Proteomics‐Informed Scaling of Irinotecan Metabolism, Transport, and Gut Microbial Activation Explain its Intestinal Toxicity after Intravenous Dose

Abstract

Objective Anticancer drug, irinotecan causes dose-limiting intestinal side effects after an intravenous dose. We hypothesized that differential tissue abundance of irinotecan metabolizing enzymes, transporters, and gut microbial activation can explain intestinal accumulation of the toxic metabolite, SN-38, and its toxicity. We integrated i) in vitro metabolism and transport data of irinotecan and its metabolites, ii) ex vivo microbiome data of SN-38 activation, and iii) tissue quantitative proteomics data of the relevant enzymes and transporters. Methods We first characterized the metabolism and transport kinetics of irinotecan using in vitro systems (recombinant proteins, cells, or membrane vesicles) expressing carboxylesterases (CESs), uridine glucuronosyltransferases (UGTs) and organic anion transporting polypeptides (OATPs), P-glycoprotein (P-gp), breast cancer resistant protein (BCRP), and multidrug resistance-associated proteins (MRPs). Then, using tissue proteomics data, we estimated the tissue-specific fractional contributions of individual enzymes and transporters. The tissue-specific metabolism was verified by performing incubations with tissue S9 fractions and hepatocytes. Finally, we estimated SN-38 reactivation by gut microbial β-glucuronidases in human fecal homogenates. Results Irinotecan and its major metabolites, SN-38, and SN-38-glucuronide (SN-38-G) were substrates of multiple uptake and efflux transporters, with OATP1B1, P-gp, and BCRP being the predominant contributors. Integration of the in vitro kinetics data with the tissue enzyme and transporter proteomics predicted that CES mediated hydrolysis of irinotecan is the rate-limiting process in the liver, whereas the toxic metabolite, SN-38 is rapidly neutralized by its hepatic glucuronidation. In contrast, the poor glucuronidation rate as compared to the efficient formation by CES2 in the enterocytes is the key mechanism of SN-38 accumulation in the intestine (Figure 1). The biliary efflux and OATP2B1 mediated enterocyte uptake are the potential mechanisms that synergize buildup of SN-38 into the enterocytes, whereas intestinal P-gp likely facilitate detoxification of the enterocytes. The higher SN-38 concentration in the intestine can be further nourished by β-d-glucuronidases leading to the higher exposure of the toxic metabolite. Conclusions: CES2, UGT1As, OATP2B1, P-gp, BCRP, and gut microbiome play crucial roles in regulating intestinal exposure and tissue-specific intestinal toxicity of irinotecan. Understanding the quantitative significance of the key disposition processes of irinotecan can be leveraged to alleviate the intestinal side effects. The proteomics-informed translational approach used here can be applied to other drugs involving complex disposition mechanisms.