Comment on a Published Paper

In a recent study, Blandizzi et al. (Br. J. Pharmacol., 132: 73 – 84, 2001) reported that the adverse cholinergic effects of the chemotherapeutic agent irinotecan (CPT-11) were unlikely to be mediated by the inhibition of acetylcholinesterase (AChE). This observation was based on results of in vitro assays performed on AChE from two sources (human erythrocyte and electric eel). They reported only modest inhibition of AChE even at the highest concentration of CPT-11 used (100 μM). 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 μM for the IC50 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. We therefore set out to replicate exactly the system used by these authors to investigate this further, given that even minor experimental differences could impact on the results obtained. In particular, Riddles et al. (1979) have recommended that reactions be carried out at a pH of 7.3 and at 25°C (rather than pH 8.0 and 37°C) 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 different 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). We repeated the experiments of Blandizzi et al. (2001) using both identical conditions and those we have used in the past. The total assay volume was 3.2 ml and included ATChI as substrate (at same concentration) and 3 u ml−1 of AChE (electric eel). The final concentrations of CPT-11 (lactone) investigated ranged from 0 – 0.78 μM and these were either spiked into the incubation mix immediately prior to the reaction or pre-incubated with AChE for 20 min (as per Blandizzi et al., 2001). The concentration resulting in a 50% reduction of enzyme activity (IC50) was estimated from the resulting plots. All assays were performed in triplicate and expressed as mean±s.d. As summarized in Table 1, there were subtle differences in the potency of the inhibition between the conditions used. At the lower temperature, CPT-11 was a more potent inhibitor. Also, pre-incubation for 20 min resulted in less inhibition of AChE, consistent with the hydrolysis of the lactone form under these conditions (Rivory et al., 1994). These effects, when combined, led to an ∼5 fold loss in potency at pH 8. However, they do not explain the >1000 lower inhibitory capacity of CPT-11 in the hands of Blandizzi et al. (2001). In contrast, the IC50 obtained by Blandizzi et al. (2001) for physostigmine in the same system is compatible with the literature. Table 1 Interaction of CPT-11 (lactone) with AChE under different assay conditions In conclusion, we are unable to explain the results of Blandizzi et al. (2001). Although we show that the classical experimental variables (temperature, pH and time of incubation) can all impact on the potency of the inhibition of AChE by CPT-11, there must remain additional and unknown factors that dramatically affect the potency of CPT-11. These factors merit further elucidation.