Europäisches Patentamt *EP000612211B1* (19) European Patent Office Office européen des brevets (11) EP 0 612 211 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.7: A01N 37/10, A01N 37/12, of the grant of the patent: A61K 31/19, A61K 31/195, 05.06.2002 Bulletin 2002/23 A61K 7/06 (21) Application number: 92924244.4 (86) International application number: PCT/US92/09438 (22) Date of filing: 04.11.1992 (87) International publication number: WO 93/08687 (13.05.1993 Gazette 1993/12) (54) ALTERATION OF RATE AND CHARACTER OF HAIR GROWTH VERÄNDERUNG DER GESCHINDIGKEIT UND DER ART DES HAARWUCHSES MODIFICATION DE LA VITESSE ET DU CARACTERE DE LA CROISSANCE DES CHEVEUX (84) Designated Contracting States: (74) Representative: AT BE CH DE DK ES FR GB GR IE IT LI NL SE Ebner von Eschenbach, Jennifer et al Ladas & Parry, (30) Priority: 05.11.1991 US 788168 Dachauerstrasse 37 80335 München (DE) (43) Date of publication of application: 31.08.1994 Bulletin 1994/35 (56) References cited: WO-A-86/02269 US-A- 4 248 861 (73) Proprietor: THE GILLETTE COMPANY US-A- 4 283 386 US-A- 4 435 419 Boston, MA 02199 (US) US-A- 4 439 432 (72) Inventors: • ENZYME, vol.24, no.1, 1979 pages 36 - 47 H.A. • HANDELMAN, Joseph, H. MILMAN ET AL ’Aminomalonic acid and its New York, NY 10023 (US) congeners as potential in vivo inhibitors of • AHLUWALIA, Gurpreet, S. L-asparagine synthetase.’ Gaithersburg, MD 20879 (US) • BIOCHEM. PHARMACOL., vol.24, no.19, 1975 pages 1787 - 1792 H.N. JAYARAM ET AL ’ethacrynic acid. Inhibitor of L-aspartate synthetase.’ Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 0 612 211 B1 Printed by Jouve, 75001 PARIS (FR) EP 0 612 211 B1 Description [0001] This invention relates to a cosmetic method for altering the rate and character of mammalian hair growth particularly androgen-stimulated hair growth, by topical application to the skin of a composition containing an inhibitor 5 of the enzyme L-asparagine synthetase. [0002] It has previously been proposed that the rate of hair growth, as well as the character of hair can be modified by topical application of inhibitors of certain enzymes such as inhibitors of 5-α-reductase or ornithine decarboxylase, or such antiandrogens as androgen receptor binding agents, as described in U.S. Patent Nos. 4,720,489 and 4,885,289. Moreover, it has been theorized that certain other enzymes, including gamma glutamyl transpeptidase, are involved 10 in various stages of hair follicle formation or of hair growth, but the relation between the various enzymes and the reactions which they control, as well as their effect upon each other and upon hair growth, has not been fully understood, as appears from Richards et al., Cancer Research, Vol. 42, 4143-4152 (1982) and DeYoung et al, Cancer Research, Vol. 38, 3697-3701 (1978); and Chase, Physiol. Zool. Vol 24, 1-8 (1951). [0003] David A. Conney et al., Int. J. Biochem., Vol. 11, 519-539 (1980) discloses a range of organic compounds 15 capable of inhibiting L-asparaginase synthetase. WO 86/02269 discloses topical compositions for altering hair growth comprising progesterone which inhibits L-asparagine synthetase (see David A. Conney et al. above, Table 11). US-A- 4,283,386 discloses topical compositions for treating skin disorders and for cosmetic purposes (hair care) comprising cysteine sulfinic acids. US-A-4,439,432 discloses topical compositions comprising progesterone, which may be used for treating abnormal hair growth. US-A-4,248,861 discloses topical compositions for preventing deleterious effects of 20 solar radiation comprising calcium pantothenate, which is a strong inhibitor of L-asparagine synthetase. (see David A. Conney et al. above, Table 9). [0004] In the present invention it has been found that the rate and character of mammalian (including human) hair growth, particularly androgen-stimulated hair growth, can be modified by topical application to the skin of a composition containing an organic inhibitor of the enzyme L-asparagine synthetase. Inorganic inhibitors such as zinc chloride are 25 undesirable because they tend to be irritants. [0005] According to the present invention there is provided a cosmetic method of reducing the rate and altering the character of mammalian hair growth comprising applying to the mammalian skin, a composition comprising a non- toxic, dermatologically acceptable vehicle and a sufficient amount of an organic inhibitor of L-asparagine synthetase, but excluding progesterone. 30 [0006] Among the known organic inhibitors of the enzyme L-asparagine synthetase which can be used in the present invention are: guanidinosuccinic acid; oxaloacetic acid; L-cysteinesulfinic acid; diethyl aminomalonate; dipeptides con- taining L-aspartic acid (L-aspartylglycine, L-aspartyl-L-leucine, L-aspartyl-L-phenylalanine, L-aspartyl-L-proline, L-α- aspartyl-L-serine and L-α-aspartyl-L-valine); N-o-nitrophenylsulfenyl-L-aspartic acid; N-o-nitrophenylsulfenyl-L- glutamine; S-adenosyl-L-methionine; L-homoserine-β-adenylate; palmitic acid; lauric acid; and ethacrynic acid. Of 35 these, guanidinosuccinic acid, ethacrynic acid, oxaloacetic acid, L-cysteinesulfinic acid and diethyl aminomalonate are preferred. [0007] The composition contains, in addition to the inhibitor, a non-toxic dermatologically acceptable vehicle or carrier which is adapted to be spread upon the skin. The concentration of the inhibitor in the composition may be varied over a wide range, either in the form of a solution or dispersion, containing from 0.1 to 30% by weight of the inhibitor, 40 preferably 2 to 15%, and the composition may be applied at a dosage rate of 10 to 25 mg/cm2 of skin. That is, the dosage of inhibitor itself is from 10 to 7,500 µg per square centimeter of skin. Penetration enhancers may also be present in the composition, including alcohol, acetone, propylene glycol, polyethylene glycol, dimethyl sulfoxide, 2-pyr- rolidone, N-methyl-2-pyrrolidone, surfactants, azone, and the like, preferably in an amount effective to cause at least 10% inhibition of hair growth when the composition is applied to the skin adjacent the hair. The maximum amount of 45 composition effectively applied is limited by the rate at which the inhibitor penetrates the skin. [0008] The following specific examples are intended to illustrate more clearly the nature of the present invention without acting as a limitation upon its scope. Example 1 50 [0009] A vehicle or carrier was prepared having the following composition: Component Percent concentration by weight Water 68% 55 Ethanol 16% Propylene Glycol 5% 2 EP 0 612 211 B1 (continued) Component Percent concentration by weight Dipropylene Glycol 5% 5 Benzyl Alcohol 4% Propylene Carbonate 2% [0010] To separate portions of the vehicle were added amounts of four different inhibitors of L-asparagine synthetase 10 as shown in Table 1 and the pH was adjusted with sodium hydroxide to achieve complete dissolution. [0011] Four groups (eight animals in each group) of male intact Golden Syrian hamsters were provided. These an- imals were considered acceptable models for human beard hair growth in that they display oval shaped flank organs, one on each side, each about 8 mm. in major diameter, which grow thick black and coarse hair similar to human beard hair. These organs produce hair in response to androgens in the hamster. The flank organs of each hamster were 15 depilated by applying a thioglycolate-based chemical depilatory (Surgex), and to one organ of each animal was applied 10µl of vehicle alone once a day, while to the other organ of each animal was applied an equal amount of vehicle containing inhibitor. After three weeks of such applications (five days a week), the flank organs were shaved and the amount of recovered hair (hair mass) from each was weighed. The extent of reduction in hair growth by the inhibitor was expressed as the percent decrease in hair mass on the organ treated with inhibitor as compared to the organ 20 treated with vehicle alone. As a control, one group of eight animals had both flank organs of each animal treated with vehicle alone. The results were as shown in Table 1 below. Table 1 Inhibition of Hair Growth by Inhibitors of L-Asparagine Synthetase 25 Hamster Flank Organ Hair Mass (mg.) Treatment Group Amount pH Untreated Mean ± SD Treated Mean ± SD Percent Inhibition Control (vehicle) 7 1.92 ± 0.19 1.81 ± 0.24 Guanidinosuccinic acid 6% 7 1.48 ± 0.09 0.64 ± 0.07 57.1% 30 Oxaloacetic acid 10% 3-4 1.44 ± 0.18 0.77 ± 0.11 38.0% Cysteinesulfinic acid 6% 3-4 1.44 ± 0.16 1.10 ± 0.19 25.5% Diethyl aminomalonate 10% 3-4 1.53 ± 0.18 1.18 ± 0.18 23.3% 35 Example 2 [0012] Compositions were prepared containing 5%, 10%, and 20% respectively of guanidinosuccinic acid in the vehicle described in Example 1 above and applied as in that Example. The results were as shown in Table 2. 40 Table 2 Inhibition of Hair Growth by Guanidinosuccinic Acid Hamster Flank Organ Hair Mass (mg.) Treatment Group Amount pH Untreated Mean ± SD Treated Mean ± SD Percent Inhibition 45 Control 7 2.17 ± 0.24 1.78 ± 0.25 Guanidinosuccinic acid 5% 7 1.52 ± 0.19 0.69 ± 0.14 52.6 ± 8.2 Guanidinosuccinic acid 10% 7 1.86 ± 0.18 0.72 ± 0.19 55.7 ± 15.1 Guanidinosuccinic acid 20% 7 1.83 ± 0.31 0.38 ± 0.12 80.1 ± 5.8 50 [0013] It was found that similar topical treatments with a 10 and 20% solution of guanidinosuccinic acid (two treat- ments over a 24 hour period using groups of 10 animals) resulted in a respective 76 and 85% reduction of L-asparagine synthetase activity in the hamster hair follicles.