THE USE OF alpha -METHYL-P-TYROSINE TO INHIBIT MELANIN PRODUCTION IN IRIS MELANOCYTES

Abstract: The use of agents for blocking the synthesis of tyrosinase to prevent permanent pigmentation of the iris caused by melanin deposit induced by pharmacological treatments or by metabolic imbalance is described.

Description: XHE USE OF α-HETHYL-P-TYROSINE TΩ INHIBIT MELANIN PRODUCTION IN IRIS MELANOCYTES The present invention refers to the use of agents for blocking the synthesis of tyrosinase to prevent permanent pigmentation of the iris caused by melanin deposit induced by pharmacological treatments or by metabolic imbalance. Background of the invention Latanoprost (13,14-dihydro-17-phenyl-18,19,20-tri- nor-PGF 2α isopropyl ester), a synthetic prostaglandin analogue, (EP-A-0364317 ) as well as naturally occurring prostaglandins such as PGF 2α and PGE2 have been shown to induce increased pigmentation of the monkey iris during chronic treatment (Selen G. , Stjernschantz J., Resul B. prostaglandin-induced iridial pigmentation in primates. Surv. Ophthalmol 1997; 41, Suppl. 2 : S125-S128) . The exact mechanism behind this response to prostaglandin treatment is not known, but increased synthesis of melanin (melanogenesis) must occur for the eye colour to become darker. Also in patients treated with latanoprost (Wistrand PJ, Stjernschantz J., Olsson K. The incidence and time-course of latanoprost-induced iridial pigmentation as a function of eye color. Surv. Ophthalmol 1997; 41, Suppl. 2:S129-S138) or with isopropyl unoprostone (13, 14-dihydro-15-keto-20-ethyl- PG 2 α isopropylester ) (Yamamoto T., Kitazawa Y. Iris- color change developed after topical isopropyl unoprostone treatment J. Glaucoma 1997; 6: 430-432) a darkening of the iris # is sometimes noted during chronic therapy. Particularly patients with heterochromic iris, i.e. blue-brown, gray-brown, green-brown or hazel eye colour seem to be predisposed to this side-effect. Since the side-effect may become cosmetically disturbing, particularly in patients with unilateral glaucoma that are treated only in one eye, and since the side-effect is irreversible, and relatively frequent, it would be advantageous to circumvent it, although it does not appear to pose a health hazard to the patients that develop it. Melanin, a large naturally occurring polymer is formed from the amino acid tyrosine. In the initial step of melanin formation tyrosine is hydroxylated to L-Dopa which is further oxidized to dopaquinone . The enzyme catalyzing both reactions is called tyrosinase. Dopaquinone is a labile compound that is converted to dopachrome, a black compound which is needed for the formation DHICA (dihydroxyindol-carboxylic acid) oligomers that are needed for the final polymerisation to yield eu elanin (black or brown melanin). Dopaquinone can alternatively react with cysteine which will lead to sulfur containing oligomers and finally pheomelanin (yellowish or reddish melanin). Important to realize is that the rate limiting step in the melanin production is the reaction catalyzed by tyrosinase. Lack of functional tyrosinase e.g. because of a mutation of the tyrosinase gene, always leads to albinism since no pigment can be formed in the body. Interestingly, the same tyrosinase enzyme is also needed in sympathetic neurons and adrenal medulla for the production of noradrenaline, a neurotransmitter , and adrenaline., a hormone, since these compounds are biosynthetized from tyrosine. Thus compounds that block the tyrosinase enzyme will have effect both on melanogenesis and on the function of the sympathetic nervous system. Summary of the invention It has now been found that inhibitors of„ the tyrosinase enzyme, particularly, α-methyl-p-tyrosine, inhibit melanin production induced by administration of PGF α and GE 2 derivatives, such as latanoprost and unoprostone. Thus, the treatment with said tyrosinase inhibitors prior, during or after administration of prostaglandin derivatives in glaucomatous patients, inhibits melanin production by iris melanocytes avoiding the eye coloring variations in these patients. Detailed description of the invention A classical agent for blocking the synthesis of tyrosinase is α-methyl-para-tyrosine, a drug known with the name of metyrosine (J. Am. Chem. Soc. 77, 700, 1958), which is a false substrate for the enzyme. Thus L-Dopa is not formed and consequently neither melanin nor adrenaline/noradrenaline can be formed. The drug has been used in the palliative treatment of pheocromo- cytoma, a tumour of the adrenal medulla leading to high concentrations of catecholamines in blood and therefore increased blood pressure. Used in concentrations high enough, α-methyl-para-tyrosine can significantly block the biosynthesis of adrenaline/noradrenaline as well as melanin. When used at clinical concentrations for the treatment of pheocromocytoma the catecholamine concentration in the body is markedly reduced (Weiner N. , Drugs that inhibit adrenergic nerves and block adrenergic receptors. In Goodman Gilman A., Goodman LS, Rail TW, Murad F., edε; Goodman and Gilman 's the Pharmacological Basis of Therapeutics, Macmillan, New York, 1985, pp 181-214), and in the in vi tro experiments of the present inventions the melanin production was significantly reduced. In essence any agent that interfers with the tyrosinase enzyme will have the same beneficial effect resulting in a blockade of the melanin production. Such agents are e.g. hydroxyquinone and substances that react with copper ions because copper is a necessary cofactor for the tyrosinase enzyme, and various kinds of false substances for the enzyme. Consequently if these agents are given either separately or in a mixture together with latanoprost, isopropyl unoprostone, or any other protaglandin that induces melanogenesis, the pigment formation in the iris can be prevented or at least significantly hindered. This activity of α-methyl-p-tyrosine has been demonstrated with the following experiments: Materials and methods Cell Culture Uveal melanocytes were isolated and cultured from adult donor eyes. The iris was excised and placed in a dish with the posterior surface upward. The iris pigment epithelium was separated from the stroma after immersion in 0.25% trypsin solution (Gibco, USA) at 37°C for 2 hour. The remaining iris stroma was placed in a 0.25% trypsin solution at 4°C for 18 hours, followed by incubation at 37 C C for 1 hour. The isolated cells were collected. The trypsin solution was replaced by collagenase solution (400 U/ml, in F-12 medium, Sigma, USA) and incubated at 37 β C. The collagenase solution was replaced, and the cells were collected, centrifuged, resuspended, and plated each hour for 3 hours. The isolated uveal melanocytes were cultured in Falcon culture dishes (Becton Dickinson, USA) with FIC medium, which consisted of F-12 medium supplemented with 10% fetal bovine serum, 2 mM glutamine (all from Gibco), 10 ng/ml cholera toxin, 0.1 mM isobutylmethylxanthine, 50 μg/ml gentamicin (all from Sigma), and 20 ng/ml basic fibroblaεt growth factor (Promega, USA). The culture dishes were incubated in a humidified 5% C0 2 atmosphere. The medium was changed three times a week. Geneticin (Sigma, USA), a cytotoxic agent, was added (100 μg/ml) for 3 to 7 days when necessary. The 8 cell strains of uveal melanocytes used in the present study were isolated from donors with different iris color (brown and brown-blue). Melanin measurement Cultured uveal melanocytes were detached by trypsin-EDTA solution and counted in a hemocytometer , the cell suspensions were centrifuged, and the pellet was dissolved in 1 N NaOH. Melanin concentration was determined by measurement of optical density at 475 nm and compared with a standard curve obtained using synthetic melanin (Sigma). Melanin content was expressed as ng/cell. Calculation of melanin production Melanin production was calculated by determining the melanin content and the cell counts at the beginning and the end of each generation by the following formula: Cp = CtP - C 0 /1.3D (P-l) where C and C*. represent the melanin content per cell at times 0 and time t, respectively; P is the population increase during time t, D is the doubling time of the uveal melanocytes; and Cp is melanin production per cell per day during time t. Tyrosinase activity Tyrosinase activity was evaluated in nine cell strains using an adaptation of the Pomerantz method, which is based on the measurement of ^H 2 0 released by the enzymatic hydroxylation of tyrosine. Results Melanin content in iris melanocytes cultured from 5 cell strains of brown irises and 3 cell strain of brown- blue irises appeared to be increased when latanoprost was added at the highest molar concentrations. The same was found for melanin production and tyrosinase activity (Table 1 and 2) . After α-methyl-p-tyrosine was added (10 ~ 5 M), a significant decrease in melanin content and production and in tyrosinase activity was found with latanoprost 10 ~ to 10 ~ 5 M both in melanocytes cultured from brown irises and brown-blue irises (Table 1 and 2). These results show that melanin production by iris melanocytes is inhibited by α-methyl-p-tyrosine. The present invention also concerns pharmaceutical compositions containing a PGF2α or PGE 2 derivative with anti-glaucoma activity and a tyrosinase inhibitor as combined preparations for simultaneous, separate or sequential use in the therapy of glaucoma. In particular, the invention concerns pharmaceutical products containing latanoprost as anti-glaucoma agent and α-methyl-p-tyrosine as combined preparations for simultaneous, separate or sequential use in the therapy of glaucoma. For the considered therapeutic uses, α-methyl-p- tyroεine will be preferably be administered by topical route or by oral route in a daily dose of about 100-500 g. Although the present description concerns particularly the inhibition of melanin production induced by latanoprost, α-methγl-p-tyrosine can be used successfully to inhibit the same side effect of pigmentation induced by other pharmacological treatments or metabolic imbalance of different origin. Table 1 5 cell strains (brown) - growth stage - Melanin content (ng/cell) no α-methyl™ α-methyl- p-tyrosine p-tyrosine 10 ~5 no latanoprost (control) 0.0138 0.0133 latanoprost 10 ~8 M 0.0141 0.0135 latanoprost 10 -7 M 0.0151 0.0136 latanoprost 10-^ M 0.0161* 0.0137 latanoprost 10 " ^ M 0.0149* 0.0131 *p<0.01 vs. control Melanin production (ng/cell/day) no α-methyl- α-methyl p-tyrosine- p-tyrosine 10 ~5 no latanoprost (control) 0.0031 0.0033 latanoprost 10 -8 M 0.0038 0.0035 latanoprost 10 -7 M 0.0046* 0.0039 latanoprost 10 ~6 M 0.0036 0.0029 latanoprost 10 -5 M 0.0047* 0.0031 *p<0.01 vs. control Tyrosinase activity (units) no α-methyl- α-methyl-p- p-tyrosine tyrosine 10 ~5 no latanoprost (control) 36.7 33.6 latanoprost 10 ~8 M 36.7 35.5 latanoprost 10 -7 M 38.3 38.7 latanoprost 10 ~6 M 42.5 34.6 latanoprost 10 ~5 M 63.1* 31.5 *p<0.01 vs. control Table 2 3 cell strain (brown-blue) - growth stage - Melanin content (ng/cell) no α-methyl- α-methyl-p- p-tyrosine tyrosine 10 ~5 no latanoprost (control) 0.0121 0.0122 latanoprost 10 ~8 M 0.0127 0.0119 latanoprost 10 ~7 M 0.0142* 0.0123 latanoprost 10 ~6 M 0.0149* 0.0125 latanoprost 10 ' M 0.0151* 0.0128 *p<0.01 vs. control Melanin production (ng/cell/day) no α-methyl- α-methγl-p- p-tyrosine tyrosine 10 ~5 no latanoprost (control) 0.0021 0.0023 latanoprost 10 ~8 M 0.0038 0.0025 latanoprost 10 " M 0.0046* 0.0029 latanoprost 10 ~6 M 0.0056* 0.0029 latanoprost 10 ' M 0.0067* 0.0031 p<0.01 vs. control Tyrosinase activity (units) no α-methyl- α-methyl-p- p-tyrosine tyrosine 10 ~5 no latanoprost (control) 32.5 32.4 latanoprost 10 ~8 M 33.7 34.6 latanoprost 10 -7 M 48.1* 31.7 latanoprost 10 ~6 M 52.5* 34.6 latanoprost 10 " ^ M 62.7* 39.5 p<0.01 vs. control

Claims: CLAIMS 1. The use of tyrosinase inhibitors for the preparation of a medicament which prevents melanin production by iris melanocytes induced by pharmacological treatments or metabolic imbalance. 2. The use according to claim 1, wherein melanin production is induced by GF2 α or PGE 2 derivatives. 3. The use according to claim 2, wherein the PGF2 α or p GE 2 derivatives are latanoprost or unoprostone. 4. The use according to any one of the preceding claim wherein the tyrosinase inhibitor is α-methyl-p-tyrosine. 5. Pharmaceutical compositions containing a PGF2α derivative as anti-glaucoma agent and a tyrosinase inhibitor as combined preparations for simultaneous, separate or sequential use in the therapy of glaucoma. 6. Pharmaceutical compositions according to claim 5, containing latanoprost as anti-glaucoma agent and α- methyl-p-tyrosine as combined preparations for simultaneous, separate or sequential use in the therapy of glaucoma. 7. A method for preventing melanin production by iris melanocyte comprising the administration of an effective amount of a tyrosinase inhibitor. 8. A method according to claim 7 wherein the tyrosinase inhibitor is α-methyl-p-tyrosine.

Inventors: DRAGO FILIPPO, DRAGO, FILIPPO
Applicants: DRAGO FILIPPO, PHARMACIA & UPJOHN, DRAGO, FILIPPO
Application year: 1998
Main classification: A61K31
IPC codes: A61K31, A61K45, A61P27, A61P43