Research paper Proline prodrug of melphalan, prophalan-L, demonstrates high therapeutic index in a murine melanoma model Sachin Mittal a,1 , Yasuhiro Tsume a , Christopher P. Landowski a,2 , Kyung-Dall Lee a , John M. Hilfinger b , Gordon L. Amidon a, * a Department of Pharmaceutical Sciences, University of Michigan, MI, USA b TSRL Inc., MI, USA Received 24 December 2006; accepted in revised form 29 March 2007 Available online 4 April 2007 Abstract The therapeutic efficacy of prophalan-L, the L-proline prodrug of melphalan that demonstrated prolidase-dependent bioactivation to melphalan, was examined in vivo in a mouse melanoma model. Prophalan-L exhibited 2- to 2.5-fold higher hydrolytic and cytotoxic activ- ity than prophalan-D, the D-analog, in B16-F10 murine melanoma cells in vitro. Prophalan-L cytotoxicity in B16-F10 cells was lower (GI 50 = 221 lM) than that of melphalan (GI 50 = 173 lM). The tumor growth profiles in C57BL/6J mice injected with B16-F10 cells and treated with melphalan (5.5 lg/g i.p.) and equimolar concentrations of the prodrugs demonstrated significant difference between the control (buffered saline) and melphalan or prophalan-L but no significant difference between control and prophalan-D or between melphalan and prophalan-L. Prophalan-L was significantly less toxic than melphalan, while no significant difference was observed in tox- icity, measured as percent weight loss, between the prodrugs and saline control. Tumor reduction efficacy at high doses (12 lg/g i.p.) was similar for melphalan and prophalan-L; however, fatal toxicity was associated with melphalan while prophalan-L exhibited significantly lower systemic toxicity. An excellent correlation between GI 50 and tumor reduction efficacy was observed for the tested drugs (r 2 = 0.95). Prophalan-L thus demonstrates higher therapeutic index than melphalan in the murine melanoma model. Ó 2007 Published by Elsevier B.V. Keywords: Prodrug; Melphalan; Mouse melanoma model; Prolidase; Therapeutic efficacy 1. Introduction Melanoma ranks amongst the top five cancers in both men and women. The incidence of melanoma has steadily increased over the last few decades and in 2004 approxi- mately 55,000 new cases will have been diagnosed and almost 10,500 deaths will have occurred [1]. Detection and treatment of early-stage disease can cure most patients but a majority of patients with deep primary tumors or tumors that metastasize to regional lymph nodes succumb to distant metastases. Median survival after the onset of distant metastases is only 6–9 months, and the 5-year sur- vival rate is less than 5% [2–4]. For the last five decades, chemotherapy has been used with varying success. Dacarbazine was approved by FDA to be used alone or in combination for treating melanoma but has not shown good response rates [5,6]. Recently, mel- phalan has been used for various malignancies including melanoma but its use has been limited due to severe side- effects [7]. The side-effects associated with chemotherapy include unacceptable damage to normal cells and organs, a narrow therapeutic index, and a relatively poor selectivity for neoplastic cells [8–10]. 0939-6411/$ - see front matter Ó 2007 Published by Elsevier B.V. doi:10.1016/j.ejpb.2007.03.024 * Corresponding author. Department of Pharmaceutical Sciences, Col- lege of Pharmacy, University of Michigan, 428, Church Street, Ann Arbor, MI 48109-1065, USA. Tel.: +1 734 764 2440; fax: +1 734 763 6423. E-mail address: glamidon@umich.edu (G.L. Amidon). 1 Present address: Biopharmaceutics and Product Enhancement Depart- ment, Merck & Co., Inc., 770 Sumneytown Pike, P.O. Box 4, West Point, PA 19486, USA. 2 Present address: Institute for Biochemistry and Molecular Medicine, University of Berne, Bu ¨ hlstrasse 28, CH-3012 Berne, Switzerland. www.elsevier.com/locate/ejpb Available online at www.sciencedirect.com European Journal of Pharmaceutics and Biopharmaceutics 67 (2007) 752–758