Abstract:
Irinotecan (CPT-11), a chemotherapeutic ester prodrug, is associated with severe toxicity in up to 36% of patients. While genetic polymorphism in the UGT1A1 enzyme (responsible for drug elimination) is considered a reliable genetic risk factor for predicting toxicity, UGT1A1 genotype explains only about half of the variation in CPT-11 induced toxicity. The remainder was hence postulated to lie in the bioactivation pathways of CPT-11 to the active metabolite (SN-38). Human plasma had been proposed by Guemei et al. (2001) and Shingyoji et al. (2004) to be a surrogate tissue for predicting total in vivo CPT-11 bioactivation, which occurs predominantly in the liver. Hence, the two major human plasma esterases, butyrylcholinesterase (BChE) and paraoxonase (HuPON1), were studied for their involvement in the hydrolysis of CPT-11 to SN-38 by high-performance liquid chromatography (HPLC). The activity of CPT-11 bioactivation in human liver S9 fraction and plasma were also examined. Additionally, ‘total’ pan-esterase activity was determined by spectrophotometry using the non-specific esterase substrate, p-nitrophenol acetate (p-NPA). It was found that BChE catalysed the formation of SN-38 from CPT-11 with a specific activity of 1.72 ± 0.42 pmol/min/mg while HuPON1 was not active in this reaction. Quantification of p-NPA hydrolysis revealed that CPT-11 was a potent non-competitive inhibitor of HuPON1 rather than a substrate. Formation of SN-38 (at 160 μM CPT-11) proceeded in the presence of human plasma and liver S9 fraction at respective rates of 0.0115 ± 0.00 and 0.135 ± 0.008 pmol/min/mg. The ‘total’ pan-esterase (p-NPA) activity was 9.3-fold higher in human liver S9 than in plasma (0.57 ± 0.01 vs. 0.061 ± 0.001 μmol/min/mg, respectively). Thus, bioactivation of CPT-11 to SN-38 was 11.7-fold greater in liver compared to plasma and this appeared to relate with ‘total’ tissue pan-esterase activity. A molecular model of HuPON1 was optimised and CPT-11 docked into this structure. The position most favoured (lowest inter-atomic distances between CPT-11 and catalytic site residues) resulted in docking of CPT-11 with the lactone rather than the carbamate moiety closest to the active site. Cleavage of the carbamate moiety is required for activity and this may explain why CPT-11 appeared to be a non-competitive inhibitor rather than a substrate of HuPON1. The data in this thesis corroborates previously limited evidence for the catalytic role of human plasma BChE in CPT-11 bioactivation. Moreover, the data suggest a novel inhibitory role of CPT-11 on HuPON1 that could potentially limit bioactivation of CPT-11. These findings may be important both in explaining part of the variation in CPT-11 induced toxicity and in further evaluation of plasma as a surrogate tissue for predicting the metabolism of CPT-11 in vivo.