Cooperativity between free and N-(2-hydroxypropyl) methacrylamide copolymer bound adriamycin and meso-chlorin e6 monoethylene diamine induced photodynamic therapy in human epithelial ovarian carcinoma in vitro.

The purpose of this study was to determine the interaction between free (unbound) and N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer bound adriamycin and meso-chlorin e6 monoethylene diamine (Mce6) induced photodynamic therapy in combination in their cytotoxic activities against human ovarian epithelial carcinoma (OVCAR-3) in vitro. The effects of each agent (free drugs and HPMA copolymer bound) alone and in combination were measured simultaneously utilizing two measures of cell viability: a) mitochondrial respiration via the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide reduction (MTT) assay; and b) thymidine incorporation via the tritiated thymidine incorporation (TI) assay. These were performed at 72 and 144 h after drug exposure. Forty-eight hours from time zero (24 h after drug addition), the cells treated with Mce6 (free and HPMA copolymer bound) and controls were exposed to 650 nm light (13 min at 15 mW/cm2, 11.7 J/cm2). The calculated ED50 values by the MTT 72 h assay for adriamycin (A) and Mce6/light (C) were 1.5 microg/ml and 209 ng/ml, respectively. Adriamycin demonstrated progressive cellular toxicity over time in both assays. Mce6/light demonstrated initial damage at 72 h by MTT and TI which recovered by 144 h. Adriamycin and Mce6/light acted cooperatively to increase the percentage of cells inhibited. In combination, 21.3+/-1.5% MTT reduction activity was observed by free adriamycin and Mce6/light compared to the expected 27+/-5% (p<0. 0001) based on additivity. Twice the ED50 of adriamycin (2A=3 microg/ml) or Mce6/light (2C=418 ng/ml) resulted in only 42+/-3.6% and 39.2+/-2.0% activity, respectively (both p<0.0001 vs. combination). When Mce6/light at 10x ED50 (10C) was combined with 1x ED50 of adriamycin (1A), or the reciprocal combination, additional cooperativity was demonstrated. Compared to free drugs, both HPMA copolymer bound adriamycin (P-A) and HPMA copolymer bound Mce6/light (P-C) required a 10-fold increase in drug concentration to show equivalency with free drugs (A or C). Dose response curves demonstrated a reduced slope compared to free drugs in the same dose ranges. When P-A was added (1-10x free adriamycin ED50) to an effective concentration of P-C (10P-C: equivalent to 10x free Mce6 ED50) an improved long-term inhibition of OVCAR-3 cell multiplication was noted in both the MTT and TI 144 h assays. P-C (1-10x free Mce6 ED50) added to an effective concentration of P-A (10P-A: equivalent to 10x free adriamycin ED50) did not appear to significantly improve the efficacy profile of P-A. A and C in vitro appear to act independently and are cooperative in their combined toxicity against the human ovarian epithelial carcinoma cell line OVCAR-3. HPMA copolymer-adriamycin and Mce6 conjugates (P-A and P-C, respectively) inhibited growth of OVCAR-3 in vitro. HPMA copolymer-adriamycin added to HPMA copolymer-Mce6 improved the efficacy of HPMA copolymer-Mce6.