Novel multifunctional acyloxyalkyl ester prodrugs of 5-aminolevulinic acid display improved anticancer activity dependent on photoactivation G. Berkovitch a , A. Nudelman b , B. Ehenberg c , A. Rephaeli d , and Z. Malik a , a The Mina and Everard Goodman Life Sciences Faculty, b Chemistry Department, c Physics Department, Bar Ilan University, Ramat Gan Israel; d Laboratory of Pharmacology and Experimental Oncology, Felsenstein Medical Research Center, Sackler Faculty of Medicine, Tel-Aviv University, ISREL ABSTRACT New approaches to PDT using multifunctional 5-aminolevulinic acid (ALA) based prodrugs activating mutual routes of toxicity are described. We investigated the mutual anti-cancer activity of ALA prodrugs which upon metabolic hydrolysis by unspecific esterases release ALA, formaldehyde or acetaldehye and the histone deacetylase inhibitor (HDACI) butyric acid. The most potent prodrug in this study was butyryloxyethyl 5-amino-4-oxopentanoate (AN-233) that stimulated a rapid biosynthesis of protoporphyrin IX (PpIX) in human glioblastoma U-251 cells and generated an efficient photodynamic destruction. AN-233 induced a considerable high level of intracellular ROS in the cells following light irradiation, reduction of mitochondrial activity, dissipation of the mitochondrial membrane potential resulting in necrotic and apoptotic cell death. The main advantage of AN-233 over ALA stems from its ability to induce photo- damage at a significantly lower dose than ALA. Keywords: ALA-prodrugs, PpIX, PDT, glioblastoma U-251 1. INTRODUCTION Glioblastoma (GBM) is the most malignant among astrocytic tumors and is associated with a poor prognosis. Improvements in overall survival of patients with malignant gliomas have not been achieved in spite of intensive treatment, including surgical resection, postoperative radiation therapy, and chemotherapy. Photodynamic therapy (PDT) is a promising type of local therapy, and favorable results have been reported in several studies of PDT in glioma patients 1-4 . The use of endogenous PpIX generated though the heme biosynthetic pathway after administration of (ALA) has led to many applications of PDT in cancer 5 . PpIX is a potent photosensitizer that accumulates excessively when ALA is given externally to cells. The enzymes porphobilinogen deaminase (PBGD) and ferrochelatase (FC) are rate limiting and contribute to the differential higher accumulation of PpIX in tumor cells 5, 6 . Subsequent activation of PpIX by light leads to the formation of highly reactive singlet oxygen that causes the destruction of target cells by a complex cascade of chemical, biological, and physiological reactions. A main disadvantage ALA-PDT is the hydrophilic nature of ALA which limits its ability to penetrate though the skin or cell membranes. One solution to the poor penetrability of ALA is to increase its bioavailability via the formation of esters 7 . Our recent investigations revealed the anticancer activity of new acyloxyalkyl ester prodrugs of ALA 8 . The examination of these prodrugs was evaluated mainly under dark conditions and showed that ALA prodrugs that release formaldehyde (AN-243 and AN-242) were substantially more effective than the ALA- Acetaldehyde prodrug AN-233 in inducing mortality of human glioblastoma, U-251 cells. These results were consistent with previous studies in which the formaldehyde was shown to be a critical antiproliferative factor 9 . However, initial investigation of these prodrugs under photo-irradiation conditions showed that both AN-233 and AN-243 that release ALA and butyric acid substantially improved induction of cell mortality compared to ALA. AN-242 that releases ALA, formaldehyde and pivalic acid had no improved activity with PDT. The enhanced effect of PDT mediated by both AN-233 and AN-243 compared to ALA alone suggests that butyric acid, which is a component of both prodrugs, plays a role in improving the activity, while the formaldehyde had no contribution. Photodynamic Therapy: Back to the Future, edited by David H. Kessel, Proc. of SPIE Vol. 7380, 73800Z · © 2009 SPIE · CCC code: 1605-7422/09/$18 · doi: 10.1117/12.822703 Proc. of SPIE Vol. 7380 73800Z-1