Inhibitor Report for: Irinotecan

The anticancer prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino-]carbonyloxycamptothecin (CPT-11) is a highly effective camptothecin analog that has been approved for the treatment of colon cancer. It is hydrolyzed by carboxylesterases to yield 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I poison. However, upon high-dose intravenous administration of CPT-11, a cholinergic syndrome is observed that can be ameliorated by atropine. Previous studies have indicated that CPT-11 can inhibit acetylcholinesterase (AChE), and here, we provide a detailed analysis of the inhibition of AChE by CPT-11 and by structural analogs. These studies demonstrate that the terminal dipiperidino moiety in CPT-11 plays a major role in enzyme inhibition, and this has been confirmed by X-ray crystallographic studies of a complex of the drug with Torpedo californica AChE. Our results indicate that CPT-11 binds within the active site gorge of the protein in a fashion similar to that observed with the Alzheimer drug donepezil. The 3D structure of the CPT-11/AChE complex also permits modeling of CPT-11 complexed with mammalian butyrylcholinesterase and carboxylesterase, both of which are known to hydrolyze the drug to the active metabolite. Overall, the results presented here clarify the mechanism of AChE inhibition by CPT-11 and detail the interaction of the drug with the protein. These studies may allow the design of both novel camptothecin analogs that would not inhibit AChE and new AChE inhibitors derived from the camptothecin scaffold.

Irinotecan (CPT-11 [Camptosar]) is an important new chemotherapeutic drug that demonstrates activity against a broad spectrum of malignancies, including carcinomas of the colon, stomach, and lung. Unfortunately, frequent and often severe gastrointestinal toxicities, particularly diarrhea, have limited its more widespread use. A cholinergic syndrome resulting from the inhibition of acetylcholinesterase activity by irinotecan is frequently seen within the first 24 hours after irinotecan administration but is easily controlled with atropine. Late diarrhea occurs in the majority of patients, however, and is National Cancer Institute (NCI) grade 3 or 4 in up to 40%. The late syndrome appears to be related to the effects on the bowel of SN-38, the active metabolite of irinotecan, which undergoes biliary excretion and inactivation. Early recognition and treatment of late diarrhea with high-dose loperamide have reduced, although not entirely eliminated, patient morbidity. Further study is needed to identify the mechanism of irinotecan-induced late diarrhea and to evaluate potential new therapies.

Irinotecan (CPT-11 [Camptosar]), a semisynthetic derivative of the plant alkaloid camptothecin, is bioactivated by carboxylesterases (EC3.1.1-) to the topoisomerase I inhibitor SN-38, a minor metabolite. Bioactivation of intravenously administered irinotecan by carboxylesterases occurs predominantly in the liver. Two human carboxylesterase isoforms responsible for SN-38 formation have been characterized. At relevant hepatic irinotecan concentrations up to 12 micrograms/mL, a low-Km isoform is responsible for irinotecan bioactivation. High concentrations of drugs commonly coadministered with irinotecan do not inhibit carboxylesterase activity. Intestinal carboxylesterases can also generate SN-38, followed by subsequent oral absorption. A second major polar metabolite of irinotecan, aminopentanecarboxylic acid (APC), is the product of CYP3A4-mediated oxidation of the terminal piperidine ring. APC is 100-fold less active than SN-38 as a topoisomerase I inhibitor and is a relatively weak inhibitor of acetylcholinesterase. SN-38 is eliminated mainly through conjugation by hepatic uridine glucuronosyltransferase (UGT*1.1), the same isoezyme responsible for glucuronidation of bilirubin. Grade 4 irinotecan-related toxicity (ie, neutropenia, diarrhea) has recently been reported in two patients with deficient UGT*1.1 activity. SN-38 glucuronide (SN-38G), which has only 1/100th the antitumor activity of SN-38, is actively secreted into the bile by a canalicular multispecific organic anion transporter. Deconjugation of SN-38G to SN-38 by beta-glucuronidase produced by the intestinal flora may contribute to enterohepatic recirculation of SN-38 and delayed intestinal toxicity.

Several mammalian carboxylesterases were shown to activate the prodrug irinotecan (CPT-11) to produce 7-ethyl-10-hydroxycamptothecin (SN-38), a topoisomerase inhibitor used in cancer therapy. However, the potential use of bacterial carboxylesterases, which have the advantage of high stability, has not been explored. We present the crystal structure of the carboxyesterase Est55 from Geobacillus stearothermophilus and evaluation of its enzyme activity on CPT-11. Crystal structures were determined at pH 6.2 and pH 6.8 and resolution of 2.0 A and 1.58 A, respectively. Est55 folds into three domains, a catalytic domain, an alpha/beta domain and a regulatory domain. The structure is in an inactive form; the side-chain of His409, one of the catalytic triad residues, is directed away from the other catalytic residues Ser194 and Glu310. Moreover, the adjacent Cys408 is triply oxidized and lies in the oxyanion hole, which would block the binding of substrate, suggesting a regulatory role. However, Cys408 is not essential for enzyme activity. Mutation of Cys408 showed that hydrophobic side-chains were favorable, while polar serine was unfavorable for enzyme activity. Est55 was shown to hydrolyze CPT-11 into the active form SN-38. The mutant C408V provided a more stable enzyme for activation of CPT-11. Therefore, engineered thermostable Est55 is a candidate for use with irinotecan in enzyme-prodrug cancer therapy.

The anticancer prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino-]carbonyloxycamptothecin (CPT-11) is a highly effective camptothecin analog that has been approved for the treatment of colon cancer. It is hydrolyzed by carboxylesterases to yield 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I poison. However, upon high-dose intravenous administration of CPT-11, a cholinergic syndrome is observed that can be ameliorated by atropine. Previous studies have indicated that CPT-11 can inhibit acetylcholinesterase (AChE), and here, we provide a detailed analysis of the inhibition of AChE by CPT-11 and by structural analogs. These studies demonstrate that the terminal dipiperidino moiety in CPT-11 plays a major role in enzyme inhibition, and this has been confirmed by X-ray crystallographic studies of a complex of the drug with Torpedo californica AChE. Our results indicate that CPT-11 binds within the active site gorge of the protein in a fashion similar to that observed with the Alzheimer drug donepezil. The 3D structure of the CPT-11/AChE complex also permits modeling of CPT-11 complexed with mammalian butyrylcholinesterase and carboxylesterase, both of which are known to hydrolyze the drug to the active metabolite. Overall, the results presented here clarify the mechanism of AChE inhibition by CPT-11 and detail the interaction of the drug with the protein. These studies may allow the design of both novel camptothecin analogs that would not inhibit AChE and new AChE inhibitors derived from the camptothecin scaffold.

CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is an anticancer prodrug that has been approved for the treatment of colon cancer. It is a member of the camptothecin class of drugs and activation to the active metabolite SN-38, is mediated by carboxylesterases (CE). SN-38 is a potent topoisomerase I poison and is highly effective at killing human tumor cells, with IC50 values in the low nM range. However, upon high dose administration of CPT-11 to cancer patients, a cholinergic syndrome is observed, that can be rapidly ameliorated by atropine. This suggests a direct interaction of the drug or its metabolites with acetylcholinesterase (AChE). Kinetic studies indicated that CPT-11 was primarily responsible for AChE inhibition with the 4-piperidinopiperidine moiety, the major determinant in the loss of enzyme activity. Structural analogs of 4-piperidinopiperidine however, did not inhibit AChE, including a benzyl piperazine derivate of CPT-11. These results suggest that novel anticancer drugs could be synthesized that do not inhibit AChE, or alternatively, that novel AChE inhibitors could be designed based around the camptothecin scaffold.

Irinotecan, a camptothecin analogue, is a prodrug which requires bioactivation to form the active metabolite SN-38. SN-38 acts as a DNA topoisomerase I poison. Irinotecan has been widely used in the treatment of metastatic colorectal cancer, small cell lung cancer and several other solid tumors. However, large inter-patient variability in irinotecan and SN-38 disposition, as well as severe but unpredictable diarrhea limits the clinical potential of irinotecan. Intense clinical pharmacology studies have been conducted to elucidate its complicated metabolic pathways and to provide scientific rationale in defining strategies to optimize drug therapy. Irinotecan is subjected to be shunted between CYP3A4 mediated oxidative metabolism to form two inactive metabolites APC or NPC and tissue carboxylesterase mediated hydrolysis to form SN-38 which is eventually detoxified via glucuronidation by UGT1A1 to form SN-38G. The pharmacology of this compound is further complicated by the existence of genetic inter-individual differences in activation and deactivation enzymes of irinotecan (e.g., CYP3A4, CYP3A5, UGT1A1) and sharing competitive elimination pathways with many concomitant medications, such as anticonvulsants, St. John's Wort, and ketoconazole. Efflux of the parent compound and metabolites out of cells by several drug transporters (e.g., Pgp, BCRP, MRP1, MRP2) also occurs. This review highlights the latest findings in drug activation, transport mechanisms, glucuronidation, and CYP3A-mediated drug-drug interactions of irinotecan in order to unlock some of its complicated pharmacology and to provide ideas for relevant future studies into optimization of this promising agent.

The anticancer agent irinotecan (CPT-11) is a prodrug converted to its active form, SN-38, by human carboxylesterase (hCE) and the SN-38 is further metabolized to its inactive form, SN-38G. We investigated the expression of hCE in human lung cancer cells as well as the ability of these cells to convert CPT-11 to SN-38 using surgically resected tumor samples and cultured cell lines. SN-38 was 40- to 3,000-fold more toxic to lung cancer cell lines than CPT-11, which acted more time-dependently than SN-38. Although human lung cancer cells expressed hCE in the cytoplasm, hCE expression levels in cancer cells were not correlated with their drug sensitivities. Although intracellular CPT-11 and SN-38 levels continuously increased within 60 min of CPT-11 exposure, SN-38 levels in cells exposed to SN-38 decreased. Cells with the ability to metabolize SN-38 to SN-38G were more resistant to extracellular SN-38 than cells lacking the ability. Of 25 squamous cell carcinomas, 15 were strongly positive for hCE and six were negative. Of 25 adenocarcinomas, four were strongly positive for hCE and 16 were positive, while five were negative. Thus, 70% of non-small cell lung cancers expressed hCE. From these results, we conclude that human lung cancer cells expressed the enzyme which can convert CPT-11 to SN-38 and that intracellular SN-38 converted from CPT-11 may act as a chemotherapeutic agent together with SN-38 absorbed from the outside and augment the dose intensity of SN-38. Therefore, to assess the effects of CPT-11 prior to chemotherapy, it is important to check if lung cancer cells express hCE.

Irinotecan (CPT-11) is a prodrug that is used to treat metastatic colorectal cancer. It is activated to the topoisomerase poison SN-38 by carboxylesterases. SN-38 is subsequently metabolised to its inactive glucuronide, SN-38G, which can however be reactivated to SN-38 by beta-glucuronidase. The purpose of this study was to examine the role of carboxylesterases and beta-glucuronidase in the in vitro production of SN-38 in human colorectal tumours. The production of SN-38 from CPT-11 and SN-38G was measured by HPLC in human colorectal tumour homogenates. Carboxylesterase and beta-glucuronidase activities were found to be lower in tumour tissues compared to matched normal colon mucosa samples. In colorectal tumour, beta-glucuronidase and carboxylesterase-mediated SN-38 production rates were comparable at clinically relevant concentrations of SN-38G and CPT-11, respectively. Therefore, tumour beta-glucuronidase may play a significant role in the exposure of tumours to SN-38 in vivo, particularly during prolonged infusions of CPT-11.

Irinotecan (CPT-11) is an anticancer prodrug. It is converted by carboxylesterase to yield an active metabolite, 7-ethyl-10-hydroxycamptothecin (SN-38), which acts as a topoisomerase I inhibitor. Several oxidative metabolites of CPT-11 have been identified in humans, including 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin (APC) and 7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecin (NPC), generated by cytochrome P-450 3A4 (CYP3A4). Other minor metabolites in which metabolic pathways and biologic activities have not been identified also exist. To further investigate the metabolism of CPT-11 in human liver, we analyzed metabolites of CPT-11 in human hepatic microsomes using a high-performance liquid chromatography/mass spectrometry (HPLC/MS) system and detected a new metabolite that was the major one produced in the microsomal system. HPLC-tandem mass spectrometry (HPLC/MS/MS) analysis indicated that this compound was an oxidation product formed by the loss of two hydrogen atoms from the terminal piperidine ring. Kinetic analyses indicated that a single enzyme generated the metabolite, and we have identified this enzyme in two in vitro systems. The formation of the new metabolite was significantly inhibited by SKF525A, ketoconazole, and an anti-CYP3A4 antibody and catalyzed specifically by CYP3A4 expressed in insect microsomes. A significant correlation was observed between the generation of this metabolite and the CYP3A4 content in individual human hepatic microsomes. These findings indicate that this newly detected metabolite is a CYP3A4-generated product that may be produced in hepatic microsomes of patients treated with CPT-11.

7-Ethyl-10[4-(1-piperidino)-1-piperidino] carbonyloxy-camptothecin (CPT-11), a DNA topoisomerase I inhibitor, undergoes several metabolic pathways to generate conjugated and unconjugated derivatives that could be excreted from the body. The objective of this study was to determine the oxidative metabolites of CPT-11 recovered in human urine samples and to identify cytochrome P450 (CYP) involved in their formation. In addition to the already known metabolites of CPT-11 [SN-38, SN-38-G, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycamptothecin (APC), and 7-ethyl-10-(4-amino-1-piperidino) carbonyloxycamptothecin (NPC)], we isolated three oxidized metabolites from the urine of two children and two adults given CPT-11. M1 and M2 (molecular weight, 602) were hydroxylated, respectively, on the CPT moiety and on the terminal piperidine ring of CPT-11. M3 had a molecular mass of 602, but its urine concentration in patients was too low to establish its chemical structure by liquid chromatography/mass spectrometry. In vitro incubations with cells expressing CYP2C8, CYP2C9, CYP1A1, CYP1A2, or CYP3A7 did not produce any detectable metabolites. Only CYP3A4 produced both APC and NPC, resulting from the oxidation of the piperidinylpiperidine side chain of CPT-11 along with metabolite M2. The metabolism of CPT-11 by CYP3A5 was markedly different because neither APC or NPC nor M2 was produced, whereas only one new metabolite, M4 (molecular weight, 558), was generated by de-ethylation of the CPT moiety. No previous study has reported the presence of the M4 metabolite. Production of APC, NPC, M2, and M4 was prevented by ketoconazole, a specific CYP3A inhibitor. The parameters of CPT-11 biotransformation into M2 and M4 were examined using cell lines expressing, respectively, with CYP3A4 and CYP3A5, indicating that CPT-11 is preferentially metabolized by CYP3A4. In conclusion, CYP3A plays a major role in the metabolism of CPT-11, with some differences of the metabolic profile exhibited by 3A4 and 3A5.

Butyrylcholinesterases (BuChEs; acylcholine acylhydrolase; EC 3.1.1.8) have been demonstrated to convert the anticancer agent CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) into its active metabolite SN-38 (7-ethyl-10-hydroxycamptothecin). In addition, significant differences in the extent of drug metabolism have been observed with BuChEs derived from different species. In an attempt to understand these differences, we have isolated the cDNA encoding a horse BuChE. Based upon the NH2-terminal amino acid sequence of a purified horse BuChE, we designed degenerate primers to amplify the coding sequence from horse liver cDNA. Following polymerase chain reaction and rapid amplification of the cDNA ends, we generated an 1850-bp DNA fragment, containing an 1806-bp open reading frame. The cDNA encodes a protein of 602 amino acid residues, including a 28-amino-acid NH2-terminal signal peptide. Furthermore, the DNA sequence and the deduced amino acid sequence revealed extensive homology to butyrylcholinesterase genes from several other species. In vitro transcription-translation of the cDNA produced a 66-kDa protein, identical to the size of native horse serum BuChE following removal of carbohydrate residues with endoglycosidase F. Additionally, transient expression of the cDNA in Cos-7 cells yielded extracts that exhibited cholinesterase activity and demonstrated a Km value for butyrylthiocholine of 106+/-9 nM. This extract converted the anticancer drug CPT-11 into SN-38, demonstrating that this drug can be activated by enzymes other than carboxylesterases.

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.

Patients treated with high doses of CPT-11 rapidly develop a cholinergic syndrome that can be alleviated by atropine. Although CPT-11 was not a substrate for acetylcholinesterase (AcChE), in vitro assays confirmed that CPT-11 inhibited both human and electric eel AcChE with apparent K(i)s of 415 and 194 nM, respectively. In contrast, human or equine butyryl-cholinesterase (BuChE) converted CPT-11 to SN-38 with K(m)s of 42.4 and 44.2 microM for the human and horse BuChE, respectively. Modeling of CPT-11 within the predicted active site of AcChE and BuChE corroborated experimental results indicating that, although the drug was oriented correctly for activation, the constraints dictated by the active site gorge were such that CPT-11 would be unlikely to be activated by AcChE.

Water-soluble 20(S)-glycinate esters of two highly potent 10,11-methylenedioxy analogues of camptothecin (CPT) have been synthesized and evaluated for their ability to eradicate human breast cancer tumor xenografts. The glycinate ester moiety increases the water solubility of the 10,11-methylenedioxy analogues 4-16-fold. However, in contrast to CPT-11, a water-soluble CPT analogue that was recently approved for second line treatment of colorectal cancer, the 20(S)-glycinate esters do not require carboxylesterase for conversion to their active forms. The glycinate esters are hydrolyzed to their parent, free 20(S)-hydroxyl active analogues in phosphate buffer (pH 7.5) and in mouse and human plasma. The glycinate esters are also 20-40-fold less potent than CPT-11 in inhibiting human acetylcholinesterase. In vivo, we examined 20(S)-glycinate-10,11-methylenedioxycamptothecin, 20(S)-glycinate-7-chloromethyl-10,11-methylenedioxycamptothecin, and CPT-11. We found that the two 10,11-methylenedioxy analogues had antitumor activity against breast cancer xenografts that was comparable to that of CPT-11. Our results indicate that water-soluble 20(S)-glycinate esters of highly potent CPT analogues provide compounds that maintain biological activity, do not require interactions with carboxylesterases, and do not inhibit human acetylcholinesterase.

CPT-11 [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin ] is a prodrug that is converted to the active metabolite SN-38 by carboxylesterases. In its active form, the drug inhibits topoisomerase I, causes DNA damage, and induces apoptosis. Data in this study show metabolism of CPT-11 to SN-38 (7-ethyl-10-hydroxycamptothecin) by a rabbit liver carboxylesterase in vitro and growth-inhibitory activity of the products of the reaction. Additionally, stable expression of the cDNA encoding this protein in Rh30 human rhabdomyosarcoma cells increased the sensitivity of the cells to CPT-11 8.1-fold. We propose that this prodrug/enzyme combination can be exploited therapeutically in a manner analogous to approaches currently under investigation with the combinations of ganciclovir/herpes simplex virus thymidine kinase and 5-fluorocytosine/cytosine deaminase.

The anticancer drug CPT-11 (7-ethyl-[4(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is a water-soluble derivative of camptothecin. We report here the conversion of APC (7-ethyl-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin), an inactive metabolite of CPT-11, to SN-38 (7-ethyl-10-hydroxycamptothecin), the active metabolite of CPT-11, by a rabbit liver carboxylesterase. This reaction is not catalyzed by any known human enzyme. The formation of SN-38 from APC was characterized by an apparent Km of 37.9 +/- 7.1 microM and a Vmax of 16.9 +/- 0.9 pmol/units/min. SN-38 was confirmed as a reaction product by high-performance liquid chromatography and mass spectrometry. A 24-h incubation of 10 microM APC with 500 units/ml of rabbit carboxylesterase produced 4 microM SN-38. The product of this reaction inhibited the growth of U373 MG human glioblastoma cells in vitro. The IC50 for a 24-h exposure of U373 MG cells to APC in the presence of 50 units/ml of rabbit carboxylesterase was 0.27 +/- 0.08 microM, whereas APC alone demonstrated no inhibition of growth at concentrations up to 1 microM. The IC50 of U373 MG cells transfected with the cDNA encoding the rabbit carboxylesterase (U373pIRESrabbit) and exposed to APC for 24 h was 0.8 +/- 0.1 microM APC, whereas the growth of cells transfected with vector control (U373pIRES) was unaffected by up to 1 microM APC. Because APC is nontoxic to human cells, we are investigating the possibility of using APC/rabbit carboxylesterase in a prodrug/enzyme therapeutic approach.

Irinotecan (CPT-11 [Camptosar]) is an important new chemotherapeutic drug that demonstrates activity against a broad spectrum of malignancies, including carcinomas of the colon, stomach, and lung. Unfortunately, frequent and often severe gastrointestinal toxicities, particularly diarrhea, have limited its more widespread use. A cholinergic syndrome resulting from the inhibition of acetylcholinesterase activity by irinotecan is frequently seen within the first 24 hours after irinotecan administration but is easily controlled with atropine. Late diarrhea occurs in the majority of patients, however, and is National Cancer Institute (NCI) grade 3 or 4 in up to 40%. The late syndrome appears to be related to the effects on the bowel of SN-38, the active metabolite of irinotecan, which undergoes biliary excretion and inactivation. Early recognition and treatment of late diarrhea with high-dose loperamide have reduced, although not entirely eliminated, patient morbidity. Further study is needed to identify the mechanism of irinotecan-induced late diarrhea and to evaluate potential new therapies.

Irinotecan (CPT-11 [Camptosar]), a semisynthetic derivative of the plant alkaloid camptothecin, is bioactivated by carboxylesterases (EC3.1.1-) to the topoisomerase I inhibitor SN-38, a minor metabolite. Bioactivation of intravenously administered irinotecan by carboxylesterases occurs predominantly in the liver. Two human carboxylesterase isoforms responsible for SN-38 formation have been characterized. At relevant hepatic irinotecan concentrations up to 12 micrograms/mL, a low-Km isoform is responsible for irinotecan bioactivation. High concentrations of drugs commonly coadministered with irinotecan do not inhibit carboxylesterase activity. Intestinal carboxylesterases can also generate SN-38, followed by subsequent oral absorption. A second major polar metabolite of irinotecan, aminopentanecarboxylic acid (APC), is the product of CYP3A4-mediated oxidation of the terminal piperidine ring. APC is 100-fold less active than SN-38 as a topoisomerase I inhibitor and is a relatively weak inhibitor of acetylcholinesterase. SN-38 is eliminated mainly through conjugation by hepatic uridine glucuronosyltransferase (UGT*1.1), the same isoezyme responsible for glucuronidation of bilirubin. Grade 4 irinotecan-related toxicity (ie, neutropenia, diarrhea) has recently been reported in two patients with deficient UGT*1.1 activity. SN-38 glucuronide (SN-38G), which has only 1/100th the antitumor activity of SN-38, is actively secreted into the bile by a canalicular multispecific organic anion transporter. Deconjugation of SN-38G to SN-38 by beta-glucuronidase produced by the intestinal flora may contribute to enterohepatic recirculation of SN-38 and delayed intestinal toxicity.

We have isolated a cDNA encoding a rabbit carboxylesterase (CE; EC 3.1.1.1) that converts the camptothecin-derived prodrug irinotecan (CPT-11) to the potent topoisomerase I inhibitor 7-ethyl-10-hydroxycamptothecin. NH2-terminal amino acid sequencing of a purified rabbit CE allowed the design of redundant oligonucleotides to perform PCR from rabbit liver cDNA. DNA sequencing of the PCR product confirmed the identity of the clone, and after both 5' and 3' rapid amplification of cDNA ends, oligonucleotide primers were designed to amplify the entire cDNA. The 1698-bp open reading frame encoded a 565-amino acid protein containing the characteristic CE B-1 and B-2 motifs, a hydrophobic NH2-terminal leader sequence, and the COOH-terminal residues HIEL that are thought to be responsible for protein localization in the endoplasmic reticulum. Transient expression of the cDNA in COS-7 cells resulted in CE activity in cell extracts and increased the sensitivity of cells to CPT-11. Additionally, stable expression of the rabbit liver CE cDNA in the human glioma U-373 MG cell line resulted in a 56-fold decrease in the IC50 value for CPT-11, whereas the expression of a human alveolar macrophage cDNA encoding a highly homologous CE produced no change in drug sensitivity.

Enzyme activation of prodrugs to improve the therapeutic index of specific anticancer agents is an attractive alternative to current chemotherapy regimens. This study addresses the potential for activating irinotecan (CPT-11) with recombinant carboxylesterases (CEs). CEs are a ubiquitous class of enzymes thought to be involved in the detoxification of xenobiotics. Their primary amino acid sequence indicates that these proteins should be localized to the endoplasmic reticulum. By PCR-mediated mutagenesis of a rabbit liver and a human alveolar macrophage CE cDNA, expression in Cos7 cells, and subsequent immunohistochemical localization, we have determined that an 18-amino acid NH2-terminal hydrophobic signal peptide is responsible for the localization of these proteins to the endoplasmic reticulum. By similar approaches, we have demonstrated that the COOH-terminal amino acids HIEL prevent secretion of the proteins from the cell. Enzymatic activity was lost by removing the NH2-terminal domain; however, active enzyme could be detected in the culture media of cells expressing the COOH-terminally truncated proteins. Secretion of CEs lacking the six COOH-terminal amino acids could be prevented with brefeldin A, confirming that these truncated enzymes were processed and released from cells by endoplasmic reticulum-mediated exocytosis. Double-truncation mutant enzymes lacking both NH2- and COOH-terminal sequences demonstrated immunostaining patterns similar to those of the NH2-terminally truncated proteins and also lacked CE activity. In all cases, metabolism of the classic esterase substrate o-nitrophenyl acetate predicted the sensitivity of cells expressing the rabbit CE to the anticancer agent CPT-11. In addition, the secreted enzyme sensitized Cos7 cells to this drug, indicating that protein association with a lipid bilayer is not required for substrate metabolism.

Irinotecan (CPT-11) is a new camptothecine derivative presently in development for the treatment of several advanced malignancies. It is converted in vivo to a highly potent metabolite, SN-38, by carboxylesterases. All camptothecine derivatives undergo lactonolysis in a pH-dependent reversible manner, generating inactive carboxylate forms. We have investigated in vitro the kinetics of transformation of CPT-11 to SN-38 by human liver microsomes originating from several donors. Microsomes from seven livers were studied individually or as a pooled preparation. CPT-11, either in its lactone or its carboxylate form, was added at a range of concentrations. The SN-38 formed was measured by HPLC with fluorometric detection. In the deacylation-limited carboxylesterase reaction, the linear steady-state kinetics between 10 and 60 min were determined. At all concentrations of CPT-11, the steady-state velocity of SN-38 formation as well as the intercept concentrations of SN-38 were about 2-fold higher when the substrate was under the lactone form than under the carboxylate form. We estimated the values (+/-SD) of K'm and Vmax to be 23.3 +/- 5.3 microM and 1.43 +/- 0.15 pmol/min/mg for the lactone and 48.9 +/- 5.5 microM and 1.09 +/- 0.06 pmol/min/mg for the carboxylate form of CPT-11, respectively. We conclude that the greater rate of conversion of CPT-11 lactone may contribute to the plasma predominance of SN-38 lactone observed in vivo. The inter-individual variation of SN-38 formation was relatively high (ratio of 4 between extreme values) but no large age- or gender-related differences were seen. The effect of twelve drugs of different therapeutic classes (antibiotics, antiemetics, antineoplastics, antidiarrhoeics, analgesics), which could be administered in association with irinotecan in the clinical setting, was evaluated in this system (drug concentration: 100 microM; CPT-11 lactone concentration: 10 microM). Loperamide and ciprofloxacine where the only drugs exerting a weak inhibition of CPT-11 conversion to SN-38.

CPT-11, a new semisynthetic derivative of camptothecin, is active in a number of tumor types in the clinic, including colon cancer. CPT-11 is a drug that is converted into the active metabolite SN-38 by a carboxylesterase. Experiments were performed to obtain more insight in the cellular characteristics in 5 unselected human colon-cancer cell lines that account for the differential sensitivity to CPT-11 and SN-38. In vitro, the sensitivity to CPT-11 and SN-38 was highest in LS174T and COLO 320 cells, intermediate in SW1398 cells and lowest in COLO 205 and WiDr cells. SN-38 was 130 to 570 times more active than CPT-11. CPT-11 induced complete remissions in 6 out of 12 COLO 320 tumors grown as subcutaneous xenografts, but was not effective in WiDr tumors. The cellular carboxylesterase activity did not relate to the sensitivity to CPT-11. The enzyme activity was higher in normal mouse tissues, i.e., serum and liver, than in COLO 320 or WiDr xenografts, indicating that tumor carboxylesterase is of minor importance for CPT-11 efficacy. The topoisomerase-1 mRNA expression in tumor cells was not predictive of the antiproliferative effects of CPT-11 or SN-38. We observed a positive relationship between the DNA topoisomerase-1 activity and the cellular sensitivity to carboxylesterase-activated CPT-11 (r = 0.75, p < 0.1) as well as to SN-38 (r = 0.89, p < 0.05). The higher topoisomerase-1 activity in COLO 320 cells and tumors when compared with that in WiDr cells and tumors reflected the differences in sensitivity to the drug(s). In conclusion, the DNA topoisomerase-1 activity was the best determinant for CPT-11/SN-38 sensitivity in this panel of unselected human colon-cancer cell lines.

Human hepatic microsomes were used to investigate the carboxylesterase-mediated bioactivation of CPT-11 to the active metabolite, SN-38. SN-38 formation velocity was determined by HPLC over a concentration range of 0.25-200 microM CPT-11. Biphasic Eadie Hofstee plots were observed in seven donors, suggesting that two isoforms catalyzed the reaction. Analysis by nonlinear least squares regression gave KM estimates of 129-164 microM with a Vmax of 5.3-17 pmol/mg/min for the low affinity isoform. The high affinity isoform had KM estimates of 1.4-3.9 microM with Vmax of 1.2-2.6 pmol/mg/min. The low KM carboxylesterase may be the main contributor to SN-38 formation at clinically relevant hepatic concentrations of CPT-11. Using standard incubation conditions, the effects of potential inhibitors of carboxylesterase-mediated CPT-11 hydrolysis were evaluated at concentrations >/= 21 microM. Positive controls bis-nitrophenylphosphate (BNPP) and physostigmine decreased CPT-11 hydrolysis to 1.3-3.3% and 23% of control values, respectively. Caffeine, acetylsalicylic acid, coumarin, cisplatin, ethanol, dexamethasone, 5-fluorouracil, loperamide, and prochlorperazine had no statistically significant effect on CPT-11 hydrolysis. Small decreases were observed with metoclopramide (91% of control), acetaminophen (93% of control), probenecid (87% of control), and fluoride (91% of control). Of the compounds tested above, based on these in vitro data, only the potent inhibitors of carboxylesterase (BNPP, physostigmine) have the potential to inhibit CPT-11 bioactivation if administered concurrently. The carboxylesterase-mediated hydrolysis of alpha-naphthyl acetate (alpha-NA) was used to determine whether CPT-11 was an inhibitor of hydrolysis of high turnover substrates of carboxylesterases. Inhibition of alpha-NA hydrolysis by CPT-11 was determined relative to positive controls BNPP and NaF. Incubation with microsomes pretreated with CPT-11 (80-440 microM) decreased alpha-naphthol formation to approximately 80% of control at alpha-NA concentrations of 50-800 microM. The inhibitors BNPP (360 microM) and NaF (500 microM) inhibited alpha-naphthol formation to 9-10% of control and to 14-20% of control, respectively. Therefore, CPT-11-sensitive carboxylesterase isoforms may account for only 20% of total alpha-NA hydrolases. Thus, CPT-11 is unlikely to significantly inhibit high turnover, nonselective substrates of carboxylesterases.

Irinotecan [7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11)] is a promising water-soluble analogue of camptothecin [S. Sawada et al., Chem. & Pharm. Bull. (Tokyo), 39: 1446-1454, 1991]. We have reported previously the presence of an important polar metabolite, in addition to 7-ethyl-10-hydroxycamptothecin (SN-38) beta-glucuronide, in samples of plasma taken from patients undergoing treatment with CPT-11 (L.P. Rivory and J. Robert, Cancer Chemother. Pharmacol. 36: 176-179, 1995; L. P. Rivory and J. Robert, J. Cromatogr., 661: 133-141, 1994). Plasma samples (0.5 ml) containing comparatively large amounts of this metabolite were extracted by solid-phase columns and subjected to high-performance liquid chromatography and mass spectrometry in parallel to fluorometric detection. The metabolite yielded [M + 1] ions with a m/z of 619, representing the addition of 32 atomic mass units to CPT-11. Purified fractions were subjected to proton nuclear magnetic resonance, and the structure determined, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]carbonyloxycampothecin (APC), was further validated following synthesis. Like CPT-11, APC was found to be only a weak inhibitor of the cell growth of KB cells in culture (IC50, 2.1 versus 5.5 micrograms/ml for CPT-11 and 0.01 microgram/ml for SN-38, the active metabolite of CPT-11) and was a poor inducer of topoisomerase I DNA-cleavable complexes (100-fold less potent than SN-38). In contrast to CPT-11, APC was not hydrolyzed to SN-38 by human liver microsomes or purified human liver carboxylesterase. Furthermore, APC did not inhibit the hydrolysis of CPT-11 in these preparations. Interestingly, APC was only a weak inhibitor of acetylcholinesterase in comparison to CPT-11 and neostigmine. It appears likely, therefore, that APC does not contribute directly to the activity and toxicity profile of CPT-11 in vivo.

We have investigated the conversion of the novel anti-topoisomerase I agent CPT-11 (irinotecan; 7-ethyl-10[4-(1-piperidino)-1-piperidno]carbonyloxycamptothecin ) to its active metabolite, SN-38 (7-ethyl-10-hydroxycamptothecin), by human liver carboxylesterase (HLC). Production of SN-38 was relatively inefficient and was enzyme deacylation rate-limited with a steady-state phase occurring after 15-20 min of incubation. This later phase followed Michaelis-Menten kinetics with an apparent Km of 52.9 +/- 5.9 microM and a specific activity of 200 +/- 10 mumol/sec/mol. However, the total enzyme concentration estimated from the intercept concentrations of SN-38 was much lower than that estimated directly from the titration of active sites with paraoxon (0.65 vs. 2.0 microM, respectively). Because deacylation rate-limiting kinetics result in the accumulation of inactive acyl-enzyme complex, we postulated that incubation of CPT-11 with HLC would result in an inhibition of the HLC-catalysed hydrolysis of p-nitrophenylacetate (p-NPA), an excellent substrate for this enzyme. Indeed, this was found to be the case although complete inhibition could not be attained. Analysis of possible kinetic schemes revealed that the most likely explanation for the disparity in estimated enzyme concentrations and the incomplete inhibition of p-NPA hydrolysis is that CPT-11 also interacts at a modulator site on the enzyme, which profoundly reduces substrate hydrolysis. Furthermore, loperamide, a drug often used for the treatment of CPT-11-associated diarrhea, was found to inhibit both CPT-11 and p-NPA HLC-catalysed hydrolysis, most likely by a similar interaction. These observations have direct implications for the clinical use of CPT-11.