Compositions Comprising Osteopontin Derivatives for the Inhibition of Hair Growth

Abstract

The present invention provides a composition for inhibiting hair production in a mammal comprising an active polypeptide component and a pharmaceutically acceptable and/or cosmetically acceptable excipient, carrier or diluent. The invention further provides methods of inhibiting hair production in a mammal, e.g. human.

Claims

1. A composition for use in inhibiting hair production in a mammal comprising: (a) a polypeptide comprising or consisting of an amino acid sequence of SEQ ID NO:1 TABLE-US-00029 SEQ ID NO: 1 VDTYDGDISVVYGLR or a fragment or variant thereof which retains an inhibitory activity on mammalian hair production; and (b) a pharmaceutically acceptable and/or cosmetically acceptable excipient, carrier, adjuvant or diluent wherein the polypeptide is between 5 and 50 amino acids in length.

2. A composition for use in inhibiting hair production according to claim 1 wherein the composition is capable of inhibiting the growth of human hair.

3. A composition for use in inhibiting hair production according to claim 1 or 2 wherein the composition is capable of inhibiting the production of hair by healthy skin.

4. A composition for use in inhibiting hair growth according to any one of the preceding claims wherein the composition is capable of inhibiting hair production on the scalp.

5. A composition for use in inhibiting hair production according to any one of the preceding claims wherein the composition is capable of inhibiting existing hair follicles.

6. A composition for use in inhibiting hair production according to any one of the preceding claims wherein the composition induces: (a) a decrease in the length of the anagen phase (e.g. by inducing an anagen to catagen phase change) in hair follicles; and/or (b) an increase in the length of the catagen phase in hair follicles; and/or (c) an increase in the length of the telogen phase in hair follicles.

7. A composition for use in inhibiting hair production according to claim 6 wherein the composition is capable of inducing the hair follicles to switch from the production of terminal hair to vellus hair.

8. A composition for use in inhibiting hair production according to any one of the preceding claims wherein the composition is capable of inhibiting the formation of new hair follicles, or stem cells for producing the same.

9. A composition for use in inhibiting hair production according to any one of the preceding claims wherein the polypeptide is fewer than 40 amino acids in length, for example fewer than 35, 30, 28, 26, 24, 22, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or fewer amino acids in length.

10. A composition for use in inhibiting hair production according to claim 9 wherein the polypeptide is between 5 and 30 amino acids in length, for example between 5 and 20, between 5 and 20, between 8 and 20, between 8 and 16, or between 10 and 15 amino acids in length.

11. A composition for use in inhibiting hair production according to claim 10 wherein the polypeptide is between 10 and 15 amino acids in length.

12. A composition for use in inhibiting hair production according to any one of the preceding claims wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:1.

13. A composition for use in inhibiting hair production according to any one of claims 1 to 11 wherein the polypeptide consists of an amino acid sequence of SEQ ID NO:2 TABLE-US-00030 SEQ ID NO: 2 VDTYDGDISVVYGLS

14. A composition for use in inhibiting hair production according to any one of claims 1 to 11 wherein the polypeptide consists of a fragment of the amino acid sequence of SEQ ID NO: 1.

15. A composition for use in inhibiting hair production according to claim 14 wherein the fragment is 14 or fewer amino acids in length, for example 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids in length.

16. A composition for use in inhibiting hair production according to claim 14 or 15 wherein the fragment comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 3 to 65 (a) 14-amino acid peptides: TABLE-US-00031 SEQ ID NO: 3 VDTYDGDISVVYGL SEQ ID NO: 4 DTYDGDISVVYGLR SEQ ID NO: 5 TYDGDISVVYGLRS (b) 13-amino acid peptides: TABLE-US-00032 SEQ ID NO: 6 VDTYDGDISVVYG SEQ ID NO: 7 DTYDGDISVVYGL SEQ ID NO: 8 TYDGDISVVYGLR SEQ ID NO: 9 YDGDISVVYGLRS (c) 12-amino acid peptides: TABLE-US-00033 SEQ ID NO: 10 VDTYDGDISVVY SEQ ID NO: 11 DTYDGDISVVYG SEQ ID NO: 12 TYDGDISVVYGL SEQ ID NO: 13 YDGDISVVYGLR SEQ ID NO: 14 DGDISVVYGLRS (d) 11-amino acid peptides: TABLE-US-00034 SEQ ID NO: 15 VDTYDGDISVV SEQ ID NO: 16 DTYDGDISVVY SEQ ID NO: 17 TYDGDISVVYG SEQ ID NO: 18 YDGDISVVYGL SEQ ID NO: 19 DGDISVVYGLR SEQ ID NO: 20 GDISVVYGLRS (e) 10-amino acid peptides: TABLE-US-00035 SEQ ID NO: 21 VDTYDGDISV SEQ ID NO: 22 DTYDGDISVV SEQ ID NO: 23 TYDGDISVVY SEQ ID NO: 24 YDGDISVVYG SEQ ID NO: 25 DGDISVVYGL SEQ ID NO: 26 GDISVVYGLR SEQ ID NO: 27 DISVVYGLRS (f) 9-amino acid peptides: TABLE-US-00036 SEQ ID NO: 28 VDTYDGDIS SEQ ID NO: 29 DTYDGDISV SEQ ID NO: 30 TYDGDISVV SEQ ID NO: 31 YDGDISVVY SEQ ID NO: 32 DGDISVVYG SEQ ID NO: 33 GDISVVYGL SEQ ID NO: 34 DISVVYGLR SEQ ID NO: 35 ISVVYGLRS (q) 8-amino acid peptides: TABLE-US-00037 SEQ ID NO: 36 VDTYDGDI SEQ ID NO: 37 DTYDGDIS SEQ ID NO: 38 TYDGDISV SEQ ID NO: 39 YDGDISVV SEQ ID NO: 40 DGDISVVY SEQ ID NO: 41 GDISVVYG SEQ ID NO: 42 DISVVYGL SEQ ID NO: 43 ISVVYGLR (h) 7-amino acid peptides: TABLE-US-00038 SEQ ID NO: 44 VDTYDGD SEQ ID NO: 45 DTYDGDI SEQ ID NO: 46 TYDGDIS SEQ ID NO: 47 YDGDISV SEQ ID NO: 48 DGDISVV SEQ ID NO: 49 GDISVVY SEQ ID NO: 50 DISVVYG SEQ ID NO: 51 ISVVYGL (i) 6-amino acid peptides: TABLE-US-00039 SEQ ID NO: 52 DTYDGD SEQ ID NO: 53 TYDGDI SEQ ID NO: 54 YDGDIS SEQ ID NO: 55 DGDISV SEQ ID NO: 56 GDISVV SEQ ID NO: 57 DISVVY SEQ ID NO: 58 ISVVYG (j) 5-amino acid peptides: TABLE-US-00040 SEQ ID NO: 59 TYDGD SEQ ID NO: 60 YDGDI SEQ ID NO: 61 DGDIS SEQ ID NO: 62 GDISV SEQ ID NO: 63 DISVV SEQ ID NO: 64 ISVVY SEQ ID NO: 65 SVVYG.

17. A composition for use in inhibiting hair production according to claim 16 wherein the fragment comprises or consists of an amino acid sequence of SEQ ID NO: 26.

18. A composition for use in inhibiting hair production according to any one of the preceding claims wherein the polypeptide comprises or consists of a variant of the amino acid sequence of SEQ ID NO: 1, or of a fragment thereof.

19. A composition for use in inhibiting hair production according to claim 18 wherein the variant comprises or consists of an amino acid sequence with at least 50% identity to the amino acid sequence of SEQ ID NO: 1, more preferably at least 60%, 70% or 80% or 85% or 90% identity to said sequence, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to said amino acid sequence.

20. A composition for use in inhibiting hair production according to claim 18 or 19 wherein the variant comprises or consists of an amino acid sequence of SEQ ID NO: 1, or a fragment thereof, in which one or more amino acids is conservatively substituted.

21. A composition for use in inhibiting hair production according to any one of claims 18 to 20 wherein the polypeptide comprises or consists of one or more additional amino acids, inserted at the N- and/or C-terminus and/or internally within the amino acid sequence of SEQ ID NO:1.

22. A composition for use in inhibiting hair production according to claim 21 wherein the polypeptide comprises or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 additional amino acids.

23. A composition for use in inhibiting hair production according to claim 22 wherein the additional amino acids are the amino acids from the corresponding positions of the wildtype human osteopontin (SEQ ID NO:66).

24. A composition for use in inhibiting hair production according to any one of claim 18 or 19 wherein the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 67, or of a fragment or variant thereof TABLE-US-00041 SEQ ID NO: 67 VDTYDGRGDSVVYGLR

25. A composition for use in inhibiting hair production according to claim 24 wherein the fragment comprises 15 or fewer amino acids in length, for example 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids in length.

26. A composition for use in inhibiting hair production according to claim 24 wherein the polypeptide comprises or consists of a variant of the amino acid sequence of SEQ ID NO:67, or of a fragment thereof.

27. A composition for use in inhibiting hair production according to claim 26 wherein the variant comprises or consists of an amino acid sequence with at least 50% identity to the amino acid sequence of SEQ ID NO:67, more preferably at least 60%, 70% or 80% or 85% or 90% identity to said sequence, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to said amino acid sequence.

28. A composition for use in inhibiting hair production according to claim 26 or 27 wherein the variant comprises or consists of an amino acid sequence of SEQ ID NO:67, or a fragment thereof, in which one or more amino acids is conservatively substituted.

29. A composition for use in inhibiting hair production according to claim 26 or 27 wherein the variant comprises or consists of an amino acid sequence of SEQ ID NO:67, or a fragment thereof, in which the RGD sequence is inactivated.

30. A composition for use in inhibiting hair production according to any one of claims 26 to 29 wherein the polypeptide comprises or consists of one or more additional amino acids, inserted at the N- and/or C-terminus and/or internally within the amino acid sequence of SEQ ID NO:67.

31. A composition for use in inhibiting hair production according to claim 30 wherein the polypeptide comprises or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 additional amino acids.

32. A composition for use in inhibiting hair production according to claim 31 wherein the additional amino acids are the amino acids from the corresponding positions of the wildtype human osteopontin.

33. A composition for use in inhibiting hair production according to any one of the preceding claims wherein the polypeptide comprises or consists of the following amino acid sequence: TABLE-US-00042 SEQ ID NO: 68 X1-X2-X3-X4-X5-X6-D-X8-I-X10-X11-X12-Y-G-X15-X16- X17 wherein X1 is V, E, G or K; X2 is D, E, S or P; X3 is any amino acid; X4 is Y, P, N or A; X5 is D, N or E; X6 is G or I; X8 is G or absent; X10 is S or E; X11 is V or L; X12 is V, A or T X15 is L or I; X16 is R or K; and X17 is R, K or absent.

34. A composition for use in inhibiting hair production according to any one of claims 18, 19 and 33 wherein the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO:69 or 70, or of a fragment or variant thereof TABLE-US-00043 SEQ ID NO: 69 VDVPNGDISLAYGLR SEQ ID NO: 70 DVPNGDISLAYGLRS

35. A composition for use in inhibiting hair production according to claim 34 wherein the fragment comprises 14 or fewer amino acids in length, for example 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids in length.

36. A composition for use in inhibiting hair production according to claim 34 or 35 wherein the fragment comprises or consists of an amino acid sequence according to any one SEQ ID NOs: 71 to 133 (a) 14-amino acid peptides: TABLE-US-00044 SEQ ID NO: 71 VDVPNGDISLAYGL SEQ ID NO: 72 DVPNGDISLAYGLR SEQ ID NO: 73 VPNGDISLAYGLRS (b) 13-amino acid peptides: TABLE-US-00045 SEQ ID NO: 74 VDVPNGDISLAYG SEQ ID NO: 75 DVPNGDISLAYGL SEQ ID NO: 76 VPNGDISLAYGLR SEQ ID NO: 77 PNGDISLAYGLRS (c) 12-amino acid peptides: TABLE-US-00046 SEQ ID NO: 78 VDVPNGDISLAY SEQ ID NO: 79 DVPNGDISLAYG SEQ ID NO: 80 VPNGDISLAYGL SEQ ID NO: 81 PNGDISLAYGLR SEQ ID NO: 82 NGDISLAYGLRS (d) 11-amino acid peptides: TABLE-US-00047 SEQ ID NO: 83 VDVPNGDISLA SEQ ID NO: 84 DVPNGDISLAY SEQ ID NO: 85 VPNGDISLAYG SEQ ID NO: 86 PNGDISLAYGL SEQ ID NO: 87 NGDISLAYGLR SEQ ID NO: 88 GDISLAYGLRS (e) 10-amino acid peptides: TABLE-US-00048 SEQ ID NO: 89 VDVPNGDISL SEQ ID NO: 91 DVPNGDISLA SEQ ID NO: 91 VPNGDISLAY SEQ ID NO: 92 PNGDISLAYG SEQ ID NO: 93 NGDISLAYGL SEQ ID NO: 94 GDISLAYGLR SEQ ID NO: 95 DISLAYGLRS (f) 9-amino acid peptides: TABLE-US-00049 SEQ ID NO: 96 VDVPNGDIS SEQ ID NO: 97 DVPNGDISL SEQ ID NO: 98 VPNGDISLA SEQ ID NO: 99 PNGDISLAY SEQ ID NO: 100 NGDISLAYG SEQ ID NO: 101 GDISLAYGL SEQ ID NO: 102 DISLAYGLR SEQ ID NO: 103 ISLAYGLRS (g) 8-amino acid peptides: TABLE-US-00050 SEQ ID NO: 104 VDVPNGDI SEQ ID NO: 105 DVPNGDIS SEQ ID NO: 106 VPNGDISL SEQ ID NO: 107 PNGDISLA SEQ ID NO: 108 NGDISLAY SEQ ID NO: 109 GDISLAYG SEQ ID NO: 110 DISLAYGL SEQ ID NO: 111 ISLAYGLR (h) 7-amino acid peptides: TABLE-US-00051 SEQ ID NO: 112 VDVPNGD SEQ ID NO: 113 DVPNGDI SEQ ID NO: 114 VPNGDIS SEQ ID NO: 115 PNGDISL SEQ ID NO: 116 NGDISLA SEQ ID NO: 117 GDISLAY SEQ ID NO: 118 DISLAYG SEQ ID NO: 119 ISLAYGL (i) 6-amino acid peptides: TABLE-US-00052 SEQ ID NO: 120 DVPNGD SEQ ID NO: 121 VPNGDI SEQ ID NO: 122 PNGDIS SEQ ID NO: 123 NGDISL SEQ ID NO: 124 GDISLA SEQ ID NO: 125 DISLAY SEQ ID NO: 126 ISLAYG (i) 5-amino acid peptides: TABLE-US-00053 SEQ ID NO: 127 VPNGD SEQ ID NO: 128 PNGDI SEQ ID NO: 129 NGDIS SEQ ID NO: 130 GDISL SEQ ID NO: 131 DISLA SEQ ID NO: 132 ISLAY SEQ ID NO: 133 SLAYG

37. A composition for use in inhibiting hair production according to claim 36 wherein the fragment comprises or consists of an amino acid sequence of SEQ ID NO:94. TABLE-US-00054 SEQ ID NO: 94 GDISLAYGLR

38. A composition for use in inhibiting hair production according to claim 34 wherein the polypeptide comprises or consists of a variant of the amino acid sequence of SEQ ID NO: 69 or 70, or of a fragment thereof.

39. A composition for use in inhibiting hair production according to claim 38 wherein the variant comprises or consists of an amino acid sequence with at least 50% identity to the amino acid sequence of SEQ ID NO: 69 or 70, more preferably at least 60%, 70% or 80% or 85% or 90% identity to said sequence, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to said amino acid sequence.

40. A composition for use in inhibiting hair production according to claim 38 or 39 wherein the variant comprises or consists of an amino acid sequence of SEQ ID NO: 69 or 70, or a fragment thereof, in which one or more amino acids is conservatively substituted.

41. A composition for use in inhibiting hair production according to any one of claims 38 to 40 wherein the polypeptide comprises or consists of one or more additional amino acids, inserted at the N- and/or C-terminus and/or internally within the amino acid sequence of SEQ ID NO:69 or 70.

42. A composition for use in inhibiting hair production according to claim 41 wherein the polypeptide comprises or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 additional amino acids.

43. A composition for use in inhibiting hair production according to claim 42 wherein the additional amino acids are the amino acids from the corresponding positions of the wildtype murine osteopontin.

44. A composition for use in inhibiting hair production according to any one of claim 38 or 39 wherein the polypeptide comprises or consists of an amino acid sequence of SEQ ID NO: 134, or of a fragment or variant thereof TABLE-US-00055 SEQ ID NO: 135 VDVPNGRGDSLAYGLR.

45. A composition for use in inhibiting hair production according to claim 44 wherein the fragment comprises 15 or fewer amino acids in length, for example 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids in length.

46. A composition for use in inhibiting hair production according to claim 44 wherein the polypeptide comprises or consists of a variant of the amino acid sequence of SEQ ID NO: 135, or of a fragment thereof.

47. A composition for use in inhibiting hair production according to claim 46 wherein the variant comprises or consists of an amino acid sequence with at least 50% identity to the amino acid sequence of SEQ ID NO: 135, more preferably at least 60%, 70% or 80% or 85% or 90% identity to said sequence, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to said amino acid sequence.

48. A composition for use in inhibiting hair production according to claim 46 or 47 wherein the variant comprises or consists of an amino acid sequence of SEQ ID NO:135, or a fragment thereof, in which one or more amino acids is conservatively substituted. A composition for use in inhibiting hair production according to claim 46 or 47 wherein the variant comprises or consists of an amino acid sequence of SEQ ID NO:135, or a fragment thereof, in which the RGD sequence is inactivated.

49. A composition for use in inhibiting hair production according to any one of claims 46 to 49 wherein the polypeptide comprises or consists of one or more additional amino acids, inserted at the N- and/or C-terminus and/or internally within the amino acid sequence of SEQ ID NO:135.

50. A composition for use in inhibiting hair production according to claim 49 wherein the polypeptide comprises or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 additional amino acids.

51. A composition for use in inhibiting hair production according to claim 49 or 50 wherein the additional amino acids are the amino acids from the corresponding positions of the wildtype murine osteopontin.

52. A composition for use in inhibiting hair production according to any one of the preceding claims wherein the polypeptide is non-naturally occurring.

53. A composition for use in inhibiting hair production according to any one of the preceding claims wherein the polypeptide comprises or consists of tandem repeats.

54. A composition for use in inhibiting hair growth according to claim 53 wherein the tandem repeats comprise or consist of the amino acid sequence of any one or more of SEQ ID NOS: 1 to 65.

55. A composition for use in inhibiting hair growth according to claim 54 wherein the tandem repeats comprise or consist of the amino acid sequence of SEQ ID NO:1 or 26.

56. A composition for use in inhibiting hair growth according to claim 53 wherein the tandem repeats comprise or consist of the amino acid sequence of any one or more of SEQ ID NOS: 69 to 133.

57. A composition for use in inhibiting hair growth according to claim 55 wherein the tandem repeats comprise or consist of the amino acid sequence of SEQ ID NO: 69 and/or 94.

58. A composition for use in inhibiting hair production according to any one of the preceding claims wherein the polypeptide is modified or derivatised at one or more amino acid positions.

59. A composition for use in inhibiting hair production according to claim 58 wherein the polypeptide is glycosylated at one or more amino acid positions.

60. A composition for use in inhibiting hair production according to claim 58 wherein the polypeptide is PEGylated at one or more amino acid positions.

61. A composition for use in inhibiting hair production according to any one of the preceding claims for topical administration.

62. A composition for use in inhibiting hair production according to any one of the preceding claims for transdermal administration.

63. A composition for use in inhibiting hair production according to any one of the preceding claims for intracutaneous administration.

64. A composition for use in inhibiting hair production according to any one of the preceding claims wherein the mammal is a human.

65. A composition for use in inhibiting hair production according to any one of claims 1 to 64 for treating or preventing a disease or disorder associated with unwanted and/or excessive hair growth in a mammal.

66. A composition for use in inhibiting hair production according to claim 65 wherein the disease or disorder associated with unwanted and/or excessive hair growth is hirsutism.

67. A composition for use in inhibiting hair production according to claim 65 or 66 wherein the disease or disorder associated with unwanted and/or excessive hair growth is selected from the groups consisting of polycystic ovary syndrome (PCOS), the most common cause, congenital adrenal hyperplasia, Cushing's disease, growth hormone excess (acromegaly), tumours in the ovaries, adrenal gland cancer, Von Hippel-Lindau disease, insulin resistance, stromal hyperthecosis (SH), obesity, porphyria cutanea tarda, side effects of medication (such as tetrahydrogestrinone, phenytoin, minoxidil), hormonal treatment and hormonal disorders.

68. A composition for use in inhibiting hair production according to claim 65 for treating or preventing ingrown hair in a mammal.

69. A composition for use in inhibiting hair production according to claim 68 wherein the ingrown hair is associated with shaving, waxing and/or acne.

70. Use of a composition according to any one of claims 1 to 64 in the preparation of a medicament for treating or preventing a disease or disorder associated with unwanted and/or excessive hair production in a mammal in a mammal.

71. The use according to claim 70 wherein the disease or disorder associated with unwanted and/or excessive hair production is hirsutism.

72. The use according to claim 70 or 71 wherein the disease or disorder associated with unwanted and/or excessive hair production is selected from the groups consisting of polycystic ovary syndrome (PCOS), the most common cause, congenital adrenal hyperplasia, Cushing's disease, growth hormone excess (acromegaly), tumours in the ovaries, adrenal gland cancer, Von Hippel-Lindau disease, insulin resistance, stromal hyperthecosis (SH), obesity, porphyria cutanea tarda, side effects of medication (such as tetrahydrogestrinone, phenytoin, minoxidil), hormonal treatment and hormonal disorders.

73. The use according to claim 70 wherein the medicament is for treating or preventing ingrown hair in a mammal.

74. The use according to claim 73 wherein the ingrown hair is associated with shaving, waxing and/or acne.

75. A method for treating or preventing a disease or disorder associated with unwanted and/or excessive hair growth in a mammal, the method comprising administering to the mammal an effective amount of a composition according to any one of claims 1 to 64.

76. A method according to claim 75 wherein the disease or disorder associated with unwanted and/or excessive hair production is hirsutism.

77. A method according to claim 75 or 86 wherein the disease or disorder associated with unwanted and/or excessive hair production is selected from the groups consisting of polycystic ovary syndrome (PCOS), the most common cause, congenital adrenal hyperplasia, Cushing's disease, growth hormone excess (acromegaly), tumours in the ovaries, adrenal gland cancer, Von Hippel-Lindau disease, insulin resistance, stromal hyperthecosis (SH), obesity, porphyria cutanea tarda, side effects of medication (such as tetrahydrogestrinone, phenytoin, minoxidil), hormonal treatment and hormonal disorders.

78. A method according to claim 76 for treating or preventing ingrown hair in a mammal.

79. A method according to claim 78 wherein the ingrown hair is associated with shaving, waxing and/or acne.

80. A method according to any one of claims 75 to 79 wherein the mammal is human.

81. A method according to any one of claims 75 to 80 wherein the mammal is male.

82. A method according to any one of claims 75 to 80 wherein the mammal is female.

83. Use of a composition according to any one of claims 1 to 64 for cosmetic hair removal in a human.

84. The use according to claim 83 wherein the human is male.

85. The use according to claim 84 wherein the hair is removed from an area of the body selected from the group consisting of face (e.g. cheeks, chin and above the upper lip), chest, shoulders, neck and back.

86. The use according to claim 83 wherein the human is female.

87. The use according to claim 86 wherein the hair is removed from an area of the body selected from the group consisting of face (e.g. cheeks, chin and above the upper lip), back, legs, arms, fingers, feet, toes and pubis.

88. A method for cosmetic hair removal in a human comprising administering to the human an effective amount of a composition according to any one of claims 1 to 64.

89. A method according to claim 88 wherein the human is male.

90. A method according to claim 88 or 89 wherein the hair is removed from an area of the body selected from the group consisting of face (e.g. cheeks, chin and above the upper lip), chest, shoulders, neck and back.

91. A method according to claim 88 wherein the human is female.

92. A method according to claim 91 wherein the hair is removed from an area of the body selected from the group consisting of face (e.g. cheeks, chin and above the upper lip), back, legs, arms, fingers, feet, toes and pubis.

93. Use of a polypeptide as defined in any one of claims 1 to 64 ex vivo or in vitro for inhibiting hair production.

94. A use according to claim 93 for inhibiting hair growth on a skin explant prior to grafting of the explant on to a mammal.

95. A use according to claim 93 for inhibiting hair growth follicles (or stem cell precursors of the same) in vitro.

96. A composition for use in the inhibition of hair production in mammals substantially as defined herein with reference to the description and figures.

97. A use substantially as defined herein with reference to the description.

98. A method substantially as defined herein with reference to the description.

Description

[0207] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0208] FIG. 1). Induction of anagen to catagen phase transition in cultured isolated human hair follicles by an exemplary polypeptide of the invention, FOL-005 (SEQ ID NO: 1; data from 3 patients pooled). Number of analysed hair follicles is between 32-45 per group.

[0209] FIG. 2). Representative cryosection photographs showing FOL-005 induces transition from anagen into catagen phase [0210] A: photo of a representative hair shaft [0211] B: magnification of the bulb area [0212] C: staining of the bulb area demonstrating strong effect on the dermal papilla

[0213] FIG. 3). Effect of FOL-005 on melatonin content in cultured isolated human hair follicles. The analysis was performed using Masson Fontana histochemistry and morphometric evaluation of each tissue section. Results presented as mean+/SEM, n=26-47 hair follicles evaluated in each group. One-way ANOVA and Bonferroni's post-hoc test, *p<0.05

[0214] FIG. 4). Representative cryosection photographs (taken at magnification 200) showing effect of FOL-005 on melanin pigmentation in cultured isolated human hair follicles. The black squares in the left panel indicated the morphometry reference area and the morphometry data is presented in FIG. 3.

[0215] FIG. 5). Induction of anagen to catagen phase transition in intact human scalp skin by FOL-005.

[0216] FIG. 6). Confirmation that FOL-005 induces catagen in intact human scalp skin at higher concentrations (mean of 2 patients).

[0217] FIG. 7). Effect of FOL-005 on proliferation of Ki67 positive cells in intact human scalp skin. The concentration of FOL-005 was 60 nM, Mean+/SEM, n=6-17 photos/punch was evaluated. D3=3 days after treatment.

[0218] FIG. 8). Effect of FOL-005 on melatonin content in intact human scalp skin. The analysis was performed using Masson Fontana histochemistry and morphometric evaluation of each tissue section. Results presented as mean+/SEM. One-way ANOVA and Bonferroni's post-hoc test, **p<0.01, *p<0.05. D3=3 days after treatment.

[0219] FIG. 9). Effect of FOL-005 on melanin pigmentation in an individual representative patient. ***p<0.001.

[0220] FIG. 10). Representative cryosection photographs (taken at magnification 200) showing effect of FOL-005 on melanin pigmentation in intact human scalp skin. Photos taken of papillary dermis, 2-5 photos/punch was evaluated.

[0221] FIG. 11). Effect of FOL-005 on emigration of Dermal Papilla (DP) cells. Only Anagen Hair follicles (HFs) analysed. Results based on pooled data from 6 patients. No significant change in the number of emigrating DP cells in HFs treated with FOL-005. Mean+/SEM, n=18-22 HFs evaluated, Mann-Whitney test, ns

[0222] FIG. 12). Effect of FOL-005 on emigration of Dermal Papilla (DP) cells. Only Anagen Hair follicles (HFs) analysed. Results based on pooled data from 6 patients. No significant change in the number of emigrating DP cells in HFs treated with FOL-005. Mean+/SEM, n=18-22 HFs evaluated, Mann-Whitney test, ns

[0223] FIG. 13). Hair Cycle staging. Treatment of HFs with FOL-005 consistently induces anagen HFs within human scalp skin to enter catagen prematurely in all six patients tested (in each vertical bar, the top region corresponds to mid-catagen, the middle region to early catagen and the bottom region to anagen). Pooled data from 6 patients.

[0224] FIG. 14). Hair Cycle staging. Treatment of HFs with FOL-005 consistently induces anagen HFs within human scalp skin to enter catagen prematurely in all six patients tested. Pooled data from 6 patients.

[0225] FIG. 15). Masson Fontana staining. No significant changes in HF pigmentation in HFs treated with FOL-005. Anagen & Catagen Hair Follices analysed. Pooled data from 6 patients. Mean+/SEM, n=41-51 HFs evaluated, unpaired Student's t-test, ns

[0226] FIG. 16). Masson Fontana staining. No significant changes in HF pigmentation in HFs treated with FOL-005. Anagen & Catagen Hair Follices analysed. Pooled data from 6 patients. Mean+/SEM, n=41-51 HFs evaluated, unpaired Student's t-test, ns

[0227] FIG. 17). Masson Fontana staining. No significant changes in HF pigmentation in HFs treated with FOL-005. Anagen HFs analysed. Pooled data from 6 patients. Mean+/SEM, n=13-22 HFs evaluated, unpaired Student's t-test, ns

[0228] FIG. 18). Masson Fontana staining. No significant changes in HF pigmentation in HFs treated with FOL-005. Anagen HFs analysed. Pooled data from 6 patients. Mean+/SEM, n=13-22 HFs evaluated, unpaired Student's t-test, ns

[0229] FIG. 19). No observable difference in the morphology of the basal membrane in hair follicles treated with FOL-005 (PAS staining).

[0230] FIG. 20). Ki67/TUNEL. Significant increase in the number of apoptotic cells in HFs treated with 15 nM FOL-005 (see right-hand bar of middle pair). No significant changes were determined in the number of proliferative cells in HFs treated with FOL-005 (see left-hand column of each pair). Anagen & Catagen HFs analysed. Pooled data from 6 patients. Mean+/SEM, n=48-59 HFs evaluated, Mann-Whitney test, *p<0.02. To reduce data distortion by outliers, Grubb's test was performed and the outlier value was eliminated from analysis.

[0231] FIG. 21). Ki67/TUNEL. Significant increase in the number of apoptotic cells in HFs treated with 15 nM FOL-005. No significant changes were determined in the number of proliferative cells in HFs treated with FOL-005. Anagen & Catagen HFs analysed. Pooled data from 6 patients. Mean+/SEM, n=48-59 HFs evaluated, Mann-Whitney test, *p<0.02. To reduce data distortion by outliers, Grubb's test was performed and the outlier value was eliminated from analysis.

[0232] FIG. 22). Toluidine blue staining for detecting Mast cells. No marked difference in the number of histochemically detectable perifollicular mast cells.

[0233] FIG. 23). MHCII staining HS 14-061 (indirect immunofluorescence. Mouse anti human Ab HLA-DP-DQ-DR from DAKO). No marked difference in the number of immunohistologically detectable perifollicular MHC class II+ cells. These MHCII+ cells are most likely macrophages.

[0234] FIG. 24). CD31 staining HS14-061 (indirect immunoflourescence, mouse anti-human CD31 Ab from DAKO). No marked differences in the number of CD31+ cells (endothelial cells).

[0235] FIG. 25). K15 staining HS14-087 (indirect immunoflourescence, mouse anti-human CK15 Ab from Chemikon) No marked differences in the number of K15+ cells. Human hair follicle (HF) epithelial stem cells are not affected negatively. Preservation of HF stem cell pool and thus regenerative potential by FOL-005.

[0236] FIG. 26). Representative photographs of grafts taken three after transplantation of human hair follicles. (A) Vehicle-treated animal, (B) FOL-005-treated animal, (C) Minoxidil-treated animal and (D) FOL-005 plus minoxidil-treated animal.

[0237] FIG. 27). Effect on mean number of human hair follicles following transplantation. (A) Vehicle-treated group, (B) FOL-005-treated group, (C) Minoxidil-treated group and (D) FOL-005 plus minoxidil-treated group.

EXAMPLES

Example A: Effects of Exemplary Peptide FOL-005 [SEQ ID NO:1] on Isolated Human Hair Follicles

[0238] (i) FOL-005 Induces a Change from Anagen to Catagen Phase

Material & Methods

[0239] Anagen VI hair follicles (see Kloepper et al., 2010, the disclosures of which are incorporated herein by reference) were micro dissected from normal human scalp skin obtained with informed patient consent from three healthy adult females undergoing routine face-lift cosmetic surgery.

[0240] Isolated hair follicles were organ-cultured for 6-10 days in insulin- and hydrocortisone-supplemented William's E medium according to Philpott et al. (see Philpott et al., 1990; Bod et al., 2009; Gspr et al., 2010, the disclosures of which are incorporated herein by reference).

[0241] Treatment with the exemplary polypeptide FOL-005 was initiated immediately upon culturing the hair follicles, and lasted 7 to 10 days.

[0242] At the end of organ-culture, hair follicles were embedded and processed for longitudinal cryosections (Bod et al., 2009; Gspr et al., 2010).

Results

[0243] Exemplary peptide of the invention FOL-005 (SEQ ID NO:1; 15 nM, 60 nM and 300 nM) induced a dose-dependent transition from anagen to catagen phase in isolated human hair follicles (see FIG. 1).

[0244] Analysis of cryosections revealed that FOL-005 promotes hair follicle regression, which indicates that FOL-005 is a strong inhibitor of human hair growth in vitro (see FIG. 2).

(ii) FOL-005 Induces Pigmentation Changes

Materials & Methods

[0245] Masson Fontana staining: Cryosections were air dried and fixed in ethanol-acetic acid. The sections were washed in tris-buffered saline (TBS) and distilled water several times. Cryosections were treated with ammoniacal silver solution (Fluka, Seelze, Germany) for 40 min at 56 C. in the dark. After washing in distilled water, the sections were treated with 5% aqueous sodium thiosulphate (Merck, Darmstadt, Germany) for 1 min. Next, the sections were washed in running tap water for 3 min and were counter-stained in 0.5% aqueous neutral red (Sigma). After washing in distilled water, sections were dehydrated and mounted in Eukitt (O. Kindler, Freiburg, Germany).

[0246] Immunohistochemistry: To compare proliferation and apoptosis of HFs in the different hair cycle stages double immunolabelling of Ki-67 mouse anti-Ki-67 antiserum (DAKO, Hamburg, Denmark) and TUNEL (ApopTag Fluorescein In Situ Apoptosis detection kit; Millipore, Berlin, Germany) was performed as described previously (see Kloepper et al., 2010, Experimental Dermatology 19:305-12, he relevant disclosures of which are incorporated by reference).

Results

[0247] Melanin pigmentation analysis results are shown in FIG. 3.

[0248] At a dose of 60 nM FOL-005, a significant reduction in pigmentation was detectable, confirming an induced transition from anagen to catagen phase in the isolated hair follicles.

[0249] Representative cryosection photographs showing effect of FOL-005 on melanin pigmentation in one subject are shown in FIG. 4. In the vehicle-treated control group, more intense pigmentation is observed close to the dermal papilla, whereas following exposure to 15 nM and especially 60 nM FOL-005 the pigmentation is seen more to the right in the figure (consistent with the movement of the hair shaft as no more melanin is synthesised near the dermal papilla).

Example B: Effects of Exemplary Peptide FOL-005 (SEQ ID NO:1) on Human Skin Pieces

[0250] (i) FOL-005 Induces a Change from Anagen to Catagen Phase

Material & Methods

[0251] Full-thickness human scalp skin organ culture was performed for six days under serum-free conditions as described in (Lu et al., 2007, the disclosures of which are incorporated herein by reference).

[0252] In brief, scalp skin of female patients was obtained from routine face-lift surgery after informed consent. Human scalp skin was washed in William's E medium (Biochrom, Cambridge, UK) supplemented with 100 IU/ml penicillin G (Sigma, St Louis, Mo., USA), 10 g/ml streptomycin (Sigma), 0.25 g/ml amphotericin B (Gibco, Karlsruhe, Germany) for 5 min. The out-growing hair shafts were shaved off to the level of the epidermis; 2 mm biopsies of intact scalp skin were then punched out (parallel to the direction of hair growth, i.e. at an oblique angle), using an Acu-puncher (STIEFEL, Offenbach am Main, Germany).

[0253] The punches of human scalp tissue were carefully placed into William's E medium [supplemented with 100 IU/ml penicillin/10 g/ml streptomycin, 10 g/ml of insulin (Sigma), 10 ng/ml of hydrocortisone (Sigma) and 2 mmol/I of I-glutamine (Invitrogen, Paisley, UK)]. The skin fragments were left to float in the medium, with the epidermis up at air/liquid interface and the dermis/subcutis down. The cultures were maintained at 37 C. in a gassed incubator with 95% air and 5% CO.sub.2. The culture medium was changed every other day.

[0254] Immediately after commencement of culturing, the skin fragments were exposed to the exemplary polypeptide FOL-005 (at a dose of 15 nM to 3 M), which lasted for the duration of culturing.

[0255] At different time points, skin specimens were embedded in O.C.T. Tissue-TEK (Sakura, Zoeterwoude, the Netherlands), frozen in liquid nitrogen, and 6 m cryosections were cut. The sections were then post-fixed in acetone, air-dried and processed for haematoxylin and eosin (H&E; Sigma), Giemsa or Masson-Fontana histochemistry. Individual hair follicles were analysed with respect to their stage of cycling as described (Whiting et al. 1999), while hair follicle melanization was assessed by standard silver nitrate histochemistry (Masson-Fontana stain). Quantification of well-defined reference areas within the proximal hair matrix was performed using the Image J software (NIH).

[0256] To evaluate apoptotic and proliferating cells, double immunofluorescence was applied. Terminal dUTP nicked label-ling (ApopTagFluorescein in situ Apoptosis Detection Kit; Chemicon, Tenecula, Calif., USA) was used as a marker for apoptosis, and Ki67 immunoreactivity (DakoCytomation, Glostrup, Denmark) as indicator of cell proliferation, as previously described (Foitzik et. al., 2006).

Results

[0257] FOL-005 induced a rapid transition from anagen to catagen phase in intact human scalp skin (see FIGS. 5 and 6).

[0258] Almost all hair follicles are observed in early catagen following addition of FOL-005 to the culture medium.

[0259] Catagen was also induced by injection of FOL-005 into the scalp skin tissue.

[0260] Analysis of cryosections revealed that FOL-005 promotes hair follicle regression, which indicates that FOL-005 is a strong inhibitor of human hair growth in vitro (see FIG. 2).

(ii) FOL-005 Inhibits the Proliferation of Ki67 Positive Cells and Increases Apoptosis

Materials & Methods

[0261] Masson Fontana staining and immunohistochemistry was performed as described above.

[0262] The proliferation of Ki67 positive cells and apoptosis therein was determined as described in Whiting et al., 1999, J Investig Dermatol Symp Proc 4:282-284 (the relevant disclosures of which are incorporated by reference).

[0263] Cells were exposed to exemplary polypeptide FOL-005 at a dose of 60 nM.

Results

[0264] There is a strong tendency towards reduced proliferation in the FOL-005 treated skin as represented by a reduced proportion of Ki67 positive cells (see FIG. 7; white bar).

[0265] In keeping with an inhibition of hair growth, there is also an increased level of apoptosis as indicated by the proportion of TUNEL positive cells following FOL-05 treatment (see FIG. 7; shaded bar).

(iii) FOL-005 Induces Pigmentation Changes

Materials & Methods

[0266] Pigmentation changes in isolated follicles treated with exemplary polypeptide FOL-005 were assessed by Masson-Fontana histochemistry (see Whiting et al., 1999, J Investig Dermatol Symp Proc 4:282-284, the relevant disclosures of which are incorporated by reference).

Results

[0267] Melanin pigmentation analysis results are shown in FIGS. 8 and 9.

[0268] Exposure of intact human scalp skin to FOL-005, either by injection or addition to the culture medium, induced a significant reduction in pigmentation, confirming a transition from anagen to catagen phase in the hair follicles.

[0269] Representative cryosection photographs showing effect of FOL-005 on melanin pigmentation in one subject are shown in FIG. 10. The vehicle-treated control group (left panel) shows strong pigmentation, in contrast to the FOL-005 treated hair follicles which display less intense pigmentation (middle and right panel).

Example C: Ex Vivo Assessment of FOL-005 [SEQ ID NO:1] on Scalp Hair Follicles (HFs)

[0270] The above ex vivo assessment of the effect of exemplary peptide FOL-005 on scalp hair follicles (HFs) was repeated in order to demonstrate the robustness of the observed inhibition of hair growth.

[0271] The following experiments were performed using human HFs in organ-cultured full-thickness human scalp skin fragments (4 mm, 6 female patients, 42-65 yrs).

a) Emigration of DP Cells

[0272] No significant effect of FOL-005 treatment was observed on the number of emigrating dermal papilla (DP) cells in HFs (pooled data from six patients; mean+/SEM, n=18-22 HFs, Mann-Whitney test).

[0273] Results are shown in FIGS. 11 and 12.

[0274] b) Analysis of HF cycling Treatment of HFs with FOL-005 consistently induced anagen HFs within human scalp skin to enter catagen prematurely in all six patients tested (pooled data from six patients).

[0275] Results are shown in FIGS. 13 and 14.

c) Hair Pigmentation

[0276] No significant changes in HF pigmentation were observed in HFs treated with FOL-005 at the doses tested (mean+/SEM, unpaired Student's t-test).

[0277] Results are shown in FIGS. 15 and 16 (anagen and catagen HFs analysed).

[0278] Further results are shown in FIGS. 17 and 18 9 (anagen HFs only analysed).

d) Hair Follicle Morphology (PAS)

[0279] No observable difference was observed in the morphology of the BM in HFs treated with FOL-005 (indicative of an absence of any toxic effect)

[0280] Results are shown in FIG. 19.

e) Proliferation/Apoptosis

[0281] A significant increase was observed in the number of apoptotic cells in HFs treated with 15 nM FOL-005 (anagen and catagen HFs analysed).

[0282] No significant changes were determined in the number of proliferative cells in HFs treated with FOL-005 at this dose.

[0283] Mean+/SEM, n=48-59 HFs evaluated, Mann-Whitney test, *p<0.02. To reduce data distortion by outliers, Grubb's test was performed and the outlier value was eliminated from analysis.

[0284] Results are shown in FIGS. 20 and 21.

f) Stain for MCs (Mast Cells), MHCII+, CD31+ Cells

[0285] No marked difference was observed in the number of histochemically-detectable perifollicular mast cells (see FIG. 22; Toluidine blue staining for detecting Mast cells)

[0286] No marked difference was observed in the number of immunohistologically-detectable perifollicular MHC class II+ cells (see FIG. 23)

[0287] No marked differences in the number of CD31+ cells (endothelial cells) (see FIG. 24).

g) Stain for K15+ Cells

[0288] No marked differences were observed in the number of K15+ cells

[0289] K15 staining was performed using HS14-087 (indirect immunoflourescence, mouse anti-human CK15 Ab from Chemikon).

[0290] Human HF epithelial stem cells were not affected negatively.

[0291] Preservation of the HF stem cell pool was achieved thus demonstrating the regenerative potential by FOL-005 (see FIG. 25)

Summary

[0292] The above findings confirm and extend the results in Example B showing that exemplary peptide FOL-005 robustly promotes catagen development in human scalp HFs, by inducing the following effects. [0293] (a) an increased % of catagen HFs after treatment with FOL-005; [0294] (b) an increase in apoptotic cells in the hair matrix of treated HFs in pooled data; and [0295] (c) a tendency towards increased emigration of DP from anagen HFs after treatment

[0296] The exemplary peptide FOL-005 appears well-tolerated, as evidenced by the following observations: [0297] (a) No significant change in HF melanin content after treatment with FOL-005; [0298] (b) No marked differences in basement membrane morphology or in the number of perifollicular macrophages and mast cells and of CD31+ cells in FOL-005-treated samples; and [0299] (c) No marked difference in the number of K15+ cells

Conclusions

[0300] FOL-005 clearly promotes HF catagen, confirming its activity as a human hair growth inhibitor: it appears well-tolerated & effective. There were no signs of any toxic effects.

Example D: Effects of Exemplary Peptide FOL-005 [SEQ ID NO:1] on Human Skin Pieces

[0301] The effect of FOL-005 on hair growth of human male scalp skin grafted onto SCID mice was studied.

[0302] The aim of the study was to investigate the effect of FOL-005 on hair growth of human male scalp skin with tendency to androgenetic alopecia. In order to address this issue, fort-two female SCID/beige mice, six weeks of age, were involved in the study. Punch grafts, 3 mm.sup.2, obtained from scalp skin of human volunteers with tendency to develop common alopecia, were transplanted onto SCID/beige mice (three grafts per mouse).

[0303] One week following transplantation, the mice were randomly divided into four treated groups as follows: [0304] 1. Vehicle (10 mice) was injected intradermally twice a week and served as a negative control. [0305] 2. Minoxidil (5%) (11 mice) was applied topically twice a day and served as appositive control. [0306] 3. FOL-005 (300 nM per graft) (11 mice) was injected intradermally twice a week. [0307] 4. Minoxidil 5%+FOL-005/Minoxidil (5%) (11 mice) was applied topically twice a day. FOL-005 (300 nM per graft) was injected intradermally twice a week. For the first 4 weeks the animals were treated with minoxidil only (in order to initiate hair growth) and then for the remaining period (2 months) FOL-005 treatment was added as injections, while minoxidil treatment was maintained as a topical application.

[0308] The number of hairs/graft was counted twice a week by two independent observers. Three month following skin transplantation, the grafts were obtained from the mice and were snapped frozen in liquid nitrogen and stored in 80 C. for further analysis.

[0309] A significant decrease in the number of hairs/graft was observed in the FOL-005 treated group (2.10.4) as compared to the vehicle one (3.70.6 respectively, p<0.05) (see FIG. 26 and FIG. 27).

[0310] Furthermore, a significant decrease in the number of hairs/graft was detected in the FOL-005+Minoxidil (3.40.7) treated group as compared to group treated with Minoxidil alone (8.10.1, p<0.001).

[0311] A similar mean number of hairs/graft was observed in the Vehicle group and FOL-005+Minoxidil (3.70.6 and 3.40.7, p=NS).

[0312] The results of this study confirm that FOL-005 possesses an inhibitory effect on hair growth. The full hair growth-inhibitory potential of FOL-005 in human scalp skin in vivo is also underscored by the fact that it strongly antagonized the hair growth-promoting effects of minoxidil (one of the best-recognized hypertrichosis-inducing agents in clinical medicine).

REFERENCES

[0313] Philpott M P, Sanders D A, Westgate G E, Kealey T. Human hair growth in vitro: a model for the study of hair follicle biology. Journal of Dermatological Science. 1994; 7:S55-S72. [0314] Bodo E, Kromminga A, Biro T, Borbiro I, Gaspar E, Zmijewski M A, et al. Human female hair follicles are a direct, nonclassical target for thyroid-stimulating hormone. The Journal of investigative dermatology. 2009; 129(5):1126-39. [0315] Gaspar E, Hardenbicker C, Bodo E, Wenzel B, Ramot Y, Funk W, et al. Thyrotropin releasing hormone (TRH): a new player in human hair-growth control. FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 2010; 24(2):393-403. [0316] Kloepper J E, Sugawara K, Al-Nuaimi Y, Gaspar E, van Beek N, Paus R. Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture. Experimental Dermatology. 2010; 19:305-12. [0317] Lu Z, Hasse S, Bodo E, Rose C, Funk W, Paus R. Towards the development of a simplified long-term organ culture method for human scalp skin and its appendages under serum-free conditions. Experimental Dermatology. 2007; 16(1):37-44. [0318] Foitzik K, Krause K, Conrad F, Nakamura M, Funk W, Paus R. Human scalp hair follicles are both a target and a source of prolactin, which serves as an autocrine and/or paracrine promoter of apoptosis-driven hair follicle regression. Am J Pathol. 2006; 168(3):748-56. [0319] Whiting D A, Waldstreicher J, Sanchez M, Kaufman K D. Measuring reversal of hair miniaturization in androgenetic alopecia by follicular counts in horizontal sections of serial scalp biopsies: results of fi-nasteride 1 mg treatment of men and postmenopausal women. J Investig Dermatol Symp Proc 1999: 4: 282-284.