title: Irinotecan-cisplatin interactions assessed in cell-based screening assays: cytotoxicity, drug accumulation and DNA adduct formation in an NSCLC cell line.
abstract: OBJECTIVE
  The use of in vitro drug cytotoxicity assays for the assessment of drug-drug interactions that lead to synergy may not take into account the many cellular determinants responsible for combination effects. Administration of the anticancer drug CPT-11, for example, is associated with rapid conversion of drug from its active lactone form to the inactive carboxylate form. Thus it is difficult to model, in vitro, the behavior of this drug when used as a single agent and when used in a combination setting, this factor may contribute to the interactions measured. Therefore, the objective of this study was to examine the influence of CPT-11 lactone ratio on the cellular accumulation of CPT-11 when used as a single agent and under conditions where it is used in combination with cisplatin.
  
  
  METHODS
  A fixed ratio experimental design was used and drug ratios of CPT-11 and cisplatin were judged to be antagonistic, additive, or synergistic to the non-small cell lung cancer cell line, H460, on the basis of the median effect analysis methodology of Chou and Talalay. The influence of extracellular pH on CPT-11 accumulation was evaluated at pH 7.4 and pH 6.6 when the drug was added immediately to the cells or first pre-equilibrated at the indicated pH. These studies were completed in the presence and absence of cisplatin.
  
  
  RESULTS
  When CPT-11 was added as a single agent to cells in pH = 7.4 media, the drug underwent hydrolysis to the carboxylate form; however, there was a rapid accumulation of the CPT-11 lactone form which peaked at 3,800 pmol/mg protein by 30 min and drops to 570 pmol/mg protein by 24 h. In pH = 6.6 media, accumulation of CPT-11 lactone was substantially lower over a 60 min timecourse; however, the cellular uptake measured at 24 h was comparable to that observed when the drug was added into pH 7.4 media. When evaluating CPT-11 lactone accumulation in a combination setting with cisplatin no significant difference in either CPT-11 lactone accumulation or cisplatin accumulation was observed, suggesting that drug interactions that led to synergy were mechanistically based. Results are presented which suggest that when cisplatin and CPT-11 are used in combination, there was a significant prolongation of platinum association with DNA compared to results obtained when cisplatin was used alone.
  
  
  CONCLUSIONS
  These results suggest that the CPT-11 lactone to carboxylate ratio does not influence the accumulation of the active CPT-11 lactone form in H460 cells and that CPT-11 does not influence cisplatin uptake when used in combination. It is argued, therefore, that the improved cytotoxicity between CPT-11 and cisplatin, as determined using cell-based assay, has the potential to be preserved in vivo assuming the optimal drug-drug ratio and concentration can be effectively delivered to the tumor.
authors.affiliation: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, ON, Canada.
authors.forename: Jason
authors.initials: J
authors.lastname: Zastre
authors.forename: Malathi
authors.initials: M
authors.lastname: Anantha
authors.forename: Euan
authors.initials: E
authors.lastname: Ramsay
authors.forename: Marcel
authors.initials: M
authors.lastname: Bally
mesh_terms: D000970:Antineoplastic Agents / Q000493:pharmacokinetics / Q000494:pharmacology; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D018736:DNA Adducts / Q000737:chemistry / Q000235:genetics; D004249:DNA Damage; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D000077146:Irinotecan; D007783:Lactones / Q000737:chemistry; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D013997:Time Factors
chemical_list: D000970:Antineoplastic Agents; D018736:DNA Adducts; D007783:Lactones; D000077146:Irinotecan; D002945:Cisplatin; D002166:Camptothecin