Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) analogues show differential substrate specificity to ABC transporters

3226 Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) monomers and dimers interact covalently with DNA to form PBD-DNA adducts (monomers) or cross-links (dimers). These DNA interactions lead to in vivo antitumour activity, and one PBD dimer (SJG136) is currently in clinical development. In this study, we have investigated whetherPBD analogues are substrates for three major ABC transporter proteins associated with multidrug resistance (MDR): P-glycoprotein (P-gp), multidrug resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP). The sulforhodamine B assay was used to assess the cytotoxicity of 10 PBD monomers and 4 dimers after 24h of drug exposure using cell lines with differential expression of ABC transporters. In addition, the effect of specific inhibitors of each transporter was used to validate substrate specificity of the PBD analogues.
 The cytotoxicity studies demonstrated that HCT 116 and A2780 cells (expressing low or no P-gp) were more sensitive than HCT 15 and A2780AD cells (expressing high P-gp) to dimers. In addition, the ratios of IC50s between P-gp high and P-gp low cells were > 2 for all the dimers and close to 1 for all the monomers suggesting that dimers are substrates of P-gp. The cytotoxicity of the dimers only was increased in HCT 15 and A2780AD in presence of a P-gp inhibitor, verapamil. Isogenic 3T3 cells expressing mdr-1 cDNA (3T3 pHamdr-1) were > 30 fold less sensitive to dimers than the parental 3T3 cells. The differential parameters listed as determinants of P-gp interaction were compared between monomers and dimers, since only the activity of the dimers appeared to be affected by P-gp expression. The number of N and O atoms, the Polar Surface Area, the MW and a number of hydrogen bonding energy can discriminate dimers and monomers. The MW correlated with the ratio of IC50s of the dimers between HCT 116 and HCT 15.
 The impact of MRP1 on the cytotoxicity of the PBD analogues was evaluated using the A549 non small cell lung cancer cell line with high MRP1 expression. IC50 values for all dimers and only 1 out of 10 monomers decreased on average ~4 fold when cells were pre-treated with the specific MRP1 inhibitor thus MK-571 enhancing cytotoxicity.
 Finally, the impact of BCRP on the cytotoxicity of the analogues was assessed using the MCF7 breast cancer cell line and the MCF7-MX cell line expressing high levels of BCRP. MCF7 cells were more sensitive than MCF7-MX cells to all dimers and several monomers: The IC50 ratios between BCRP high and BCRP low cells were > 2 for all dimers and for 8 out of 10 monomers. IC50 values decreased up to 13 fold for dimers when MCF7-MXcells were pre-treated with FTC, a specific Brcp inhibitor.
 In conclusion, PBD dimers appear to be substrates of all three transporters; however, some monomers showed a degree of selectivity against them. The Caco-2 transwell system is currently being used to confirm differential substrate specificity of these PBD analogues.