A Combined Experimental and Mathematical Approach for Molecular-based Optimization of Irinotecan Circadian Delivery

Abstract

Circadian timing largely modifies efficacy and toxicity of many anticancer drugs. Recent findings suggest that optimal circadian delivery patterns depend on the patient genetic background. We present here a combined experimental and mathematical approach for the design of chronomodulated administration schedules tailored to the patient molecular profile. As a proof of concept we optimized exposure of Caco-2 colon cancer cells to irinotecan (CPT11), a cytotoxic drug approved for the treatment of colorectal cancer. CPT11 was bioactivated into SN38 and its efflux was mediated by ATP-Binding-Cassette (ABC) transporters in Caco-2 cells. After cell synchronization with a serum shock defining Circadian Time (CT) 0, circadian rhythms with a period of 26 h 50 (SD 63 min) were observed in the mRNA expression of clock genes REV-ERBα, PER2, BMAL1, the drug target topoisomerase 1 (TOP1), the activation enzyme carboxylesterase 2 (CES2), the deactivation enzyme UDP-glucuronosyltransferase 1, polypeptide A1 (UGT1A1), and efflux transporters ABCB1, ABCC1, ABCC2 and ABCG2. DNA-bound TOP1 protein amount in presence of CPT11, a marker of the drug PD, also displayed circadian variations. A mathematical model of CPT11 molecular pharmacokinetics-pharmacodynamics (PK-PD) was designed and fitted to experimental data. It predicted that CPT11 bioactivation was the main determinant of CPT11 PD circadian rhythm. We then adopted the therapeutics strategy of maximizing efficacy in non-synchronized cells, considered as cancer cells, under a constraint of maximum toxicity in synchronized cells, representing healthy ones. We considered exposure schemes in the form of an initial concentration of CPT11 given at a particular CT, over a duration ranging from 1 to 27 h. For any dose of CPT11, optimal exposure durations varied from 3h40 to 7h10. Optimal schemes started between CT2h10 and CT2h30, a time interval corresponding to 1h30 to 1h50 before the nadir of CPT11 bioactivation rhythm in healthy cells.

Identification of Circadian Determinants of Cancer Chronotherapy through In Vitro Chronopharmacology and Mathematical Modeling

“…Quantitative PCR was performed with LightCycler 480 using LightCycler 480 SYBR Green I master kit (Roche). Primers used for gene amplification were previously described (24,28). Hybridization temperature for all the primers was 60 C. Relative quantification of target RNA using 36B4 as reference were realized with Relquant software (Roche).…”

“…24; Supplementary Table S1). We removed the first 2 data points at 0 and 4 hours from the parameter estimation procedure as purely circadian variations may be perturbed by the seric shock during the first 4 hours.…”

“…Irinotecan was added to the culture medium 6 hours before incubation with cells in order to reach the lactone-carboxylate equilibrium. CPT11 and SN38 extra-and intracellular concentrations were measured by the method of high-performance liquid chromatography (HPLC) described (24).…”

“…In contrast with chronopharmacology studies in whole organisms, investigations in circadian synchronized cell culture models could enable systematic and quantitative information to be gathered, so as to precisely determine and model molecular chronopharmacology (24). Here, we conduct such an in vitro-in silico circadian investigation of irinotecan PK-PD in synchronized colorectal cancer cells for the first time.…”

Cells as Machines: Towards Deciphering Biochemical Programs in the Cell

“…The same strategy for parameter optimization can also be used to compute control parameters to achieve a desired behavior at the single cell of cell population levels. This has been used for the model-based real-time control of gene expression in yeast cells using a microfluidic device in [49], and at the whole body scale, to couple models of cell cycle, circadian clock, drug effects, DNA repair system, and optimize anti-cancer drug chronotherapeutics in [16,3].…”