Inhibition of acetylcholinesterase by the anticancer prodrug CPT-11
Introduction
The development of effective anticancer agents in recent years has been limited, in part, due to the failure to identify agents whose spectrum of activity, and/or antitumor efficacy is any better than agents that are currently available. With the notable exception of Gleevec, virtually all chemotherapeutic agents in clinical use are non-selective poisons, resulting in toxicity to normal tissues as well as to tumor cells. However, within these classes of compounds, there are some very effective drugs whose toxicity can be managed by extensive supportive care. This includes the camptothecins, of which the parent compound (camptothecin (CPT)) was originally identified as potential antitumor agent in 1966 [1]. Subsequent drug development has produced two highly effective CPT analogs, topotecan and CPT-11 that are currently used for the treatment of ovarian and colon cancer, respectively [2], [3], [4], [5]. These drugs have shown dramatic responses in the treatment of a variety of solid tumors. For example in a Phase I trial with CPT-11 in pediatric patients with a spectrum of tumor histiotypes, responses were observed in 21 out of 23 individuals [6]. Such efficacy in these small scale trials, that a designed to assess safety and toxicity, are very uncommon.
As with all cytotoxic chemotherapeutic agents, side effects occur following CPT-11 administration. These include a cholinergic syndrome that occurs within 1 h of drug infusion resulting in lacrimation, miosis, increased salivation diaphoresis, flushing, rhinitis and intestinal hyperperistalsis that usually leads to diarrhea [7], [8], [9], [10]. These toxicities can be rapidly alleviated with atropine. The dose limiting toxicity for CPT-11 however is delayed diarrhea that occurs 48–96 h following drug administration. This is thought to arise from two different mechanisms. Firstly, since CPT-11 is eliminated via the bile into the duodenum, and this region of the gut has high levels of carboxylesterase activity, direct conversion of the drug to SN-38 will result in direct injury to the gut epithelia and hence diarrhea [11], [12]. Secondly, the SN-38 is subject to glucuronidation in the liver and also expelled into the intestine though the bile. Glucuronidases present within the bacterial flora of the gut can the convert the glucuronide back to SN-38, resulting in direct toxicity to dividing cells within the crypts and villi [13], [14], [15]. Measures to prevent both of these mechanisms and hence reduce the delayed diarrhea associated with CPT-11 are currently being explored [12], [13].
Since a cholinergic syndrome rapidly occurs following high dose i.v. CPT-11 administration that can be ameliorated by atropine, this suggests that the drug directly interacts with AChE. To confirm if this was the case, and to identify the domains within the drug that might be responsible for AChE inhibition, we have performed a series of detailed kinetic analyses with CPT-11 and a variety of analogs. These studies demonstrate that the 4-piperidinopiperidino moiety present at the 10-position of the molecule (Fig. 1) is primarily responsible for enzyme inhibition. In addition, we demonstrate that butyrylcholinesterase (BChE) can convert CPT-11 to SN-38, and may play a role in the production of the active metabolite in vivo.
Section snippets
Enzymes and drugs
hAChE, Electrophorus electricus AChE (EeAChE) and hBChE were all obtained from Sigma Biochemicals (St. Louis, MO). Torpedo californica AChE (TcAChE) was prepared as previously described [16], [17], [18]. CPT-11 and SN-38 were gifts kindly provided by Dr. J.P. McGovren (Pfizer, New York, NY). Donepezil was obtained from the St. Jude Children's Research Hospital pharmacy as Aricept tablets. An analog of CPT-11 that contains a piperidinobenzyl moiety at the 10-position of the molecule (PB-CPT,
Activation of CPT-11 by acetylcholinesterase and butyrylcholinesterase
As AChE is a member of the esterase class of proteins, potentially AChE could convert CPT-11 to SN-38. To assess whether these enzymes can activate the drug, we incubated either 20 U or 200 U of hAChE or hBChE with 25 μM CPT-11 for up to 24 h, and determined the amounts of SN-38 produced. As indicated in Table 1, no SN-38 was seen in samples incubated with hAChE. In contrast, readily detectable levels of the active metabolite were present following incubation with hBChE. Therefore, hBChE can
Discussion
CPT-11 is probably the best new broad spectrum anticancer drug to be identified in the last 30 years. It demonstrates remarkable antitumor activity in animal models [24], [25] and these results have been translated into effective responses in patients diagnosed with a variety of solid tumors [6]. However, as with all chemotherapeutic agents, severe toxicity is frequently seen with CPT-11 administration. While the cholinergic syndrome is not life threatening, it is very unpleasant for the
Acknowledgements
This work was supported in part by an NIH Cancer Center Core Grant P30-CA-21765, the American Lebanese Syrian Associated Charities, by the US Army Medical Research & Materiel Command to I.S. and J.L.S., the Kimmelman Center for Biomolecular Structure and Assembly, the Benoziyo Center for Neurosciences and the Kalman and Ida Wolens Foundation. The structure was determined in collaboration with the Israel Structural Proteomics Center, supported by the Israel Ministry of Science & Technology, the
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