Patent Publication Number: US-6037326-A

Title: Reduction of hair growth

Description:
The invention relates to reducing hair growth in mammals. 
     A main function of mammalian hair is to provide environmental protection. However, that function has largely been lost in humans, in whom hair is kept or removed from various parts of the body essentially for cosmetic reasons. For example, it is generally preferred to have hair on the scalp but not on the face. 
     Various procedures have been employed to remove unwanted hair, including shaving, electrolysis, depilatory creams or lotions, waxing, plucking, and therapeutic antiandrogens. These conventional procedures generally have drawbacks associated with them. Shaving, for instance, can cause nicks and cuts, and can leave a perception of an increase in the rate of hair regrowth. Shaving also can leave an undesirable stubble. Electrolysis, on the other hand, can keep a treated area free of hair for prolonged periods of time, but can be expensive, painful, and sometimes leaves scarring. Depilatory creams, though very effective, typically are not recommended for frequent use due to their high irritancy potential. Waxing and plucking can cause pain, discomfort, and poor removal of short hair. Finally, antiandrogens--which have been used to treat female hirsutism--can have unwanted side effects. 
     It has previously been disclosed that the rate and character of hair growth can be altered by applying to the skin inhibitors of certain enzymes. These inhibitors include inhibitors of 5-alpha reductase, ornithine decarboxylase, S-adenosylmethionine decarboxylase, gamma-glutamyl transpeptidase, and transglutaminase. See, for example, Breuer et al., U.S. Pat. No. 4,885,289; Shander, U.S. Pat. No. 4,720,489; Ahluwalia, U.S. Pat. No. 5,095,007; Ahluwalia et al., U.S. Pat. No. 5,096,911; Shander et al., U.S. Pat. No. 5,132,293; and Shander et al., U.S. Pat. No. 5,143,925. 
     DNA topoisomerase are a family of enzymes that are involved in DNA replication and metabolism. The enzymes tie or untie DNA knots so that the DNA can replicate effectively. They regulate the helical structure of the double-stranded DNA by breaking one (topoisomerase type I) or both (topoisomerase type II) strands of the DNA helix (See Wang J. Adv. Pharmacol. 29A:1-19, 1994). Topoisomerase I appears to sense and relieve the torsional pressure that accumulates during DNA transcription. Topoisomerase II is involved in the separation of intertwined DNA strands. 
     DNA topoisomerase types I and II can be inhibited by direct interaction at the catalytic or active site of the protein. In addition, DNA topoisomerase type II is dependent upon ATP and requires the formation of an intermediate complex with the DNA substrate. Therefore, prevention of ATP binding or stabilization of the intermediate enzyme-DNA complex are known mechanisms of topoisomerase inhibition. 
     It has now been found that unwanted mammalian (including human) hair growth--particularly androgen-stimulated hair growth--can be reduced by applying to the skin a composition including an inhibitor of a DNA topoisomerase, e.g., DNA topoisomerase I or DNA topoisomerase II, in an amount effective to reduce hair growth. The unwanted hair growth which is reduced may be normal hair growth, or hair growth that results from an abnormal or diseased condition. 
     Classes of topoisomerase inhibitors include coumarins, quinolones, epipodophyllotoxins, flavanoids, and actinomycins. 
     Specific inhibitors include novobiocin and nalidixic acid (Drlica, K. and Franco, R., J. Biochem. 27:2253-2259, 1988); etoposide (Mizumoto et al., Mol. Pharm. 46:860-895, 1994); ellagic acid (Constantinou et al., Nutrition and Cancer 23:121-130, 1995); bisbenzamide (or Hoechst 33342) (Sumner, A. T. Exp. Cell. Res. 217:440-447, 1995); bufalin (Jap J. Cancer Res. 85:645-651, 1994); terpentecin (J. Antiobiot. 48:211-216, 1995); bisdioxopiperizine analogs such as dexrazoxane (ICRF-187) and razoxane (ICRF-159) (Hasinoff et al., Biochem. Pharmacol. 50:953-958, 1995); 1,2-bis(3,5-dioxopiperazinyl-1-yl)propane (Gorbsky, G., Cancer Res. 54:1042-1048, 1994); 1,2,3,4-tetrahdyro-β-carboline derivatives (Lehnert et al., 4:2411-2416, 1994); pyridoacridine alkaloids such as dehydrokuanoniamine B and shermilamine C (McDonald et al., J. Med Chem. 37:3819-3827, 1994); azatoxin (Solary et al., Biochem. Pharmacol. 45:2449-2456, 1993); 2-.hydroxy-3,8,9-trimethoxy-5-methylbenzo[c]phenanthridine also known as fagaronine (Larsen et al. Biochem. Pharmacol. 46:1403-1412, 1993); 4-hydroxymethyl-4&#39;,5&#39;-benzopsoralen (Mutation Res.: Fund. Mol. Mech. Mut. 311:277-285, 1994); saintopin (J. Biol. Chem. 269:28702-28707, 1994); makaluvamines, isolated from a sponge of the genus Zyzzya (Barrows et al., Anti-Cancer Drug Des. 8:333-347, 1993); acridines, such as amsacrine (Nelson et al., Proc. Natl. Acad. Sci. 81:1361-1365, 1984); ellipticines (Pommier et al., Biochem. 24:6410-6416, 1985); amonafide (Corbett, A. H. and Osheroff, N., Chem. Res. Toxicol. 6:585-597, 1993); epipodophyllotoxins (Ross et al., Cancer Res. 44:5857-5860, 1984); anthracenediones (De Isabella et al., Mol. Pharmacol. 43:715-721, 1993); merbarone and teniposide (Chen, M and Beck, W. T., Cancer Res. 55:1509-1516, 1995); indole-4,7-quinones (J. Org. Chem. 60:3543-3545, 1995); elenic acid (Juagdan et al. Tetrahedron Lett. 36:2905-2908, 1995); 1-cyclopropyl-6,8-difluro-1,4-dihydro-7-(2,6-dimethyl-4-pyridinyl)-4-substituted quinolines (Kuo et al., Bioorg. Med. Chem. Lett. 5:399-404, 1995); and 3-benzylquinolines (Eissenstat et al., Bioorg. Med. Chem. Lett. 5:1021-1026, 1995). 
     The inhibitors of a DNA topoisomerase preferably are incorporated in a topical composition which preferably includes a non-toxic dermatologically acceptable vehicle or carrier which is adapted to be spread upon the skin. Examples of suitable vehicles are acetone, alcohols, or a cream, lotion, or gel which can effectively deliver the active compound. One such vehicle is disclosed in co-pending application PCT/US93/0506A. In addition, a penetration enhancer may be added to the vehicle to further enhance the effectiveness of the formulation. 
     The concentration of the inhibitor in the composition may be varied over a wide range up to a saturated solution, preferably from 0.1% to 30% by weight or even more; the reduction of hair growth increases as the amount of inhibitor applied increases per unit area of skin. The maximum amount effectively applied is limited only by the rate at which the inhibitor penetrates the skin. The effective amounts may range, for example, from 10 to 3000 micrograms or more per square centimeter of skin. 
     The composition should be topically applied to a selected area of the body from which it is desired to reduce hair growth. For example, the composition can be applied to the face, particularly to the beard area of the face, i.e., the cheek, neck, upper lip, and chin. The composition can also be applied to the legs, arms, torso or armpits. The composition is particularly suitable for reducing the growth of unwanted hair in women suffering from hirsutism or other conditions. In humans, the composition should be applied once or twice a day, or even more frequently, for at least three months to achieve a perceived reduction in hair growth. Reduction in hair growth is demonstrated when the frequency of hair removal is reduced, or the subject perceives less hair on the treated site, or quantitatively, when the weight of hair removed by shaving (i.e., hair mass) is reduced. 
     Male intact Golden Syrian hamsters are 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. To evaluate the effectiveness of a composition including an inhibitor of a DNA topoisomerase, the flank organs of each of a group of hamsters are depilated by applying a thioglycolate based chemical depilatory (Surgex) and/or shaved. To one organ of each animal 10 μl. of vehicle alone once a day is applied, while to the other organ of each animal an equal amount of vehicle containing an inhibitor of a DNA topoisomerase is applied. After thirteen applications (one application per day for five days a week), the flank organs are shaved and the amount of recovered hair (hair mass) from each is weighed. Percent-reduction of hair growth is calculated by subtracting the hair mass (mg) value of the test compound treated side from the hair mass value of the vehicle treated side; the delta value obtained is then divided by the hair mass value of the vehicle treated side, and the resultant number is multiplied by 100. 
     The above-described assay will be referred to herein as the &#34;Golden Syrian hamster&#34; assay. Preferred compositions provide a reduction in hair growth of at least about 30%, more preferably at least about 50%, and most preferably at least about 60% when tested in the Golden Syrian hamster assay. A number of known inhibitors of DNA topoisomerase were tested in the Golden Syrian hamster assay; the results are provided in Table 1: 
     
                       TABLE I                                                     
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Effect of Topoisomerase Inhibitors on Hair Mass                           
Ve-                  Hair Mass (mg) Vehicle                               
Compounds                                                                 
        hicle  .sup.b pH                                                  
                      Dose % Reduction                                    
                                   Treated                                
                                          Control                         
______________________________________                                    
novobiocin                                                                
        A      8.0    10%  75 ± 6                                      
                                   0.67 ± .17                          
                                          2.90 ± .42                   
nalidixic                                                                 
        B      5.5    10%  63 ± 6                                      
                                   0.67 ± .05                          
                                          1.93 ± .58                   
acid                                                                      
etoposide                                                                 
        C      6.0    10%  60 ± 6                                      
                                   0.96 ± .17                          
                                          2.19 ± .58                   
ellagic acid.sup.a                                                        
        D             20%   39 ± 10                                    
                                   1.13 ± .10                          
                                          2.14 ± .33                   
kaempferol                                                                
        E      4.5    10%  75 ± 5                                      
                                   0.50 ± .12                          
                                          1.92 ± .26                   
gossypol                                                                  
        C      4.0    10%  59 ± 6                                      
                                   0.50 ± .12                          
                                          1.31 ± .26                   
morina  E      4.5    7.5% 48 ± 6                                      
                                   0.74 ± .11                          
                                          2.50 ± .47                   
bis-    A      4.0    10%  58 ± 5                                      
                                   1.33 ± .14                          
                                          3.18 ± .18                   
benzamide.sup.a                                                           
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 .sup.a Type I and type II inhibitor;                                     
 .sup.b vehicle A = water (68%), ethanol (16%), propylene glycol (5%),    
 dipropylene glycol (5%), benzyl alcohol (4%) and propylene carbonate (2%)
 Vehicle B = ethanol (70%), propylene glycol (30%); Vehicle C = dimethyl  
 sulfoxide (50%), ethanol (35%), dipropylene glycol (15%); Vehicle D =    
 water 80.84%), glyceryl stearate SE (4.24%), polyethylene glycol stearate
 (4.09%), cetearyl alcohol (3.05%), ceteareth20 (2.5%), mineral oil  #    
 (2.22%), stearyl alcohol (1.67%) and dimethicone (0.56%); Vehicle E =    
 acetone (40%), ethanol (30%), dipropylene glycol (30%).                  
 
    
     Hair follicle extracts from hamsters flank organs were used as a topoisomerase source for in vitro experiments that demonstrate the presence of topoisomerase II in flank organ hair follicles, and that the inhibition of topoisomerase II by inhibitors tested in the Golden Syrian hamster assay using a kit supplied by TopoGen, Inc., Columbus, Ohio. The assay measures the decatenation of catenated DNA (kDNA) following incubation at 37° C. with flank organ-derived hair follicle extract and the appropriate buffer containing MgCl 2  and ATP. Inhibitors of a DNA topoisomerase were tested at concentrations listed in Table II, FIG. 1. The reaction mixture is analyzed by agarose gel electrophoresis, and the migration pattern of the decatenated DNA is determined using standards provided in the kit. Relative quantitation of decatenated DNA bands representing topoisomerase II catalytic activity was performed by scanning a polaroid photograph of the gel followed by subsequent computer-assisted analysis using Adobe Photoship (Adobe Systems, Inc., Mountain View, Calif.) and IPGEL Lab (Signal Analytics, Vienna, Va.) software packages. 
    
    
     DETAILED DESCRIPTION OF THE DRAWINGS 
     FIG. 1. Dose-dependent inhibition of flank organ topoisomerase II activity. 
    
    
     Both in-vitro and in-vivo studies suggest that inhibition of DNA topoisomerase, particularly type II, is a mechanism for reducing the growth of hair. 
     Other embodiments are within the claims.