A Mechanistic Study on Reduced Toxicity of Irinotecan by Coadministered Thalidomide, a Tumor Necrosis Factor- Inhibitor

Dose-limiting diarrhea and myelosuppression compromise the success of irinotecan (7-ethyl-10-[4-[1-piperidino]-1-piperidino]carbonyloxycamptothecin) (CPT-11)-based chemotherapy. A recent pilot study indicates that thalidomide attenuates the toxicity of CPT-11 in cancer patients. This study aimed to investigate whether coadministered thalidomide modulated the toxicities of CPT-11 and the underlying mechanisms using several in vivo and in vitro models. Diarrhea, intestinal lesions, cytokine expression, and intestinal epithelial apoptosis were monitored. Coadministered thalidomide (100 mg/kg i.p. for 8 days) significantly attenuated body weight loss, myelosuppression, diarrhea, and intestinal histological lesions caused by CPT-11 (60 mg/kg i.v. for 4 days). This was accompanied by inhibition of tumor necrosis factor, interleukins 1 and 6 and interferon, and intestinal epithelial apoptosis. Coadministered thalidomide also significantly increased the systemic exposure of CPT-11 but decreased that of SN-38 (7-ethyl-10-hydroxycampothecin). It significantly reduced the biliary excretion and cecal exposure of CPT-11, SN-38, and SN-38 glucuronide. Thalidomide hydrolytic products inhibited hydrolysis of CPT-11 in rat liver microsomes but not in primary rat hepatocytes. In addition, thalidomide and its major hydrolytic products, such as phthaloyl glutamic acid (PGA), increased the intracellular accumulation of CPT-11 and SN-38 in primary rat hepatocytes. They also significantly decreased the transport of CPT-11 and SN-38 in Caco-2 and parental MDCKII cells. Thalidomide and PGA also significantly inhibited P-glycoprotein (PgP/MDR1), multidrug resistance-associated protein (MRP1)and MRP2-mediated CPT-11 and SN-38 transport in MDCKII cells. These results provide insights into the pharmacodynamic and pharmacokinetic mechanisms for the protective effects of thalidomide against CPT-11-induced intestinal toxicity. Irinotecan (CPT-11, 7-ethyl-10-[4-[1-piperidino]-1-piperidino]carbonyloxycamptothecin), a potent DNA topoisomerase I inhibitor, has been widely used for the treatment of advanced colorectal cancer as a first-line therapy in combination with 5-fluororacil and other malignancies (Pizzolato and Saltz, 2003). As a prodrug, CPT-11 is activated by carboxylesterases (CEs) to SN-38 (7-ethyl-10-hydroxycampothThis work was supported by National University of Singapore Academic Research Funds R-148-000-054-112, R-148-000-047-101, R-148-066-112, and R-148-000-067-112. 1 Recipients of the Ph.D. scholarship of the National University of Singapore. These authors contributed equally to this work. 2 Recipient of the postdoctoral fellowship of the National University of Singapore. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.103606. ABBREVIATIONS: CPT-11, irinotecan, 7-ethyl-10-[4-[1-piperidino]-1-piperidino]carbonyloxycamptothecin; CE, carboxylesterase; SN-38, 7-ethyl10-hydroxycampothecin; SN-38G, SN-38 glucuronide; AUC, area under the concentration time curve; Mdr, multidrug resistance; MRP, multidrug resistance-associated protein; TNF, tumor necrosis factor; RT-PCR, reverse transcription polymerase chain reaction; IL, interleukin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; TEER, transepithelial electric resistance; PGA, phthaloyl glutamic acid; IFN, interferon; PgP, P-glycoprotein; MK-571 (or L-660,711), 3-[[[3-[2-(7-chloro-2-quinolinyl)-(E)-ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid; BNPP, bis(p-nitrophenyl) phosphate sodium salt; UGT, uridine diphosphate glucuronosyltransferase; i.p., intraperitoneal; i.v., intravenous; HPLC, high-performance liquid chromatography; DMSO, dimethyl sulfoxide; MDCK, Madin-Darby canine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide kidney; CL, clearance; AP, apical; BL, basolateral; HBSS, Hanks’ balanced salt solution; ANOVA, analysis of variance; RT, reverse transcription; PCR, polymerase chain reaction; Brij 58, polyoxyethylene 20 cetyl ether; Brij 38, polyoxyethylene 23 cetyl ether. 0022-3565/06/3191-82–104$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 319, No. 1 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 103606/3138509 JPET 319:82–104, 2006 Printed in U.S.A. 82 at A PE T Jornals on Sptem er 6, 2017 jpet.asjournals.org D ow nladed from ecin), which is 100to 1000-fold more cytotoxic than CPT-11. SN-38 is further converted to its glucuronide (SN-38G) by uridine diphosphate glucuronosyltransferase (UGT) 1A isoforms (Fig. 1) (Gupta et al., 1994). SN-38G can be converted back to SN-38 by intestinal microbial -glucuronidase and undergo enterohepatic recycling (Mathijssen et al., 2001). A second less important metabolic pathway of CPT-11 is cytochrome P450 (CYP3A)-catalyzed bipiperidine side chain oxidation, giving rise to 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin and 7-ethyl-10[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin (Gupta et al., 1994). Biliary excretion is the major elimination route for CPT-11 and its major metabolites, with the urinary excretion being a less important pathway. In vitro and animal studies have indicated that CPT-11, SN-38, and SN-38G are actively transported by P-glycoprotein (PgP/MDR1), multidrug resistance-associated proteins (MRP1, MRP2, and MRP4), and the breast cancer resistance protein (Chu et al., 1998; Tian et al., 2005). However, dosing-intensified regimens of CPT-11-based chemotherapy are often limited by severe myelosuppression and intestinal mucositis characterized by severe diarrhea. In Fig. 1. The metabolic scheme of CPT-11. APC, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin; NPC, 7-ethyl-10-[4(1-piperidino)-1-amino]-carbonyloxycamptothecin; CYP3A, cytochrome P450 3A. Thalidomide Reduced the Toxicity of CPT-11 83 at A PE T Jornals on Sptem er 6, 2017 jpet.asjournals.org D ow nladed from particular, the incidence of grade 3 or 4 diarrhea was up to 40% of patients treated with CPT-11, which may cause dehydration, renal failure, and thromboembolic events (Rothenberg et al., 1996). High-dose loperamide is recommended to manage CPT-11-induced diarrhea (Benson et al., 2004). However, a number of patients do not respond to this agent. Therefore, new and effective agents are needed to alleviate CPT-11-induced intestinal mucositis. The increasing understanding of the molecular events that lead to chemotherapy-induced intestinal mucosal injury may allow us to identify new approaches to manage chemotherapy-induced intestinal damage. The mucosal lesions seem to be associated with intestinal exposure to these cytotoxic drugs, which induce epithelial apoptosis, decreased crypt cell renewal, complicated inflammatory responses, and destruction of the mucosal architecture (Thompson, 1995). Cytotoxic drugs, including CPT-11, induce severe intestinal inflammatory responses with hypersecretion of proinflammatory cytokines, including tumor necrosis factor (TNF)and interleukins (ILs) (Sonis, 2004). TNF, mainly produced by activated macrophages, is a key cytokine in initiation of mucosal immunoinflammatory responses and lesions. It is a critical cytokine that could orchestrate inflammatory responses by activating a wide range of cells, including neutrophils, macrophages, and natural killer cells. Activation of these cells in turn induces the production of proinflammatory cytokines, such as IL-1 and IL-6, and up-regulation of adhesion molecules on cell surface. There exists a close interaction between intestinal epithelial cellular apoptosis and intestinal proinflammatory cytokine expression. TNFhas been proven to play an essential role in regulating intestinal epithelial cell apoptosis and/or survival during chronic inflammation (Marini et al., 2003). Therefore, TNFplays a critical role in initiation of chemotherapy-induced primary mucosal damage responses, including early lesion to connective tissue and endothelium, reduction of epithelial oxygenation, and ultimately, epithelial basal-cell death and injury (Sonis, 2004). Thalidomide [2-(2,6-dioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione], a derivative of glutamic acid, has been widely used in the treatment of various inflammatory and autoimmune disorders, as well as a variety of tumors because of its immunomodulating and antiangiogenic effects (Franks et al., 2004). It has shown inhibitory effects on TNFproduction by enhancing the degradation of TNFmRNA (Moreira et al., 1993). Thalidomide is often used in combination with other cytotoxic agents aimed at generating additive/synergistic activity, alleviating toxicity, and overcoming tumor resistance (Catley et al., 2005). A recent pilot clinical study demonstrated that concomitant thalidomide almost eliminated CPT-11-induced gastrointestinal toxicities, including nausea and diarrhea (Govindarajan et al., 2000). However, the mechanisms underlying this striking protective effect are not clear. Spontaneous hydrolysis is the major elimination pathway of thalidomide, whereas CYP2C-mediated metabolism plays only a minor role in its elimination (Ando et al., 2002). In the present study, we investigated whether combination of thalidomide modulated the toxicities of CPT-11 using a rat model and explored the underlying pharmacokinetic and pharmacodynamic mechanisms using a variety of in vivo and in vitro models. The rats were chosen in this study because of the similarity in the metabolic and disposition pathways of CPT-11 and SN-38 between rats and humans (Yang et al., 2005a) and because the rat is relatively sensitive to CPT-11 and has been widely used in the pharmacological and toxicological studies of CPT-11. In an established rat toxicity model induced by CPT-11, the effects of coadministered thalidomide on the intestinal and blood toxicities, expression of TNFand other proinflammatory cytokines, including IL-1 , IL-2, IL-6, and interferon (IFN), and intestinal epithelial apoptosis were monitored. The effects of coadministered thalidomide on the plasma and intestinal (cecal) pharmacokinetics and biliary excretion of CPT-11 and its major metabolites were also investigated. Rat liver microsomes were used to study the possible metabolic interactions between CPT-11 and thalidomide and its hydrolytic products. Herein we also included freshly isolated primary rat hepatocytes to study the effects of thalidomide, its total hydrolytic products, and one of its major hydrolytic products, phthaloyl glutamic acid (PGA), on the metabolism and intracellular accumulation of CPT-11 and SN-38. In addition, the effects of thalidomide, its total hydrolytic products, and PGA on the transport in Caco-2 cells and MDCKII cells overexpressing MDR1, MRP1, or MRP2 were investigated. Moreover, the effects of thalidomide, its total hydrolytic products, and PGA on rat plasma protein binding of CPT-11 and SN-38 were examined to identify possible displacement of the substrates from the binding sites of plasma proteins. As such, the roles of both pharmacodynamic and pharmacokinetic components in the protective effect of thalidomide against CPT-11-induced toxicity were comprehensively explored in the present study. Materials and Methods Chemicals and Reagents. Camptothecin (CPT) analogs, including irinotecan (CPT-11), CPT, and SN-38 in lactone form [all compounds with a purity 99.0% as determined by high-performance liquid chromatography (HPLC)], were purchased from SinoChem Ningbo Import and Export Co. (Ningbo, China). An injectable formulation of CPT-11 was prepared as described previously (Kurita et al., 2003). Sodium 1-heptane-sulfonate, lyophilized type IX-A -glucuronidase (from Escherichia coli, activity 1,724,400 U/g solid form), probenecid, nifedipine, Brij 58, Brij 38, verapamil, probenecid, bilirubin, bis(p-nitrophenyl) phosphate sodium salt (BNPP), uridine diphosphate glucuronic acid (UDPGA), Dulbecco’s modified Eagle’s medium, D-sorbitol, and D-lactic acid were all purchased from SigmaAldrich Chemical Co. (St. Louis, MO). Fetal bovine serum was obtained from Hyclone Lab Inc. (Logan, UT). Thalidomide (purity 99.0%, determined by HPLC) was provided by Celgene Co. (Summit, NJ). The leukotriene D4 receptor antagonist 3-[[[3-[2-(7chloro-2-quinolinyl)-(E)-ethenyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid (MK-571 or L-660,711) was a gift from Dr. Ford Hutchinson (Merck Frosst Canada Ltd., Kirkland, QC, Canada). PGA, a hydrolysis product of thalidomide, with a purity 99.0% as determined by HPLC was a gift provided by Dr. Tom Pui-Kai Li (School of Pharmacy, Ohio State University, Cincinnati, OH). The control (wild-type) MDCKII cell line with empty vector and its human MDR1-, MRP1-, or MRP2-recombinantly transfected derivatives, MDR1-MDCKII, MRP1-MDCKII, and MRP2-MDCKII cells, were obtained as a kind gift from Professor Piet Borst (Netherlands Cancer Institute, Amsterdam, The Netherlands). Mouse monoclonal antibody to MDR1 (C219), mouse monoclonal antibody against MRP1 (m6), and mouse monoclonal [M2III-5] antibody to MRP2 were purchased from Abcam Co. (Cambridge, UK). The stable overexpression of MDR1, MRP1, and MRP1 in transfected cells were monitored and confirmed every two to four passages by Western blotting analysis. The water was purified by a Milli Q water purification System (Millipore, Bedford, MA). All other chemicals 84 Yang et al. at A PE T Jornals on Sptem er 6, 2017 jpet.asjournals.org D ow nladed from were of analytical grade or HPLC grade obtained from commercial