid extract from about 1 wt. % to about 20 wt. % dry solid extract from birch bark is dispersed in a non-polar liquid to obtain an oleogel. In certain embodiments, the non-polar liquid is sunflower oil.

[59] In certain embodiments, the oleogel is sterile. The oleogel can be sterilized by suitable methods known to those skilled in the art, for example using ionizing radiation such as electron beam (EB), x-rays or gamma rays. In some embodiments, the oleogel is sterilized with ionizing (e.g., gamma) radiation at doses 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 with ionizing (e.g., gamma) radiation at doses that range from about 5 to about 25 kGy, from about 9 to about 25 kGy, from about 10 to about 25 kGy, from about 11 to about 25 kGy. kGy, from about 12 to about 25 kGy, or from about 11 to about 20 kGy.

[60] In some embodiments, the implementation of the oleogel contains from about 1 wt. % to about 30 wt. % solid extract from birch bark, dispersed in from about 70 wt. % to about 99 wt. % of one or more non-polar liquids, while the oleogel, in addition to particles of a solid extract from birch bark, contains at least one oleogel-forming agent. In some embodiments, the implementation of the oleogel contains from about 1 wt. % to about 20 wt. % solid extract from birch bark, dispersed in from about 80 wt. % to about 99 wt. % of one or more non-polar liquids, while the oleogel, in addition to particles of a solid extract from birch bark, contains at least one oleogel-forming agent.

[61] In other embodiments, the implementation of the oleogel contains from about 1 wt. % to about 30 wt. % particles of a solid extract from birch bark, dispersed in from about 70 wt. % to about 99 wt. % of one or more non-polar liquids, while in the oleogel the dispersed particles of the solid extract from the birch bark are the only agent that forms the oleogel. In some embodiments, the implementation of the oleogel contains from about 1 wt. % to about 20 wt. % solid extract from birch bark, dispersed in from about 80 wt. % to about 99 wt. % of one or more non-polar liquids, while the oleogel, in addition to particles of a solid extract from birch bark, contains at least one oleogel-forming agent.

[62] In certain embodiments, the implementation of the oleogel contains approximately 5 wt. % particles of solid extract from birch bark, dispersed in approximately 95 wt. % of one or more non-polar liquids; approximately 10 wt. % particles of solid extract from birch bark, dispersed in approximately 90 wt. % of one or more non-polar liquids; approximately 15 wt. % particles of solid extract from birch bark, dispersed in approximately 85 wt. % of one or more non-polar liquids; or about 20 wt. % particles of solid extract from birch bark, dispersed in approximately 80 wt. % of one or more non-polar liquids.

[63] In the above embodiments, the amount of birch bark solid extract particles (eg, from about 1 wt. % to about 20 wt. %) includes up to about 0.5 wt. % particles of solid extract from birch bark, which are dissolved in a non-polar liquid.

[64] The load on the oleogel, which it is subjected to by centrifugation, provides information about the tendency of the oleogel to separate (or exfoliate) non-polar liquid. In some embodiments of the oleogels of the present invention, non-polar liquid shedding in the oleogel is 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% after centrifugation at 25° C. for about 30 minutes at 2750 g (eg 4400 rpm).

[65] The oleogels of the present invention can be characterized by their viscosity. In certain embodiments, the viscosity at 200/s of the oleogel is in the range of about 0.5 to about 4.0 Pa s and the thixotropy index of the oleogel is in the range of about 200 to about 1200 Pa s as measured according to the method rotational viscometry, described in monograph 2.2.10 of the European Pharmacopoeia, using a cone-plate viscometer.

[66] The oleogels of the present invention can also be characterized by their density values, which are determined using a texture analyzer. Texture analysis is predominantly associated with the measurement of the mechanical properties of the product, calculated from the results of the test with the analysis of the texture profile with two cycles. Texture analyzers perform this test by applying controlled forces to the product and recording its response in terms of force, strain, and time.

[67] In the present invention, density is the force [mN] required to penetrate 1 cm into a sample (e.g., oleogel), which is measured using a material testing device (texture analyzer) with a cylindrical penetrating object (0.5 inch = 1.27 cm). The penetration rate is 0.4 mm/s.

[68] The oleogels of the present invention can be distinguished from simple thickened mixtures of birch bark solid extracts based on their density values. In particular, the oleogels of the present invention may have a density value of less than about 3000 mN and at least about 250 mN as measured by a texture analyzer. In some embodiments, the density value of the oleogels is approximately 300-2000 mN as measured with a texture analyzer.

[69] The present invention also provides methods for producing oleogels. In some embodiments, the method involves dispersing a solid birch bark extract in a suitable non-polar liquid.

[70] In some embodiments, a sterile wound dressing is provided comprising a liner and a therapeutically effective layer comprising the oleogel of the present invention. In certain embodiments, the pad is an absorbent pad. In other embodiments, the liner is any dense material suitable for covering a wound, including cotton gauze. In some embodiments, a therapeutically effective layer of oleogel is applied to one or more pad surfaces (eg, a pad surface intended for direct contact with a patient's skin or wound). In other embodiments, the liner is immersed in the oleogel so that the oleogel is located on at least a portion of the outer surfaces of the liner and optionally on at least portions of the interior of the liner.

[71] In some embodiments, the liner may comprise a wound dressing material that dissolves upon application to release the oleogel. Such materials may include absorbable materials containing collagen, alginate, etc.

[72] The term "emulsion" refers to heterogeneous systems consisting of two liquids that do not mix with each other or mix only to a limited extent, which are usually referred to as phases. In an emulsion, one of the two liquids is dispersed in the other liquid in the form of fine droplets.

[73] In some embodiments, an emulsion comprising a solid birch bark extract of the present invention is provided. In other embodiments, emulsions containing the oleogels of the present invention are provided.

[74] In some embodiments, the implementation of the emulsion of the present invention include an emulsifier. In certain embodiments, the emulsifier is (hydropropyl)methylcellulose. In certain other embodiments, the emulsions are substantially free of an emulsifier.

[75] Foams can be superior to oleogels in the treatment of skin wounds because foams can be applied to wounds in a substantially non-contact manner, while oleogels require contact. Foams are usually based on emulsions in which the propellant is mixed with the dispersed lipid phase of the emulsion.

[76] The present invention provides foams containing the above-described emulsion containing a solid birch bark extract.

[77] In certain embodiments, the implementation of the foam contains an oleogel, consisting of from about 5 wt. % to about 10 wt. % solid extract from birch bark, and the emulsion is a water-in-oil type emulsion consisting of an oleogel and from about 20 wt. % to about 30 wt. % water.

[78] In certain other embodiments, the implementation of the foam contains an oleogel, consisting of approximately 7 wt. % solid extract from birch bark, and the emulsion is a water-in-oil type emulsion consisting of an oleogel and approximately 25 wt. % water.

[79] In certain embodiments, the implementation of the foam of the present invention additionally contain an emulsifier. Emulsifiers are well known in the art as emulsion stabilizers and include surfactants. Emulsifiers useful in the present invention are emulsifiers that are acceptable for pharmaceutical use, in particular for skin or wound contact. In some embodiments, suitable emulsifiers include emulsifying waxes, cetearyl alcohol, polysorbate 20, ceteareth 20, etc. In certain additional embodiments, the emulsifier is selected from the group consisting of phosphatidylcholine, polyglyceryl-3-methylglucose distearate, and combinations thereof.

[80] In certain embodiments, the implementation of the oleogels, emulsions and foams of the present invention additionally contain a disinfectant. In some additional 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 invention further comprise an antibiotic, in particular lipophilic antibiotics such as fluoroquinolones, macrolides, tigecycline, lincosamides, rifampin, linezolid, tetracyclines and chloramphenicol.

[81] In certain embodiments, the foams of the present invention have certain physical properties. In some embodiments, the foam index is greater than about 2. In other embodiments, the emulsion used in the foam has an interfacial tension greater than about 4 nM/m as measured using methods known in the art. As used herein, the term "foaming index" refers to the ratio of the densities of the specified material in the foamed and non-foamed states. For example, the foam index for a particular foam is the ratio of the density of the foamed material to the density of the material before foaming.

[82] The present invention also provides pressurized containers filled with an emulsion of the present invention and a pharmaceutically acceptable propellant, wherein the emulsion forms a foam upon release of at least a portion of the mixture from the container.

Ways to use solid extracts from birch bark

[83] The present invention also provides methods for treating a wound in a patient by topically applying an effective amount of the oleogel, emulsion or foam of the present invention to at least a portion of the wound.

[84] In certain embodiments, the wound to be treated is selected from the group consisting of burns (such as mild or severe burns), surgical skin lesions, superficial injuries, chronic wounds (such as bedsores, diabetic ulcers). foot, chronic trophic venous ulcers, ulcers due to arterial circulatory insufficiency, etc.), wounds associated with aesthetic skin treatments (such as those associated with ablative laser skin treatments, chemical peels, dermabrasion, etc.). etc.), wounds caused by adverse drug reactions (such as toxic epidermal necrolysis, Lyell's syndrome, Stevens-Johnson syndrome, radiation dermatitis, chemotherapy-induced dermatitis, etc.), rare skin diseases (such as , epidermolysis bullosa, pemphigus vulgaris, pemphigoid, bullous pemphigoid, pemphigus foliaceus, pyoderma gangrenosum, etc.) and combinations thereof.

[85] The present invention also provides methods for treating epidermolysis bullosa in a patient in need thereof, comprising topically applying an effective amount of the oleogel, emulsion or foam of the present invention to the patient's epidermolysis bullosa site.

INCLUSION BY LINK

[86] All references, articles, publications, patents, patent publications and patent applications referred to in this document are incorporated by reference in their entirety for any purpose. However, the citation of any reference, article, publication, patent, patent publication, and patent application referred to in this document does not constitute and should not be taken as an endorsement or any form of suggestion that they represent the current state of the art. or are part of the public domain in any country in the world.

Claims (13)

1. Sterile oleogel containing from 1 to 20 wt. % particles of a solid extract from birch bark, characterized by an average particle size of less than 50 microns, dispersed in one or more non-polar liquids; where the solid birch bark extract contains at least 70 wt. % betulin and one or more triterpenes selected from the group consisting of betulinic acid, oleanolic acid, erythrodiol and lupeol; And where the oleogel is sterilized using ionizing radiation at doses ranging from 11 to 40 kGy. 2. Sterile oleogel according to claim. 1, where the oleogel is sterilized using ionizing radiation in doses ranging from 11 to 20 kGy. 3. The sterile oleogel according to claim 1, wherein the ionizing radiation is X-ray or gamma radiation. 4. Sterile oleogel according to claim 1, wherein the solid birch bark extract particles dispersed in the oleogel are the only oleogel forming agent. 5. Sterile oleogel according to claim 1, containing 10 wt. % particles of solid extract from birch bark. 6. Sterile oleogel according to claim 1, wherein the non-polar liquid contains at least one hydrocarbon with C7 or more carbon atoms. 7. Sterile oleogel according to claim 1, wherein the non-polar liquid contains one or more vegetable oils. 8. Sterile oleogel according to claim 7, wherein the non-polar liquid contains sunflower oil. 9. Sterile oleogel according to claim 1, wherein the oleogel is substantially free of birch bark solid extract particles larger than 50 microns. 10. Sterile oleogel according to claim 1, wherein the separation of the non-polar liquid from the oleogel is less than 10% after centrifugation at 25° C. for 30 minutes at 2750 g. 11. The sterile oleogel according to claim 1, wherein the viscosity at 200/s of the oleogel is in the range of 0.5 to 4.0 Pa⋅s and the thixotropy index of the oleogel is in the range of 200 to 1200 Pa⋅s as measured according to with the method of rotational viscometry described in monograph 2.2.10 of the European Pharmacopoeia, using a cone-plate viscometer.

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