The Mechanism for the Inhibition of Acetylcholinesterases by Irinotecan (CPT-11)

Abstract

Irinotecan (CPT-11) is an anticancer drug that occasionally produces acute cholinergic side effects. Preliminary findings suggest that these are mediated through the inhibition of acetylcholinesterase (AChE). In this study, the inhibition of various AChEs by CPT-11 was studied. The lactone form of CPT-11 resulted in apparent noncompetitive inhibition of electric eel and both human recombinant and erythrocyte AChE with K(i) values of 0.065, 0.19, and 0.29 microM, respectively. The carboxylate form of CPT-11 was approximately 10 times less potent. Apparent noncompetitive inhibition of AChE may arise through several mechanisms, and those relevant to CPT-11 were identified from key experimental findings. First, the inhibition by CPT-11 was found to be instantly reversible in dilution studies. Second, incubation of the enzyme with CPT-11 before the introduction of neostigmine protected the enzyme from inactivation. Third, regeneration of the active enzyme after preincubation with neostigmine was totally suppressed by the addition of 2 microM CPT-11, indicating that CPT-11 is a potent inhibitor of decarbamoylation and, by inference, deacylation. Additional experiments with tacrine revealed functional differences between these two inhibitors. Also, preliminary molecular modeling of the interaction between AChE and CPT-11 indicated that the latter does not bind at the same site as tacrine. Displacement studies with the peripheral site-specific ligand, propidium, confirmed that CPT-11 binds at this site. The rapid reversibility of the inhibition of AChE by CPT-11 and the lower activity of the carboxylate form are likely reasons for the transient nature of the cholinergic toxicity observed clinically.

Comment on a Published Paper

“…As pointed out by the authors, these data are not consistent with our own results (Dodds & Rivory, 1999), and those of others using very similar systems in which values of 0.2 mM for the IC 50 have been consistently reported (Kawato et al, 1993;Rivory et al, 1996;Morton et al, 1999). In our most recent study, we demonstrated that CPT-11 is a potent inhibitor of AChE at clinically relevant concentrations and revealed its mechanism of inhibition as being instantly reversible and apparently non-competitive (Dodds & Rivory, 1999). On reading the paper by Blandizzi et al (2001), we could not explain the reported discrepancy.…”

“…However, despite the e orts made to reproduce our experiments on the in vitro acetylcholinesterase (AChE) assay, we note that these authors still fail to propose any convincing explanation accounting for the discrepancy between our data (Blandizzi et al, 2001) and their repeated observations (Dodds & Rivory, 1999). Therefore, it is conceivable that in vitro assays, carried out on puri®ed preparations of AChE extracted from various sources, do not o er su cient levels of reliability to allow a clear assessment of the putative anti-AChE properties of irinotecan.…”

“…In conclusion, much work remains to be done, at experimental level, to clarify the di erences of Dodds and Rivory (1999) data on irinotecan-induced inhibition of AChE, compared with our results. However, in order to better elucidate the pathophysiological mechanisms which subserve the cholinergic side e ects of irinotecan, we do not believe that in vitro pharmacological assays of AChE can be regarded as critical tests until they are uncon®rmed on functional basis by means of more integrated methodological procedures.…”

“…In particular, Riddles et al (1979) have recommended that reactions be carried out at a pH of 7.3 and at 258C (rather than pH 8.0 and 378C) to ensure the maximum stability of the 5,5'-dithio-bis(2-nitrobenzoic acid) during incubation. Also, CPT-11, as with all camptothecins, hydrolyses in a rapid but reversible reaction to open-ring carboxylate forms with very di erent pharmacological properties, including their ability to inhibit AChE (Dodds & Rivory, 1999). We have previously shown the lactone form of CPT-11 to be *10 fold more potent at inhibiting both human and electric eel AChE (Dodds & Rivory, 1999).…”

“…Also, CPT-11, as with all camptothecins, hydrolyses in a rapid but reversible reaction to open-ring carboxylate forms with very di erent pharmacological properties, including their ability to inhibit AChE (Dodds & Rivory, 1999). We have previously shown the lactone form of CPT-11 to be *10 fold more potent at inhibiting both human and electric eel AChE (Dodds & Rivory, 1999).…”