Time of administration and cryptochrome-dependent changes in tolerability and pharmacokinetics of oxaliplatin
The circadian clock controls many physiological functions in mammals, including responses to cancer therapy, by influencing the pharmacokinetics, efficacy, and toxicity of anticancer drugs. Our objective was to investigate how the timing of administration, as well as the influence of cryptochrome (Cry), a key gene in the circadian clock, impact oxaliplatin's pharmacokinetics and toxicity. Additionally, we aimed to shed light on the underlying mechanism through RNA sequencing. Overall design: Cryptochrome (Cry) 1/2 heterozygous mice in C57BL/6J background were generously provided by Aziz Sancar (University of North Carolina, Chapel Hill, North Carolina, USA) and used to obtain Cryptochrome double-knockout (Cry1-/-Cry2-/-, DKO) mice. Animals were kept in Koc University Research Center for Translational Medicine (KUTTAM) and genotyping was done to determine the knockouts. Animals (12-16 weeks old) were housed under 12 h of light- 12 h of dark cycle (LD 12:12) and synchronized for 3 weeks prior to the beginning of experiments. Mice were kept in constant darkness (DD) throughout the experiments, starting 3 days before drug administration A single dose of oxaliplatin (12 mg/kg, i.p.) was administered to wild-type and CRYDKO mice over two circadian times (CT8/ CT16, Circadian Time). Drug-induced toxicity; assessed by death rate and weight loss. Oxaliplatin pharmacokinetics were studied by ICP-MS. 12 mg/kg i.p. for three days. Following administration of oxaliplatin, RNA obtained from liver of mice was sequenced. Total RNA from liver samples (WT and CRYDKO mice treated with oxaliplatin at CT8 and CT16) were isolated by using RNeasy Mini Kit (QIAGEN, Germany). Quality of amount of RNA were assessed by using Qubit (Thermo Fisher Scientific, USA). High quality RNA samples (OD260/280 1.8-2.0) were used to prepare library for RNA Sequencing analysis. Briefly, adenylated mRNAs were isolated by using Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, US). Then mRNAs were fragmented, converted to cDNA and second strand synthesis was performed. The 150bp paired-end sequencing of libraries were carried out via Illumina platform (Macrogen, Netherlands).