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|||||IIII USOC538416A United States Patent (19) 11 Patent Number: 5,384,116 Pawelek Et Al |||||IIII USOC538416A United States Patent (19) 11 Patent Number: 5,384,116 Pawelek et al. 45) Date of Patent: Jan. 24, 1995 54 SYNTHETIC MELANINASA SUNSCREEN reductase and Metal Ions in Dopachrome Conversion in AND TANNING AGENT the Eumelanin Pathway,” Biochemistry 27:6156-6159. Palumbo et al., 1987, "Effect of Metal Ions on the Rear 75 Inventors: John Pawelek; Michael P. Osber, rangement of Dopachrome,” Biochemical et Bio both of Hamden, Conn.; Seth J. physica Acta 925:203-209. Orlow, Long Island City, N.Y. Korner and Gettins, 1985, "Synthesis in vitro of 73 Assignee: Yale University, New Haven, Conn. 5,6-Dihydroxyindole-2-Carboxylic Acid by Dopa chrome Conversion Factor From Cloudman S91 Mela 21 Appl. No.: 16,418 noma,” Biological Abstracts 81 (1):AB 601-602, Refer 22 Filed: Mar. 25, 1993 ence 5362. Pawelek et al., 1982, "The Biosynthesis of Mammalian Related U.S. Application Data Melanin,” American Scientist Mar.-Apr. 70(2):136-144. Korner and Pawelek, 1981, "Dopachrome Conversion: 60 Division of Ser. No. 867,851, Apr. 13, 1992, Pat. No. A Possible Control Point in Melanin Biosynthesis,” 5,227,459, which is a continuation-in-part of Ser. No. 674,489, Mar. 25, 1991, Pat. No. 5,225,435, which is a Biological Abstracts 71(1):389, Reference 3755. continuation of Ser. No. 603,111, Oct. 25, 1990, Pat. Ito et al., 1974, “Isolation of Oligomers of 5,6-Dihy No. 5,218,079, which is a continuation of Ser. No. droxyindole-2-Carboxylic Acid From the Eye of the 525,944, May 18, 1990, Pat. No. 5,216,116. Catfish,” Chen. J. 143:207-217. 51) Int. Cl............................................... CO8G 63/06 Primary Examiner-Douglas W. Robinson 52 U.S. C. ........................................ 424/63; 424/59; Assistant Examiner-Jean C. Witz 424/78.03; 514/415 Attorney, Agent, or Firm-Pennie & Edmonds 58 Field of Search ........................ 424/59, 63, 78.03; 57 ABSTRACT 514/415 A melanin that is soluble in an aqueous solution at a pH (56) References Cited between 5 and 9 at a temperature of 0 to 100° C. Ad U.S. PATENT DOCUMENTS vantageously, the melanin is capable of being filtered through at least a 0.45 micron size filter, and has a mo 4,515,773 5/1985 Herlihy ................................. 424/59 lecular weight of greater than 10,000 kilodaltons. The 4,968,497 11/1990 Wolfram et al. ...................... 424/59 melanin is useful for providing a naturally-appearing tan 5,017,194 5/1991 Arifoglu et al. ........................ 8/111 to mammalian skin and hair. Such melanin can be pro OTHER PUBLICATIONS duced by combining dopachrome and an appropriate enzyme, or by incubating 5,6-dihydroxyindole-2-car Orlow et al., 1992, "Synthesis and Characterization of boxylic acid alone or with 5,6-dihydroxyindole, or with Melanins from Dihydroxyindole-2-Carboxylic Acid 3-amino-tyrosine. The melanin is also useful for provid and Dihydroxyindole,” Pigment Cell Research ing a sun-screen to mammalian skin and hair, to treat 5:113-12. post-inflammatory hypo- and hyperpigmentation, to Pawelek J. M., 1992, "Synthesis and Characterization of tint glass and plastic, to protect industrial materials Melanins from Dihydroxyindole-2-Carboxylic Acid against ultraviolet damage, and as a coloring agent in and Dihyroxyindole,” in Pigment Cell Research, pp. foodstuffs such as coffee, tea, soda, whiskey and liquors. 113-121. Leonard et al., 1988, "Function of Dopachrome Oxido 4 Claims, 10 Drawing Sheets U.S. Patent Jan. 24, 1995 Sheet 2 of 10 5,384,116 s K. SueO ept do U.S. Patent Jan. 24, 1995 Sheet 3 of 10 5,384,116 s An ISuaO Leo I do U.S. Patent Jan. 24, 1995 Sheet 4 of 10 5,384,116 3. An ISueO leo do U.S. Patent Jan. 24, 1995 Sheet 5 of 10 5,384,116 O O O C C L E O CU S 5 CldE. C) d H |- 3. C C C O O C KISueO Leo Indo U.S. Patent Jan. 24, 1995 Sheet 6 of 10 5,384,116 e 1.0 0.8 a 0.6 g FIG. 3A (phs) is s 0.2 z -o s O. O 1-- O 4 8 24 Time hours e 1.0 0.8 0.6 g 2-0 FIG. 3B (phs) is ... 4 3 0.2 S OO -o-o-o- 7%.1-- 0 4 8 24 Time hours e 0 Z-O 0.8 a 0.6 g FIG. 3C (ph7) 3 ... 4 s 0.20.0 ... -- 1-T- O A 8 24 Time hours 5,384,116 o GOJee UW of GOJeW 00900900700€00,2 I9,|7 U.S. Patent Jan. 24, 1995 Sheet 8 of 10 5,384,116 C C C und Ka SuaO go do U.S. Patent Jan. 24, 1995 Sheet 9 of 10 5,384,116 WUO82) eoUeqJOSQW 5,384,116 1. 2 King, "Function of Dopachrome Oxidoreductase and SYNTHETIC MELANNASA SUNSCREEN AND Metal Ions in Dopachrome Conversion in the Eumela TANNING AGENT nin Pathway', Biochemistry, 27, 6156-6159, 1988, pres ent similar results to those of Palumbo et al. regarding This application is a division of application Ser. No. metal ions and the formation of 5,6-dihydroxyindole-2- 07/867,851 filed Apr. 13, 1992, now U.S. Pat. No. carboxylic acid from dopachrome. Like Palumbo et al., 5,227,459 which is a continuation-in-part of Ser. No. Leonard et al. also do not teach or suggest that such 07/674,489 filed Mar. 25, 1991, now U.S. Pat. No. metal ions could be used to affect the color for forma 5,225,435 which in turn is a continuation of Ser. No. tion of soluble melanin. 07/603,111 filed Oct. 25, 1990, now U.S. Pat. No. O Korner and Pawelek, J. Invest Dernatol. 75, 192-195, 5,218,079 which in turn is a continuation of Ser. No. 1980, report the presence of an enzymic activity, "dopa 07/525,944 filed May 18, 1990, now U.S. Pat. No. chrome conversion factor', in melanocytes which cata 5,216,116. lyzes the conversion of dopachrome to 5,6-dihydrox yindole-2-carboxylic acid. Pawelek, Biochem. Biophys. FIELD OF THE INVENTION 15 The present invention concerns the synthesis of solu Res. Conn., 166, 1328-1333, 1990, showed that this ble forms of melanin and their composition, and meth conversion reaction is an isomerization and that dopa ods of using such compositions to provide a natural chrome conversion factor is an isomerase. Aroca, appearing tan to mammalian skin and hair and to pro Garcia-Borron, Solano, and Lozano, Biochem. Biophys. vide a sun-screen, to treat hypopigmentation, to tint 20 Res. Acta, 1035, 266-275, 1990, suggested that the mech glass and plastic, to provide color to foods and bever anism is indeed an isomerization which involves a tauto ages, and to protect industrial materials against ultravio meric shift and proposed that the enzyme be named let damage. "dopachrome tautomerase' with the official Enzyme Commission number of E.C.5.3.2.3. Tsukamoto, Jack BACKGROUND INFORMATION 25 son, Urabe, Motague, and Hearing, EMBO.J., in press, In biology, melanins are heteropolymers consisting of 1992, reported identification of a gene for dopachrome L-dopa and its enzymatic derivatives. They are ubiqui tautomerase. The above information is reviewed by tous in living organisms and are produced throughout Pawelek, Pigment Cell Research, 4, 53-62, 1991. the zoological and botanical phyla. In mammalian skin, Wolfram et al. (U.S. Pat. No. 4,968,497) disclose melanins are produced through enzymatic processes in 30 tanning by applying a composition comprising melanin specialized cells known as "melanocytes'. Melanins are precursors or melanin precursor-like materials to the the pigments of mammalian skin and hair. skin. They do not teach or claim that applying melanins, Mammalian melanins are highly insoluble and can be either natural or synthetic, to the skin will mimic skin dissolved (solubilized) only through non-physiological tanning. Rather they teach that such melanins achieve treatments such as boiling in strong alkali, or through 35 "only superficial external tanning results which is the use of strong oxidants such as hydrogen peroxide. readily removed by rinsing with water or rubbing with Tyrosinase, a key enzyme in the melanin biosynthetic a towel'. pathway, can catalyze the formation of melanin in a test Herlihy (U.S. Pat. No. 4,515,773) discloses a skin tube using L-tyrosine, L-dopa or 5,6-dihydroxyindole tanning composition containing a melanin precursor as substrates; however, the product is an insoluble pre and a tyrosinase enzyme in a cosmetic base. He does cipitate as described above. not, however, teach or claim the application of mela Ito, "Reexamination of the Structure of Eumelanin', nins, either natural or synthetic to the skin. Biochinica et Biophysica Acta, 833, 155-161, 1986, men Gaskin (U.S. Pat. No. 4,806,344) discloses a sun pro tions that natural melanin may be a polymer of 5,6-dihy tectant composition comprising "melanin' as one of the droxyindole and 5,6-dihydroxyindole-2-carboxylic 45 ingredients. Gaskin further discloses that the melanin is acid. Ito, however, does not teach or suggest combining “synthesized from tyrosinase and DOPA'. She does not these chemicals to form melanin. teach or suggest that such melanin is synthesized non Ito and Nicol, "Isolation of Oligimers of 5,6-Dihy enzymatically or that it is comprised of precursors droxyindole-2-carboxylic Acid from the Eye of the which confer either negative or positive charges on the Catfish', Biochemical Journal, 143, 207-217, 1974, men 50 polymer, rendering it soluble in aqueous solutions.
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