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

Abstract

Cancer chronotherapy aims at enhancing tolerability and efficacy of anticancer drugs through their delivery according to circadian clocks. However, mouse and patient data show that lifestyle, sex, genetics, drugs, and cancer can modify both host circadian clocks and metabolism pathways dynamics, and thus the optimal timing of drug administration. The mathematical modeling of chronopharmacology could indeed help moderate optimal timing according to patient-specific determinants. Here, we combine in vitro and in silico methods, in order to characterize the critical molecular pathways that drive the chronopharmacology of irinotecan, a topoisomerase I inhibitor with complex metabolism and known activity against colorectal cancer. Large transcription rhythms moderated drug bioactivation, detoxification, transport, and target in synchronized colorectal cancer cell cultures. These molecular rhythms translated into statistically significant changes in pharmacokinetics and pharmacodynamics according to in vitro circadian drug timing. The top-up of the multiple coordinated chronopharmacology pathways resulted in a four-fold difference in irinotecaninduced apoptosis according to drug timing. Irinotecan cytotoxicity was directly linked to clock gene BMAL1 expression: The least apoptosis resulted from drug exposure near BMAL1 mRNA nadir (P < 0.001), whereas clock silencing through siBMAL1 exposure ablated all the chronopharmacology mechanisms. Mathematical modeling highlighted circadian bioactivation and detoxification as the most critical determinants of irinotecan chronopharmacology. In vitro-in silico systems chronopharmacology is a new powerful methodology for identifying the main mechanisms at work in order to optimize circadian drug delivery.

Systems Chronotherapeutics

“…TOP1 is also the target enzyme of the anticancer drug irinotecan, whose inhibition results in DNA breaks and cellular apoptosis. TOP1 mRNA and cytoplasmic protein levels displayed circadian rhythms in synchronized cell cultures of a human colorectal cancer model at confluence (Dulong et al, 2015). Although it has been shown that the protein dimer CLOCK-BMAL1 promotes TOP1 transcription, the circadian regulation of TOP1 expression remains still mostly unknown.…”

“…The chronoefficacy rhythms may at least partly originate from the circadian control of drug metabolism and distribution at the whole-body level, which influences tumor drug concentrations. In contrast, in vitro studies have shown that some cancer cell lines, such as human breast cancer MCF-7, have lost circadian rhythmicity in clock gene transcription (Xiang et al, 2012), whereas others such as human colon cancer Caco-2 at confluence have retained coordinated circadian expression (Dulong et al, 2015). However, most studies in vivo have shown the lack of consistent 24-hour patterns in experimental cancer tissues, especially for poorly differentiated carcinomas (Lévi et al, 2010).…”

“…Irinotecan PK-PD, toxicities, and efficacy displayed circadian rhythms in synchronized cell culture, in mice and in cancer patients (Ballesta et al, 2012; Dulong et al, 2015). Irinotecan is usually administered to cancer patients mostly in combination with other cytotoxic drugs and/or targeted molecules (Lévi et al, 2010; Sasine et al, 2010).…”

“…An in vitro study of irinotecan chronopharmacology led to the design of a cellular chronoPK-PD model (A) incorporating multitype experimental data, including the extra- and intracellular concentrations of active metabolite SN 38 and irinotecan-induced apoptosis after irinotecan exposure at three CTs (B–D) Dulong et al, 2015). This cellular investigation provided the basis for a mouse study and the development of a whole-body model of irinotecan chronoPK-PD explicitly incorporating the cellular model in relevant organs (E) (Ballesta et al, 2012).…”

“…Recent in vitro investigations have characterized irinotecan chronopharmacology at the molecular scale in human colorectal adenocarcinoma Caco-2 cells (Ballesta et al, 2011; Dulong et al, 2015). Experimental design was guided by an ODE-based physiologic PK-PD model that considered the following: 1) irinotecan passive diffusion through the cell membrane and bioactivation into SN38 through carboxylesterase; 2) SN38 detoxification into SN38G through UGT1As; 3) irinotecan, SN38, and SN38G efflux outside of the cells by ABC transporters.…”