Effects of Inflammation and Soy Isoflavones on Irinotecan Pharmacokinetics and Toxicity
Chityala, Pavan Kumar
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Colorectal cancer (CRC) is a form of cancer that starts in the colon or rectum. According to the GLOBOCON statistics, there are over 1.8 million new colorectal cancer cases and 881,000 deaths are estimated to occur in 2018. Colorectal cancer is also the 3rd most common type of cancer diagnosed in both men and women in the USA and worldwide. Metastatic colorectal cancer (mCRC) is an advanced stage where the cancer cells from the colon or rectal tumor detach and spread to other parts of the body, such as the liver or lungs through the bloodstream. Irinotecan is an intravenous (i.v.) infusion chemotherapy drug that was approved by FDA in 1996 and it is a topoisomerase I inhibitor indicated for first-line therapy in combination with 5-fluorouracil and leucovorin for patients with metastatic colorectal cancer and Patients with metastatic colorectal cancer whose disease has recurred or progressed after initial fluorouracil-based therapy. Irinotecan belongs to a class of topoisomerase I inhibitors and it is a prodrug to its cytotoxic metabolite SN-38 (7-ethyl-10-hydroxy-camptothecin), which is about 100-1000-fold more cytotoxic than irinotecan. Although irinotecan is a very potent chemotherapy drug, elevated blood concentrations of its active metabolite, SN-38 leads to increased gastrointestinal (GI) toxicity and diarrhea in patients. Irinotecan is metabolized by Cyp3a4, carboxylesterase and Ugt1a1 enzymes and these enzymes are known to be downregulated during inflammation. Therefore, there is a possibility of reduced metabolism and clearance of irinotecan during inflammation, which may lead to increased toxicity in patients. Along with diarrhea, irinotecan chemotherapy is also associated with hepatotoxicity in patients undergoing liver resection for colorectal liver metastasis. The irinotecan associated hepatotoxicity is in the form of steatosis or steatohepatitis. These toxicities result in life-threatening complications in patients and reduce the use of irinotecan as a chemotherapeutic agent. Therefore, there is an urgency to better understand the effects of inflammation on irinotecan PK and to develop new interventions to prevent diarrhea and steatosis associated with irinotecan. To achieve these goals, we pursued the following 3 aims. Aim 1: To determine the effects of inflammation on irinotecan pharmacokinetics and the development of a best-fit PK model. In this aim, we investigated the effects of inflammation on the pharmacokinetics (PK) of irinotecan (CPT-11) and its active metabolite, SN-38. Mice were i.p.-injected with either saline or lipopolysaccharide (LPS) to induce inflammation. After 16 h, irinotecan was administered orally. Blood was collected from the tail vein of mice from 0-24 h after dosing. Concentrations of irinotecan, SN-38 and SN-38G were analyzed using LC-MS/MS. The AUC, Cmax, and tmax were derived using WinNonlin® 5.2. A PK model was developed using Phoenix NLME® to describe the PK of irinotecan and SN-38 during inflammation. Results indicated a significant increase in the blood concentrations of irinotecan and SN-38 in mice during inflammation. The AUC of irinotecan and SN-38 in the LPS group were 2.6 and 2-folds, respectively, of those in control saline-treated mice. The Cmax of irinotecan and SN-38 in LPS treated mice were 2.4 and 2.3-folds of those in saline-treated mice. The PK model was successfully developed and validated. The best-fit plots of individual PK analysis showed a good correlation between observed and predicted concentrations of irinotecan and SN-38. Together, this study reveals that SN-38 concentrations are elevated during inflammation, which may increase the GI toxicity and diarrhea in patients who receive irinotecan; and the developed PK model can quantitatively describe the PK of irinotecan and SN-38 during inflammation. Aim 2: To determine the role of TLR2 in irinotecan-induced diarrhea and steatosis. Evidence from the literature strongly suggests that toll-like receptors (TLRs), especially TLR2 is involved in the pathogenesis of gastrointestinal and hepatotoxicity. Therefore, in this aim, we investigated the role of TLR2 in irinotecan-induced diarrhea and steatosis. Specifically, we used TLR2 WT and KO mice and treated with either saline (control) or irinotecan (treatment group) for 8 days and sacrificed the mice on day 9. To measure the extent of GI toxicity in TLR2 WT and KO mice, we conducted body weight measurements, the incidence of diarrhea, blood concentration analysis, and histological analysis of intestinal damage. Similarly, to determine the role of TLR2 in irinotecan-induced hepatotoxicity, H&E and Oil-Red-O histological analysis of liver sections, gene expression of enzymes and pro-inflammatory cytokines, and enzyme activity assays were conducted. The results indicate that the TLR2 KO mice showed significantly lower damage both in terms of diarrhea and steatosis after irinotecan treatment. Together, the results indicate a key role of TLR2 in the pathogenesis of irinotecan-induced toxicity. In the future, pharmacological inhibition of TLR2 may help in healing irinotecan-induced GI and hepatotoxicity. Aim 3: To determine the effects of soy isoflavones on irinotecan-induced diarrhea and steatosis. Soy isoflavones have been shown to have beneficial effects on both gastrointestinal (GI) and hepatotoxicity caused by chemotherapy. Here, we studied the effects of NovaSoy, a soy-based isoflavone concentrate with a total isoflavone of 40% (20% genistein, ~18% daidzein, ~2% other isoflavones), on irinotecan-induced diarrhea and steatosis in mice. Results showed that the mice treated with Novasoy and irinotecan (NS/IRI) showed significantly less bodyweight loss and incidence of diarrhea compared to mice treated with saline and irinotecan (Sal/IRI). Histopathological analysis of liver and intestine sections revealed a less severe fat accumulation (<10% steatosis) and intestinal damage in NS/IRI group. Analysis of liver and intestine concentrations showed less accumulation of SN-38 concentrations in the NS/IRI group compared to the Sal/IRI group. Enzyme activity assays exhibited a significant increase in the activity of carboxylesterase, whereas ugt1a1 activity is reduced. Toxicokinetic (TK) studies showed no significant changes in blood concentrations of irinotecan and metabolites. Together, the results suggested that soy isoflavone treatment could delay the incidence of diarrhea and steatosis caused by irinotecan and can be used as an intervention to reduce the toxicities associated with irinotecan.