Carboxylesterase-Mediated Activation of Irinotecan
By Philip M. Potter, PhD, and Mary K. Danks, PhD
During the last 30 years, many new anticancer agents have been identified; however, the majority of these have failed in human trials either because of lack of efficacy or severe toxicities. Camptothecin is an example of an agent that demonstrated good antitumor activity in vitro, but produced marked hemorrhagic cystitis in patients undergoing treatment with this drug.1-3 The low therapeutic index of camptothecin now is known to be due, at least in part, to a pH-dependent shift in the chemical form of the drug, resulting in precipitation, primarily in the urine.4 Recently, chemical modification of the parent camptothecin structure has yielded several novel agents that have demonstrated considerable promise in the treatment of human malignancies. These include topotecan, 9-nitrocamptothecin, and the prodrug irinotecan (CPT-11, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin).
Irinotecan is activated by carboxylesterases (CE) to yield the potent topoisomerase I inhibitor SN-38 (7-ethyl-10-hydroxycamptothecin; see Figure).5 Irinotecan has demonstrated remarkable antitumor activity in animals bearing human tumor xenografts and currently is in clinical trials.6-8 The FDA recently approved irinotecan for front-line therapy in colon adenocarcinoma, since this is the most active agent thus far identified for the treatment of this disease. This review will focus on the efficacy and toxicity of irinotecan as related to its activation in vivo.
Expression of Carboxylesterases
CEs are a ubiquitous class of enzymes thought to be involved in the detoxification of xenobiotics;9 therefore, one would expect the expression of these proteins to be detected in tissues exposed to such agents. Consistent with this hypothesis, CE activity has been identified in extracts of lung, gut, and liver of mice.10 Until recently, the cDNAs encoding these proteins had not been isolated and the relative ability of each to activate irinotecan was unknown. With the increasing use of this drug in both animal models and humans, considerable effort has been expended in identifying the CEs responsible for irinotecan activation in vivo. Consequently, several mammalian CE cDNAs have been isolated and characterized. These include human liver CE 1 (hCE1), human liver CE 2 (hCE2), human small intestinal CE (hiCE), and rabbit liver CE (rCE).
Carboxylesterases that Activate Irinotecan
To identify CEs that can activate irinotecan, a simple fluorescence assay was developed that allows the rapid detection of SN-38, irinotecan’s active metabolite. A series of commercially available esterases was screened for the ability to efficiently convert irinotecan to SN-38, and rCE was identified. Following N-terminal amino acids sequencing, 5’ and 3’ RACE, and PCR, a full-length cDNA encoding the protein was isolated.11 rCE expression sensitized human tumor cells to irinotecan, both in vitro and when grown as xenografts in immune-deprived mice.12 Additionally, cell extracts expressing rCE efficiently converted irinotecan to SN-38.
Searches of the Genbank database indicated that a human alveolar macrophage CE (hCE1) was greater than 86% similar to the active rCE; it was assumed that hCE1 was responsible for irinotecan activation in humans.13 However, biochemical studies and analyses indicated that hCE1 was very inefficient at drug metabolism and cells expressing this protein were not sensitized to irinotecan.12
Recently, two independent lines of investigation identified a human CE that can activate irinotecan. When analysis of esterase-deficient mice identified the small intestine as a major site of irinotecan activation, searches were performed of the Genbank database.10 These searches indicated that a human CE cDNA (iCE) had been isolated from intestinal epithelium.14 (We have designated this iCE as hiCE to indicate that this protein is derived from humans.) The ability of hiCE to activate irinotecan, however, was not reported at the time the cDNA was isolated. We subsequently showed that hiCE expression in COS7 cells resulted in efficient drug activation by whole cell sonicates and that cells expressing hiCE were sensitized to irinotecan.15
In a second study, purification and analysis of the kinetic properties of hCE1 and hCE2, two CE proteins from human liver, indicated that the latter protein was very efficient at irinotecan activation.16 Interestingly, sequence comparisons of rCE, hiCE, hCE1, and hCE2 show that hiCE and hCE2 are highly homologous and that each can efficiently activate irinotecan. However, hCE1, which is greater than 86% similar to the rCE, metabolizes the drug poorly if at all (see Table).12 Therefore, sequence alone does not predict the ability of CEs to convert irinotecan to SN-38.
Northern analyses of human tissues with hiCE cDNA identify the intestine as having the highest level of CE expression, at least 10-fold greater than that observed in the liver.14 Additional experiments using RNA derived from different regions of the human intestinal tract indicate that duodenum and jejunum demonstrate the highest hiCE expression. Further, since hiCE and hCE2 are highly homologous and probably represent the same gene, the data suggest that the major source of irinotecan activation in humans occurs not in the liver as expected, but in the small intestine.
Toxicities Associated With Irinotecan
Molecular studies may explain the toxicities associated with this agent. The dose-limiting side effect of irinotecan is diarrhea, and this occurs in two forms. An initial form likely results from the direct inhibition of acetylcholinesterase (AcChE), as it can be alleviated by the administration of atropine.17-19 Computer modeling studies indicate that irinotecan binds within the active site of AcChE; however, the drug cannot be activated by this enzyme because of a displacement of the ester linkage from the catalytic amino acids.17
A second form of diarrhea occurs between 24 and 96 hours following drug administration. This can be severe and frequently requires extended hospitalization and supportive care. Data from studies in patients with cannulated bile ducts treated with radiolabeled irinotecan indicate that free drug is secreted unchanged into the bile following IV dosing.20 Biliary irinotecan then becomes a substrate for hiCE present in the small intestine, resulting in very high local concentrations of SN-38. Potentially, SN-38 may produce severe cytotoxicity to the gut epithelia, resulting in diarrhea.
| Table-Sequence Identity (%) Between Carboxylesterases | |||||
| Rabbit | hiCE | hCE1 | hCE2 | Activates irinotecan | |
| Rabbit | — | 47 | 81 | 48 | Yes |
| hiCE | — | 49 | 99 | Yes | |
| hCE1 | — | 49 | No | ||
| hCE2 | — | Yes | |||
Optimizing the Therapeutic Index of Irinotecan
Although the optimal schedule for irinotecan administration has not been identified, two specific possibilities are mentioned here as approaches to improve the agent’s clinical utility. First, in several studies an inverse relationship was observed between the amount of irinotecan administered to patients and the percent of the drug converted to SN-38.8 One interpretation of this observation is that once drug-activating enzymes have been saturated, no further activation can occur. Further, since irinotecan itself is toxic, high doses of parent compound likely contribute to toxicity but have little therapeutic benefit. Logically, the therapeutic index would be lowest when irinotecan is given in high doses over short infusion times and, conversely, may improve if peak plasma levels of the parent drug are minimized.
Second, irinotecan has demonstrated good antitumor activity despite the relatively low levels of conversion to the active metabolite, SN-38. On this basis, it may be a useful prodrug for novel enzyme prodrug therapy approaches such as viral-directed enzyme prodrug therapy (VDEPT). With such approaches, selective expression of an enzyme that can efficiently activate non-toxic prodrugs in tumor cells results in enhanced antitumor activity and an improved therapeutic index. Irinotecan-based VDEPT approaches using rCE to purge tumor cells from hematopoietic cells prior to autologous stem cell rescue and to treat minimum residual disease following surgical resection of bulk tumor currently are under investigation.
Conclusion
The recent isolation and biochemical and molecular analyses of CEs have yielded considerable information detailing irinotecan activation. Extension of these studies may provide oncologists and pharmacokineticists the data necessary to tailor drug dosing to individual patients and may increase irinotecan’s efficacy and therapeutic index. Ongoing clinical trials involve the modulation of drug uptake from the intestine using breast cancer resistance protein (BCRP) antagonists and the inhibition of biliary excretion using cyclosporine A and phenobarbital.21 Such procedures aim to increase or prolong the area under the curve for irinotecan/SN-38 in the hope of increasing its antitumor effect. What impact these manipulations will have on therapeutic index is unknown.
Additionally, future approaches include the use of this drug in an enzyme/prodrug therapy approach to selectively convert irinotecan to SN-38 in tumor cells, resulting in selective cytotoxicity. Overall, the knowledge gained from studying the expression, biochemistry, and structure of CEs may lead to improved use of irinotecan and its novel derivatives in the clinic setting. (Drs. Potter and Danks are in the Department of Molecular Pharmacology at St. Jude Children’s Research Hospital in Memphis, TN.) v
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