Discovery of Novel Selective Inhibitors of Human Intestinal Carboxylesterase for the Amelioration of Irinotecan-Induced Diarrhea: Synthesis, Quantitative Structure-Activity Relationship Analysis, and Biological Activity

Abstract

The dose-limiting toxicity of the highly effective anticancer agent 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (irinotecan; CPT-11) is delayed diarrhea. This is thought to be caused by either bacteria-mediated hydrolysis of the glucuronide conjugate of the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) or direct conversion of CPT-11 to SN-38 by carboxylesterases (CE) in the small intestine. After drug administration, a very high level of CPT-11 is present in the bile; this is deposited into the duodenum, the region of the gut with the highest levels of CE activity. Hence, it is likely that direct conversion of the drug to SN-38 is partially responsible for the diarrhea associated with this agent. In an attempt to ameliorate this toxicity, we have applied Target-Related Affinity Profiling to identify novel CE inhibitors that are selective inhibitors of the human intestinal enzyme (hiCE). Seven inhibitors, all sulfonamide derivatives, demonstrated greater than 200-fold selectivity for hiCE compared with the human liver CE hCE1, and none was an inhibitor of human acetylcholinesterase or butyrylcholinesterase. Quantitative structure-activity relationship (QSAR) analysis demonstrated excellent correlations with the predicted versus experimental K i values (r 2 ϭ 0.944) for hiCE. Additionally, design and synthesis of a tetrafluorine-substituted sulfonamide analog, which QSAR indicated would demonstrate improved inhibition of hiCE, validated the computer predictive analyses. These and other phenyl-substituted sulfonamides compounds are regarded as lead compounds for the development of effective, selective CE inhibitors for clinical applications.

Intestinal glucuronidation protects against chemotherapy-induced toxicity by irinotecan (CPT-11)

“…Human intestinal CES2 exhibits a higher affinity and velocity than human hepatic CES1, demonstrating the significant ability to hydrolyze CPT-11 (10,26,27). Intestinal selective CES inhibitors have been suggested as a new clinical target to minimize CPT-11-induced late diarrhea (35,36). However, we note that Ces1/2 gene expression is already greatly reduced in tumor tissue, indicating that further reduction in activity would minimize therapeutic efficacy.…”

Intracellular inhibition of carboxylesterases by benzil: modulation of CPT-11 cytotoxicity

“…In contrast, the hiCE-specific inhibitor 2 (25) or the benzil analogues 3 or 4 (26) did not significantly reduce the levels of intracellular fluorescence (Fig. 3D -F).…”

“…Because all of the selective carboxylesterase inhibitors that have been recently identified act in a reversible fashion (25,26,31), a novel in situ assay was required to ensure that enzyme inhibition was occurring intracellularly. This was necessary because, during the preparation of cell extracts to measure carboxylesterase activity, the concentration of inhibitor would be vastly diluted, and hence, inhibition of carboxylesterase would not be apparent.…”

“…However, the doselimiting toxicity for CPT-11 is diarrhea, and because high levels of carboxylesterase are expressed in the small intestine (5,24), we have proposed that this toxicity may be due to direct activation of CPT-11 to SN-38 in gut epithelium. Based on this hypothesis, we have identified a series of specific carboxylesterase inhibitors (25) that may be used in conjunction with CPT-11 to ameliorate the diarrhea that occurs 48 to 96 hours following drug administration. We envisage that the inhibitor could be administered 8 to 96 hours after CPT-11, with the goal of inhibiting the intracellular conversion of CPT-11 to SN-38 in the intestinal cells, thereby reducing both the levels of active metabolite in the gut and this toxicity associated with CPT-11 therapy.…”

“…In this study, we evaluated the ability of these inhibitors to accumulate within human tumor cells and directly inhibit carboxylesterases. Because these inhibitors act in a partially competitive and reversible fashion (25,26), we have developed a novel assay that examines enzyme inhibition in situ.…”