[CANCER RESEARCH 50, 3743-3747, June 15, 1990] Melanocytotoxicity and Antimelanoma Effects of Phenolic Amine Compounds in Mice in Vivo1 Frank Alena, Kowichi Jimbow,2 and Shosuke Ito3 Division of Dermatology and Cutaneous Sciences, University of Alberta, Edmonton, Canada T6G 2C2 ABSTRACT A phenolic amine compound, 4-S-cysteaminylphenol (4-S-CAP), is a potent depigmenting agent. To develop more efficacious antimelanoma agents, we synthesized four homologues of 4-S-CAP: W-acetyW-S-CAP (W-Ac-4-S-CAP), a-methyl-4-S-CAP, 4-S-homo-CAP, and N,N'-di- methyl-4-S-CAP. We tested these five compounds in mice in vivo. After s.c. or i.p. injection of saline solution (in control groups) or one of the compounds, follicular melanocytes were examined by light and electron microscopy to assess the degree of melanocytotoxicity; /V-Ac-4-S-CAP induced the most depigmentation (98%), whether given ¡.p. or s.c. After injection of 4-S-CAP or W-Ac-4-S-CAP, the number of murine B16F10 melanoma colonies formed in the lungs was determined; 4-S-C\P and A-Ac-4-S-CAP were almost equally effective, reducing the colonies to 32 and 25% of mean control, respectively. Metabolic studies of the urine showed 9% of 4-S-CAP and 20% of 7V-Ac-4-S-CAP injected i.p. were excreted unchanged in 24 h; 1.3% of the A'-Ac-4-S'-CAP was excreted as 4-S-CAP, indicating some conversion. We conclude that /V-Ac-4-S-CAP is a suitable model for developing chemotherapy to treat melanoma characterized by high tyrosinase activity and melanin synthesis. INTRODUCTION Melanin pigment is synthesized solely within melanocytes and melanoma cells. The enzyme tyrosinase (EC 1.14.18.1) converts the amino acid tyrosine to L-DOPA and thence to dopaquinone, which gives rise to a melanin polymer. This process occurs in a specific secretory granule, the melanosome (1, 2). There have been several attempts to use this unique melanin pathway as a basis for developing chemotherapeutic agents selectively toxic to melanoma cells, in most cases based on L-DOPA and related catechols (3-7), but chemical instability and severe toxicity due to nonspecific oxidation have precluded success. Because phenolic compounds seem to possess fewer drawbacks and appear more promising for use in chemotherapy to combat melanoma, we developed a new subgroup of phenols, combining phenols with cysteine and cysteamine to yield cys- teinylphenol and cysteaminylphenol, respectively (8). Both 4-5-CP4 and 4-5-CAP are substrates for mammalian tyrosinase, can depigment black hair, and possess an anti- melanoma property in vivo, whereas their 2-5-isomers are not tyrosinase substrates and evidence no melanocytotoxic effects (8). Most importantly, 4-5-CAP has a significant antimelanoma effect (9). Padgette et al. (10), however, in developing hypoten- sive agents discovered that 4-5-CAP is a substrate of plasma MAO. Our earlier study confirmed this result in vitro and Received 7/25/89; revised 1/30/90. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This study was supported by a grant from the National Cancer Institute of Canada. 2To whom requests for reprints should be addressed. 3 Professor of Chemistry at the School of Hygiene, Fujita-Gakuen Health University, Toyoake, Aichi, Japan; Visiting Professor of the Alberta Heritage Foundation for Medical Research during this study. 'The abbreviations used are: 4-S-CAP, 4-5-cysteaminylphenol; Ar-Ac-4-5- CAP, A'-acetyl-4-5-cysteaminylphenol; a-Me-4-S-CAP, a-methyl-4-5'-cysteami- nylphenol; 4-S-HomoCAP, 4-5-homocysteaminylphenol; N,N-D\Me-4-S-CAP, A',A''-dimethyl-4-5-cysteaminylphenol; HPLC, high performance liquid chroma- tography; L-DOPA, L-dihydroxyphenylalanine; MAO, monoamine oxidase; PTCA, pyrrole-2,3,5-tricarboxylic acid. indicated that 4-5-CAP could produce a cytotoxic aldehyde through interaction with plasma MAO (11). To overcome these difficulties, we synthesized four homologues of 4-5-CAP; N- Ac-4-S-CAP, a-Me-4-S-CAP, 4-5-homo-CAP, and N,N-DiMe- 4-5-CAP. We found that all four compounds are substrates for tyrosinase and that 4-5-HomoCAP is also a substrate for MAO (12). The depigmentation potency in black follicles of test com pounds given s.c. is a reliable screening method for evaluating melanocytotoxicity (13). In the present study, in mice in vivo we compared this property (visible change and chemical mela nin content) of the four new phenolic amine compounds and the mother compound, 4-5-CAP, given both i.p. and s.c., and assessed the antimelanoma effect of 4-5-CAP and the most efficacious depigmenting compound by assaying the formation of melanoma colonies in the lungs. MATERIALS AND METHODS Animals. Breeding pairs of C57BL/6J black mice were purchased from The Jackson Laboratory (Bar Harbor, ME), and their female progeny were used in the study when 8 weeks old and weighing 17.0 g. All procedures were approved by the University's Health Sciences Animal Welfare Committee. Chemicals. The methods used to synthesize the phenolic amine compounds [4-5-CAP, W-Ac-4-S-CAP, a-Me-4-S-CAP, 4-S-Homo- CAP, and A',A'-DiMe-4-5-CAP] were as reported previously (8, 9, 11). Briefly, the basic compound, 4-S-CAP, was prepared through refluxing a mixture of phenol and cysteamine with IIBr (Fig. 1). In all experi ments, the agent injected into controls was l N saline solution. I .DSU. The LD50 for each compound (n = 6 mice each) was established by a single i.p. injection of the test drug dissolved in normal saline solution, in a dose range of 100 to 1300 mg/kg body weight. The LD50 was 600 mg/kg for 4-5-CAP, 1200 mg/kg for W-Ac-4-S-CAP, 500 mg/ kg for a-Me-4-S-CAP, 350 mg/kg for 4-S-HomoCAP, and 300 mg/kg for N,N-DiMe-4-S-CAP. Melanoma Cell Line. The murine B16F10 melanoma cell line, with a strong metastatic property to form tumor colonies in lungs, was kindly supplied by Dr. B. M. Longenecker (Department of Immunol ogy, University of Alberta). Cells were grown in T-75 flasks in Dulbec- co's modified Eagle's medium (GIBCO, Grand Island, NY) supple mented with 10% fetal calf serum (GIBCO), penicillin (100 units/ml), and streptomycin (100 jig/ml). Cells were incubated at 37°Cin a humidified atmosphere of 5% CO2 in air. Melanocytotoxicity in Vivo. Black hairs were plucked manually from the back of the mice, to initiate new anagen growth and activate follicular melanocytes with increased tyrosinase activity. Thirty mice were randomized into six groups of five (one control and one for each compound). Starting on day 1, daily for 14 days the agent was injected i.p. or was infiltrated s.c. in an area where hair follicles had been plucked; the dose was 300 mg/kg body weight. Melanin Content. In the same 30 mice, hair follicles were harvested on day 22 (early telogen phase) and their eumelanin content was analyzed. Details of the assay, including chemical degradation of mel anin and HPLC, were as before (14). Briefly, a 10-mg hair sample is homogenized in water at a concentration of 10 mg/ml; the 200-^1 homogenate is transferred to a screw-capped test tube, mixed with l M HjSO4 (800 n\), and oxidized with 3% KMnO4. The product, PTCA, is analyzed by HPLC with a UVL detector; samples are measured in duplicate. The eumelanin content is expressed as PTCA (ng/mg) versus 3743 Research. on December 12, 2021. © 1990 American Association for Cancer cancerres.aacrjournals.org Downloaded from