Investigations into the Mechanisms that Are Responsible for Reduction in Colonic SN-38 Exposure

Statement of the Problem: Irinotecan is used as a single agent or in combination with other drugs to treat metastatic colorectal cancer. However, its usage is largely limited as it exhibits late onset diarrhea in 30-40% of the patient population due to the high accumulation of SN-38 in colon. It has been hypothesized that multiple factors, such as higher biliary excretion of SN-38 and SN-38 glucuronide mediated through different efflux transporters, deglucuronidation of SN-38 glucuronide to SN-38 by bacterial β-glucuronidase secreted by commensal microbiota, generation of toxic active metabolite SN-38 from irinotecan mediated by intestinal carboxylesterse enzymes in gut and enterohepatic recycling of irinotecan and SN-38, etc., can contribute to the elevation of colonic SN-38 content resulting in severe late onset diarrhea. In this particular study, we aimed to investigate the mechanisms that are responsible to reduce the colonic content of SN-38. Towards our goal, three major specific aims were proposed to: (1) develop a modified in situ rat perfusion model for determination of the disposition of irinotecan, SN-38 and SN-38 glucuronide in major metabolic organs and bio-matrices; (2) investigate the potential of certain chemicals and natural products to reduce colonic exposure of SN-38 by inhibiting biliary and intestinal excretions of SN-38 and SN-38 glucuronide employing the modified in situ rat perfusion model; and (3) determine if the selected chemicals and natural products can decrease the colonic exposure of SN-38 by decreasing fecal content of SN-38 employing in vivo pharmacokinetic study. Methods: Surgeries and perfusion experiments were carried out on three different groups of wistar rats (n=4) at different dose of intravenous irinotecan (0.5, 5 and 50 mg/kg) for 150 minutes. Bile, perfusate, urine and plasma were collected at each 30 min interval. Irinotecan, SN-38 and SN-38 glucuronide concentrations from all the collected bio-matrices were quantified by our developed and validated LC-MS/MS assay. Chemical inhibition study using different chemical inhibitors of efflux transporters have been performed to explore the impacts on efflux transporters for the disposition of irinotecan. Traditional Chinese herbal formulations have been coadministered with irinotecan to observe if they can alter the disposition of irinotecan significantly. Lastly, in vivo rat pharmacokinetic study was conducted to evaluate the effectiveness of chemical inhibitor and Chinese herbal formulation to decrease the fecal content of SN-38 without altering the efficacy of chemotherapeutic treatment. Results: Dose response study using the modified in situ rat perfusion model revealed that predominant excretion of irinotecan is biliary route (45 to 60%) in comparison to urinary (4 to 15%) and intestinal excretion (3 to 10%). In addition, the high steady state concentrations of irinotecan and its metabolites in bile and urine compared to their plasma concentrations (5 to 5000 fold) displayed that the excretion of these compounds at different metabolic organs was mainly governed by predominant efflux transporters. Chemical inhibition study using the same perfusion model demonstrated the possible involvement of MATE-1 transporter alongwith P-gp and MRP2 on the biliary and renal excretions of irinotecan. Inhibition study using Chinese herbal medicine indicated that Xiao Chao Hu Tang (XCHT) could decrease the biliary and intestinal excretions of SN-38 and SN-38 glucuronide by 60 -90%. The in vivo rat pharmacokinetic study using cimetidine and XCHT revealed that both XCHT and cimetidine could decrease the fecal content of SN-38 by 40-50% without altering the efficacy of the chemotherapeutic treatment. Conclusions: In this particular study, we demonstrated that biliary route is the predominant excretion route for irinotecan, SN-38 and SN-38 glucuronide compared to other excretion routes. In addition, effluxes of irinotecan and its metabolites in biliary and urinary routes, respectively, are mainly governed by efflux transporters. Chemical inhibition study utilizing the modified in situ rat perfusion model demonstrated the possible involvement of MATE-1 transporter on the disposition of irinotecan through biliary and urinary routes. We have also demonstrated using the modified perfusion model as a screening tool that Chinese herbal medicine Xiao Chao Hu Tang (XCHT) has the ability to decrease the biliary and intestinal excretions of SN-38 and SN-38 glucuronide. Lastly, to the best of our knowledge, this is the first time cimetidine and Chinese herbal formulation XCHT have displayed their abilities to decrease fecal excretion of SN-38 which may lead to amelioration of irinotecan induced late onset diarrhea. However, further investigations are warranted to prove their effectiveness in alleviating irinotecan toxicity using a preclinical diarrhea model.