701 Nanoparticulate monobenzone (MBEH) as a potential drug candidate for melanoma

Background Monobenzone (MBEH) is a skin depigmenting agent FDA-approved for topical applications. 1 It specifically interacts with tyrosinase, 2 a key enzyme in melanogenesis, to form reactive quinones that are toxic to pigmented cells, including melanoma cells. As melanoma cells express abundant tyrosinase activity, 3 repurposing MBEH to target melanoma cells might serve as a treatment strategy. Furthermore, quinones can haptenize tyrosinase, 4 supporting neo-antigen formation. 5 Modified tumor antigens then initiate an immune cascade, engaging T cells to target tumor cells. This biphasic effect of MBEH makes it a suitable candidate to target melanoma. Although topical treatment of MBEH can suppress subcutaneous melanoma growth in vivo, 6 systemic administration of the drug was toxic, 7 limiting the application of MBEH for metastatic disease. To overcome this limitation, we encapsulated MBEH and its derivatives into nanoscale liposomes (~100 nm) and evaluated its anti-tumor efficacy. Methods Liposomes were prepared 8 and MBEH was loaded into the liposomes. Loading was evaluated using mass spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. In vitro cytotoxicity of liposomal MBEH to mouse and human melanoma cell lines was evaluated by MTT assays. Meanwhile, in vivo trafficking of fluorescent liposomes to B16-F10 tumors and vital organs was evaluated in tissue homogenates by flow cytometry. The anti-tumor effects of liposomal MBEH towards subcutaneously injected B16-F10 melanoma cells were evaluated in C57BL/6 mice (n=4 per group) over time. Results Mass spectroscopy and NMR data revealed that MBEH was encapsulated into the liposomes at 2.3%mol MBEH per liposome. Liposomal MBEH was toxic to both mouse and human melanoma cells with lower half maximal inhibitory concentration (IC50) values in B16-F10 and A375 and higher IC50 values in 888-A2 and 624.38 cells. In vivo trafficking in mice revealed ~ 55% uptake of liposomal fluorescence by the tumor. In contrast to empty liposomes (mean tumor volume on day 21: 467.5 mm 3 ), MBEH loaded liposomes significantly (P<0.0001) reduced B16-F10 tumor growth mice (mean tumor volume on day 21: 107 mm 3 ). Skin depigmentation was not observed over the 21 day period of follow up after liposomal MBEH treatment, supporting that liposomal MBEH can be safely administered. Conclusions We provide a proof-of-concept to use nanoparticulate MBEH to target the melanogenic pathway in melanoma. A detailed study of MBEH loading, nanoparticle stability, and tumor infiltrating lymphocyte can further establish nanoparticulate MBEH as a potential drug candidate for melanoma. Acknowledgements This work was supported by a grant from the Sherman Fairchild Foundation. References Bolognia JL, Lapia K, and Somma S, Depigmentation therapy. Dermatologic Therapy 2001. 14(1): p. 29–34. McGuire J and Hendee J, Biochemical Basis for Depigmentation of Skin by Phenolic Germicides. Journal of Investigative Dermatology 1971. 57(4): p. 256–261. Michaeli Y, et al., Melanoma cells present high levels of HLA-A2-tyrosinase in association with instability and aberrant intracellular processing of tyrosinase. European Journal of Immunology , 2012. 42(4): p. 842–850. Manini P, et al., A reactive ortho-quinone generated by tyrosinase-catalyzed oxidation of the skin depigmenting agent monobenzone: self-coupling and thiol-conjugation reactions and possible implications for melanocyte toxicity. Chem Res Toxicol , 2009. 22(8): p. 1398–405. 5. Westerhof W, et al ., The haptenation theory of vitiligo and melanoma rejection: a close-up. Experimental Dermatology 2011. 20(2): p. 92–96. Hariharan V, et al., Topical application of bleaching phenols; in-vivo studies and mechanism of action relevant to melanoma treatment. Melanoma Res , 2011. 21(2): p. 115–26. Kelly KH, Bierman HR, and Shimkin MB, Negative effects of oral monobenzyl ether of hydroquinone in malignant melanoma in man. proceedings of the society for experimental Biology and Medicine 1952. 79(4): p. 589–590. Stefanick JF, et al., A systematic analysis of peptide linker length and liposomal polyethylene glycol coating on cellular uptake of peptide-targeted liposomes. ACS Nano 2013. 7(4): p. 2935–2947.