Schedule-dependent pharmacodynamic effects of gemcitabine and cisplatin in mice bearing Lewis lung murine non-small cell lung tumours

The combination of 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) and cis-diammine-dichloroplatinum(II) (cisplatin, CDDP) is increasingly applied in clinical oncology. We studied the underlying mechanisms of the in vivo schedule dependency and supra-additive interaction between dFdC and CDDP in C57/Bl6 mice bearing Lewis lung (LL) tumours. Mice were treated with CDDP (6 mg/kg) and dFdC (60 mg/kg) either simultaneously or in a 4 or 24 h interval with dFdC preceding CDDP or vice versa. Four, 8 (in some cases 12) and 24 h after treatment mice were sacrificed and tumours, kidneys, blood and bone marrow (BM) were collected. Since CDDP acts by formation of Platinum (Pt)-DNA adducts and dFdC by incorporation of its triphosphate (dFdCTP) into DNA, we measured total Pt levels, dFdCTP accumulation and Pt-DNA adducts by atomic absorption spectrometry (AAS), high performance liquid chromatography (HPLC) and ³²P-postlabelling, respectively. These levels were related to the previously determined antitumour efficacy and toxicity of the dFdC/CDDP combination. Peak dFdCTP accumulation in tumours (11 pmol/mg) was found 4 h after dFdC treatment, while CDDP tended to reduce this in a time-dependent way. Peak levels of total Pt in tumours were found 4 h after CDDP treatment (581 fmol/mg) and dropped 1.8-fold after simultaneous treatment with dFdC (P = 0.04). Treatment with dFdC 4 h after or simultaneously with CDDP increased Pt retention (level 24 h after CDDP treatment) 1.4- and 1.6-fold (P = 0.04 and P = 0.03, respectively). Peak Pt-DNA adduct levels in tumours were also found 4 h after CDDP treatment (7 fmol/μg DNA) and were decreased 3-fold by dFdC treatment 24 h prior to CDDP (P = 0.04). Pt-DNA adduct retention was only decreased when dFdC was given 4 h before CDDP (8-fold (P <0.01)). The retention and the area-under the concentration-time curve of Pt-DNA adducts were related to decreased tumour doubling time (linear regression coefficient (R) = 0.95; P <0.05, 0.96 P = 0.04 and 0.90; P = 0.04. Pt-DNA adduct levels in the BM cells reached a plateau level 4-24 h after CDDP treatment (approximately 10 fmol/μg DNA), which was increased by dFdC when given either simultaneously with, 4 h before or 4 h after CDDP (6-, 3- and 5-fold at 28 h, 8 h and 28 h, respectively (P ≤ 0.04)). Peak Pt-DNA adduct formation (24 h: 8 fmol/μg DNA) in kidneys was enhanced by dFdC when given simultaneously with or 4 h before CDDP (4 h timepoint) (P <0.01). However, retention was 4- and 6-fold decreased when dFdC was given 4 or 24 h after CDDP, respectively (P≤0.01). dFdC given 24 h before CDDP decreased all Pt-DNA adduct levels in kidneys 3-fold or more (P≤0.03). Pt-DNA adduct levels were inversely related to kidney toxicity when the most toxic schedule was excluded from the analysis. Peak levels of total Pt in kidneys were reached 24 h after CDDP treatment (4.3 fmol/mg) and the 8 h levels were increased 2-fold by dFdC when given 4 h after CDDP (P = 0.07). (C) 2000 Elsevier Science Ltd.