Inhibitor Report for: SN-38

Introduction: CPT11 (Irinotecan; 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is an important camptothecin-based broad-spectrum anticancer prodrug. The activation of its warhead, SN38 (7-ethyl-10-hydroxycamptothecin), requires hydrolysis by carboxylesterases. NPC (7-ethyl-10-[4-(1-piperidino)-1-amino] carbonyloxycamptothecin) is a metabolic derivative of CPT11 and is difficult to be hydrolyzed by human carboxylesterase. Microbial carboxylesterase with capability on both CPT11 and NPC hydrolysis is rarely reported. A marine microbial carboxylesterase, E93, was identified to hydrolyze both substrates in this study. This enzyme was an appropriate subject for uncovering the catalytic mechanism of carboxylesterases to CPT11 and NPC hydrolysis. Methods: X-ray diffraction method was applied to obtain high-resolution structure of E93. Molecular docking was adopted to analyze the interaction of E93 with p -NP ( p -nitrophenyl), CPT11, and NPC substrates. Mutagenesis and enzymatic assay were adopted to verify the binding pattern of substrates. Results: Three core regions (Region A, B, and C) of the catalytic pocket were identified and their functions on substrates specificity were validated via mutagenesis assays. The Region A was involved in the binding with the alcohol group of all tested substrates. The size and hydrophobicity of the region determined the binding affinity. The Region B accommodated the acyl group of p -NP and CPT11 substrates. The polarity of this region determined the catalytic preference to both substrates. The Region C specifically accommodated the acyl group of NPC. The interaction from the acidic residue, E428, contributed to the binding of E93 with NPC. Discussion: The study analyzed both unique and conserved structures of the pocket in E93, for the first time demonstrating the discrepancy of substrate-enzyme interaction between CPT11 and NPC. It also expanded the knowledge about the substrate specificity and potential application of microbial Family VII carboxylesterases.

BACKGROUND: Irinotecan is widely used to treat various types of solid and metastatic cancer. It is an ester prodrug and its hydrolytic metabolite (SN-38) exerts potent anticancer activity. Irinotecan is hydrolyzed primarily by carboxylesterase-2 (CES2), a hydrolase abundantly present in the intestine such as the duodenum. We have identified several potent and covalent CES2 inhibitors such as remdesivir and sofosbuvir. Remdesivir is the first small molecule drug approved for COVID-19, whereas sofosbuvir is a paradigm-shift medicine for hepatitis C viral infection. Irinotecan is generally well-tolerated but associated with severe/life-threatening diarrhea due to intestinal accumulation of SN-38. OBJECTIVE: This study was to test the hypothesis that remdesivir and sofosbuvir protect against irinotecan-induced epithelial injury associated with gastrointestinal toxicity. METHODS: To test this hypothesis, formation of organoids derived from mouse duodenal crypts, a robust cellular model for intestinal regeneration, was induced in the presence or absence of irinotecan +/- pretreatment with a CES2 drug inhibitor. RESULTS: Irinotecan profoundly inhibited the formation of intestinal organoids and the magnitude of the inhibition was greater with female crypts than their male counterparts. Consistently, crypts from female mice had significantly higher hydrolytic activity toward irinotecan. Critically, remdesivir and sofosbuvir both reduced irinotecan hydrolysis and reversed irinotecan-reduced formation of organoids. Human duodenal samples robustly hydrolyzed irinotecan, stable CES2 transfection induced cytotoxicity and the cytotoxicity was reduced by CES2 drug inhibitor. CONCLUSION: These findings establish a therapeutic rationale to reduce irinotecan-gastrointestinal injury and serve as a cellular foundation to develop oral formulations of irinotecan with high safety.

PURPOSE: Irinotecan, a drug widely used in the treatment of advanced colorectal cancers, is a prodrug requiring activation to 7-ethyl-10-hydroxycamptothecin (SN-38) by carboxylesterase 2 (hCE2). The existence of functional polymorphisms in the gene encoding this enzyme could explain the individual variability in drug efficacy and toxicity. We have explored this possibility in looking for single nucleotide polymorphisms and their functional consequence.
METHODS: In a series of 115 human deoxyribonucleic acid samples, we have explored the 12 exons of the hCE2 gene, the intron-exon junctions, and the 5'- and 3'-untranslated regions, by denaturing HPLC and sequencing of polymerase chain reaction products. The functionality of the variations identified was studied in 60 human liver samples by measuring hCE2 gene expression by real-time reverse transcriptase-polymerase chain reaction of messenger ribonucleic acid extracts and carboxylesterase activity by use of irinotecan as a substrate.
RESULTS: We have identified a total of 11 single nucleotide polymorphisms, none of them able to alter the amino acid sequence of the protein. They are distributed in 10 distinct genotypes in addition to the wild type. The most frequent variation (localized in IVS10) has an allele frequency of 0.17 and has been identified at the homozygous state in 1 sample. hCE2 gene expression and carboxylesterase activity in the variants identified were not significantly different from those measured in wild-type samples. CONCLUSION: The hCE2 gene presents several polymorphisms, none of which seems to be involved in significant variations in protein activity and, therefore, in irinotecan activation.

Introduction: CPT11 (Irinotecan; 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is an important camptothecin-based broad-spectrum anticancer prodrug. The activation of its warhead, SN38 (7-ethyl-10-hydroxycamptothecin), requires hydrolysis by carboxylesterases. NPC (7-ethyl-10-[4-(1-piperidino)-1-amino] carbonyloxycamptothecin) is a metabolic derivative of CPT11 and is difficult to be hydrolyzed by human carboxylesterase. Microbial carboxylesterase with capability on both CPT11 and NPC hydrolysis is rarely reported. A marine microbial carboxylesterase, E93, was identified to hydrolyze both substrates in this study. This enzyme was an appropriate subject for uncovering the catalytic mechanism of carboxylesterases to CPT11 and NPC hydrolysis. Methods: X-ray diffraction method was applied to obtain high-resolution structure of E93. Molecular docking was adopted to analyze the interaction of E93 with p -NP ( p -nitrophenyl), CPT11, and NPC substrates. Mutagenesis and enzymatic assay were adopted to verify the binding pattern of substrates. Results: Three core regions (Region A, B, and C) of the catalytic pocket were identified and their functions on substrates specificity were validated via mutagenesis assays. The Region A was involved in the binding with the alcohol group of all tested substrates. The size and hydrophobicity of the region determined the binding affinity. The Region B accommodated the acyl group of p -NP and CPT11 substrates. The polarity of this region determined the catalytic preference to both substrates. The Region C specifically accommodated the acyl group of NPC. The interaction from the acidic residue, E428, contributed to the binding of E93 with NPC. Discussion: The study analyzed both unique and conserved structures of the pocket in E93, for the first time demonstrating the discrepancy of substrate-enzyme interaction between CPT11 and NPC. It also expanded the knowledge about the substrate specificity and potential application of microbial Family VII carboxylesterases.

BACKGROUND: Irinotecan is widely used to treat various types of solid and metastatic cancer. It is an ester prodrug and its hydrolytic metabolite (SN-38) exerts potent anticancer activity. Irinotecan is hydrolyzed primarily by carboxylesterase-2 (CES2), a hydrolase abundantly present in the intestine such as the duodenum. We have identified several potent and covalent CES2 inhibitors such as remdesivir and sofosbuvir. Remdesivir is the first small molecule drug approved for COVID-19, whereas sofosbuvir is a paradigm-shift medicine for hepatitis C viral infection. Irinotecan is generally well-tolerated but associated with severe/life-threatening diarrhea due to intestinal accumulation of SN-38. OBJECTIVE: This study was to test the hypothesis that remdesivir and sofosbuvir protect against irinotecan-induced epithelial injury associated with gastrointestinal toxicity. METHODS: To test this hypothesis, formation of organoids derived from mouse duodenal crypts, a robust cellular model for intestinal regeneration, was induced in the presence or absence of irinotecan +/- pretreatment with a CES2 drug inhibitor. RESULTS: Irinotecan profoundly inhibited the formation of intestinal organoids and the magnitude of the inhibition was greater with female crypts than their male counterparts. Consistently, crypts from female mice had significantly higher hydrolytic activity toward irinotecan. Critically, remdesivir and sofosbuvir both reduced irinotecan hydrolysis and reversed irinotecan-reduced formation of organoids. Human duodenal samples robustly hydrolyzed irinotecan, stable CES2 transfection induced cytotoxicity and the cytotoxicity was reduced by CES2 drug inhibitor. CONCLUSION: These findings establish a therapeutic rationale to reduce irinotecan-gastrointestinal injury and serve as a cellular foundation to develop oral formulations of irinotecan with high safety.

BACKGROUND AND PURPOSE: Irinotecan-induced diarrhea (IID) results from intestinal damages by its active metabolite SN-38. Alleviation of these damages has focused on lowering luminal SN-38 concentrations. However, it is unclear if the enteric bioavailability of SN-38 is mostly dependent on luminal SN-38 concentrations. EXPERIMENTAL APPROACH: Irinotecan (50mg/kg, i.p. once daily for 6days) was administered to female wildtype FVB, Mdr1a (-/-), Mrp2 (-/-) and Bcrp1 (-/-) mice for pharmacokinetic (PK), toxicokinetic (TK) and biodistribution studies. Plasma PK/TK profiles and tissues drug distribution were determined after first or sixth daily doses, along with activities of blood and gut esterases and intestinal Ugts. Caco-2 cells and bile-cannulate mice were used to further investigate intestinal and biliary disposition of irinotecan and its metabolites. KEY RESULTS: Significant differences in IID severity were observed with the susceptible rank of Bcrp1(-/-)>wildtype FVB>Mdr1a(-/-)>Mrp2(-/-). This rank order did not correlate with biliary excretion rates of SN-38/SN-38G. Rather, the severity was best correlated (R=0.805) with the intestinal ratio of Css SN-38/SN-38G, a measure of gut Ugt activity. On the contrary, IID was poorly correlated with plasma AUC ratio of SN-38/SN-38G (R=0.227). Increased intestinal esterase activities due to repeated dosing and gut efflux transporter functionality are the other key factors that determine SN-38 enteric exposures. CONCLUSION AND IMPLICATIONS: Intestinal SN-38 exposure is mainly affected by intestinal Ugt activities and blood esterase activities, and strongly correlated with severity of IID. Modulating intestinal SN-38 concentration and gut Ugt expression should be the focus of future studies to alleviate IID.

PURPOSE: Pharmacokinetics and pharmacodynamics of irinotecan have been reported to be altered in cancer patients with end-stage kidney disease (ESKD). Carboxylesterase (CES) has an important role in metabolism of irinotecan to its active metabolite, SN-38, in human liver. The purpose of the present study was to investigate whether CES activity was altered in ESKD patients. METHODS: The present study investigated the effects of uremic serum, uremic toxins, and fatty acids on the hydrolysis of irinotecan and a typical CES substrate, p-nitrophenyl acetate (PNPA), in human liver microsomes. Normal and uremic serum samples were deproteinized by treatment with methanol were used in the present study. RESULTS: The present study showed that both normal and uremic serum significantly inhibited CES-mediated metabolism of both irinotecan and PNPA. The inhibition by uremic serum was weaker than that by normal serum, suggesting that CES activity may be higher in ESKD patients. Although four uremic toxins did not affect PNPA metabolism, arachidonic acid inhibited it. There was no difference in inhibitory effect of PNPA metabolism between both mixtures of seven fatty acids used at concentrations equivalent to those present in 10% normal or uremic serum. Interestingly, those mixtures had a more pronounced effect than either 10% normal or uremic serum. CONCLUSIONS: The present study showed that the inhibition of CES activity by uremic serum was weaker than that by normal serum, suggesting that an increase in maximum plasma concentration of SN-38 in cancer patients with ESKD can be attributed to an accelerated CES-mediated irinotecan hydrolysis.

The aim of the present study was to search for an effective regimen among existing chemotherapies for head and neck squamous cell carcinoma (HNSCC). Among the tested drugs, we focused on combined SN-38, which is the active metabolite produced from irinotecan hydrochloride - a type I DNA topoisomerase inhibitor - after it is metabolized by carboxylesterase in the liver and gefitinib, an EGFR tyrosine kinase inhibitor treatment, based on the ability of this combination to inhibit HNSCC cell growth. Contrary to our expectation, in vivo, there was no significant difference in tumor growth suppression between gefitinib-only treatment and gefitinib plus SN-38. However, when tumor measurements were continued after treatment ceased, we found that several tumors showed renewed growth in the gefitinib-only group. The tumors that resumed growing after treatment showed increased CD44 expression compared with tumors from the combined treatment group. Next, we investigated the mechanism whereby SN-38 and gefitinib inhibited CD44 expression in vitro. These studies revealed that the combined treatment promoted lysosomal degradation of CD44. The present study revealed that combined gefitinib and SN-38 treatment inhibits CD44 expression by promoting its lysosomal degradation in HNSCC cells. However, it is still unclear whether inhibition of CD44 expression in HNSCC cells can directly suppress tumor regrowth after therapy. Thus, it may be necessary to elucidate the relationship between the effects of these chemotherapeutic agents on CD44 expression and tumor recurrence/metastasis in future studies.

BACKGROUND: DTS-108 is a hydrosoluble prodrug, where the SN-38 moiety is covalently linked to a 20-amino acid vector peptide by a specific esterase-sensitive cross-linker, releasing 7-ethyl-10-hydroxycampthotecin (SN-38) by esterase bond cleavage.
METHODS: The pharmacokinetics of DTS-108, adverse events graded according to NCI-CTCv3.1, dose-limiting toxicities at cycle 1, the maximum tolerated dose (MTD), and the recommended Phase II dose (RP2D) of intravenous DTS-108 (1-2 hours) every 2 weeks were evaluated in a first-in-human Phase I study in patients with advanced/metastatic carcinomas, according to an accelerated dose escalation design. SN-38 and SN-38 glucuronide (SN-38G) levels were evaluated with fluorescence high-performance liquid chromatography (HPLC) test, then liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods.
RESULTS: Forty-two patients received DTS-108 across 14 dosing cohorts (range 3-416 mg/m2). At 416 mg/m2, three out of six patients had grade 4 neutropenia thereby defining the MTD and the RP2D at 313 mg/m2. Fluorescence HPLC was inaccurate to quantify DTS-108 and its metabolites (SN-38 and SN-38G). New processes and analytical LC/MS/MS methods for testing SN-38 were implemented. At a dose of 313 mg/m2, mean DTS-108, SN-38, and SN-38G area under the plasma concentration-time curve to infinity (coefficients of variation %) were 439,293 (24%), 1,992 (34%), and 4,538 (46%) h.ng/mL. Stable disease (according to Response Evaluation Criteria in Solid Tumors) was observed in nine patients. CONCLUSION: Assessing SN-38 concentration using fluorescence HPLC is questionable since this method failed to monitor dose escalation of DTS-108, a new topoisomerase I inhibitor, due to ex vivo degradation. LC/MS/MS methods were consistent in evaluating SN-38 exposures allowing drug monitoring. The maximum tolerated dose of DTS-108 was 416 mg/m2. The RP2D for intravenous DTS-108 was 313 mg/m2 every 2 weeks in patients with advanced/metastatic solid tumors.

AIM: Our aim is to investigate the impact of high fat diet-induced obesity on plasma concentrations of the toxic irinotecan metabolite, SN-38, in mice. MAIN
METHODS: Diet-induced obese (DIO, 60% kcal fed) and lean mice (10% kcal fed) were treated orally with a single dose of 10mg/kg irinotecan to determine pharmacokinetic (PK) parameters. Feces and livers were collected for quantification of irinotecan and its metabolites (SN-38 & SN-38G). SN-38G formation by Ugt1a1 enzyme was analyzed in liver S9 fractions. Expression of the pro-inflammatory cytokine, TNF-alpha was determined in liver and plasma. Hepatic beta-glucuronidase and carboxylesterase enzymes (CES) were also determined. KEY FINDINGS: AUC0-8 and Cmax of SN-38 increased by 2-fold in DIO mice compared to their lean controls. This was accompanied by a~2-fold reduction in AUC0-8 and Cmax of SN-38G in DIO mice. There were no differences in the PK parameters of irinotecan in DIO or lean mice. Conversion of SN-38 to SN-38G by Ugt1a1 enzyme was reduced by ~2-fold in liver S9 fractions in DIO mice. Furthermore, in DIO mice, beta-glucuronidase activity increased by 2-fold, whereas there was no change in CES activity. TNF-alpha mRNA expression was 3 fold higher in DIO mice. SIGNIFICANCE: Our study demonstrates that reduced hepatic Ugt1a activity during obesity likely contributes to reduced glucuronidation, and results in higher levels of the toxic metabolite, SN-38. Thus, irinotecan dosage should be closely monitored for effective and safe chemotherapy in obese cancer patients who are at a higher risk of developing liver toxicity.

Organic anion-transporting polypeptides (OATP) mediate the hepatic uptake of many drugs, thus codetermining their clearance. Impaired hepatic clearance due to low-activity polymorphisms in human OATP1B1 may increase systemic exposure to SN-38, the active and toxic metabolite of the anticancer prodrug irinotecan. We investigated the pharmacokinetics and toxicity of irinotecan and SN-38 in Oatp1a/1b-null mice: Plasma exposure of irinotecan and SN-38 was increased 2 to 3-fold after irinotecan dosing (10 mg/kg, i.v.) compared with wild-type mice. Also, liver-to-plasma ratios were significantly reduced, suggesting impaired hepatic uptake of both compounds. After 6 daily doses of irinotecan, Oatp1a/1b-null mice suffered from increased toxicity. However, Oatp1a/1b-null mice had increased levels of carboxylesterase (Ces) enzymes, which caused higher conversion of irinotecan to SN-38 in plasma, potentially complicating pharmacokinetic analyses. Ces inhibitors blocked this increased conversion. Interestingly, liver-specific humanized OATP1B1 and OATP1B3 transgenic mice had normalized hepatic expression of Ces1 genes. While irinotecan liver-to-plasma ratios in these humanized mice were similar to those in Oatp1a/1b-null mice, SN-38 liver-to-plasma ratios returned to wild-type levels, suggesting that human OATP1B proteins mediate SN-38, but not irinotecan uptake in vivo. Upon direct administration of SN-38 (1 mg/kg, i.v.), Oatp1a/1b-null mice had increased SN-38 plasma levels, lower liver concentrations, and decreased cumulative biliary excretion of SN-38. Mouse Oatp1a/1b transporters have a role in the plasma clearance of irinotecan and SN-38, whereas human OATP1B transporters may only affect SN-38 disposition. Oatp1a/1b-null mice have increased expression and activity of Ces1 enzymes, whereas humanized mice provide a rescue of this phenotype. Mol Cancer Ther; 13(2); 492-503. (c)2013 AACR.

In an attempt to improve the antitumor activity and reduce the side effects of irinotecan (2), novel prodrugs of SN-38 (3) were prepared by conjugating amino acids or dipeptides to the 10-hydroxyl group of SN-38 via a carbamate linkage. The synthesized compounds completely generated SN-38 in pH 7.4 buffer or in human plasma, while remaining stable under acidic conditions. All prodrug compounds demonstrated much greater in vitro antitumor activities against HeLa cells and SGC-7901 cells than irinotecan. The most active compounds, 5h, 7c, 7d, and 7f, exhibited IC50 values that were 1000 times lower against HeLa cells and 30 times lower against SGC-7901 cells than those of irinotecan, and the inhibitory activities of these prodrugs against acetylcholinesterase (AchE) were significantly reduced, with IC50 values more than 6.8 times greater than that of irinotecan. In addition, compound 5e exhibited the same level of tumor growth inhibitory activity as irinotecan (CPT-11) in a human colon xenograft model in vivo.

In this study, single-walled carbon nanotubes (SWNTs) conjugated with antibody C225 were used to achieve targeted therapy against EGFR over-expressed colorectal cancer cells. In addition, the control release of the chemotherapeutic drug, 7-Ethyl-10-hydroxy-camptothecin (SN38), was studied. We used three different colorectal cancer cell lines, HCT116, HT29, and SW620, listed in the order of decreasing expression levels of EGFR. Our results showed that SWNT could use C225 to specifically bind to EGFR-expressed cells. The cellular uptakes of SWNT of EGFR over-expressed cells (HCT116 and HT29) were much higher than that of the negative control (SW620). We, next, demonstrated that receptor-mediated endocytosis was the primary cell entry route for SWNT. As a consequence, abundant amount of SN38 was released and EGFR over-expressed cells were killed. The drug control release process was studied by utilizing human carboxylesterase enzyme (hCE) that would break the bond linking SN38 and SWNT-carrier in cytoplasm. The intracellular SN38 release observed by confocal microscopy showed that SN38 actually dissociated from the SWNT-carrier first. SN38's entry to nucleus was then followed while the SWNT-carrier still remained in the cytoplasm. Overall, all these data suggested that SWNT could be a good carrier for targeting controlled release therapy.

A sensitive and accurate liquid chromatography method with mass spectrometry detection using MRM in positive ion mode was developed and validated for the simultaneous quantification of irinotecan (CPT-11) and 7-ethyl-10-hydroxycamptothecin (SN-38) in mouse plasma and brain. Camptothecin (CPT) was used as internal standard. A single step protein precipitation was used for plasma sample preparation, and a liquid-liquid extraction was needed for brain sample preparation. The method was validated with respect to selectivity, extraction recovery, linearity, intra- and inter-day precision and accuracy, limit of quantification and stability. Limits of quantification were 5 ng/mL for CPT-11 and SN-38 in plasma samples and 1.25 ng/g in brain. Linear calibration curves were obtained over concentration ranges of 5-5000 ng/mL in plasma and 1.25-1250 ng/g in brain for CPT-11 and SN-38. The intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 15% for both substances in both media. Stability studies showed that plasma carboxylesterase had to be inactivated in order to prevent in vitro conversion of CPT-11 into SN-38. Zinc sulfate (1 M) was used to inactivate the enzyme before sample storage. Brain samples did not require enzyme inactivation. This method was successfully applied to perform brain and plasma pharmacokinetic studies of CPT-11 and SN-38 in mice after intraperitoneal administration.

The targeted delivery of therapeutics to the tumor site is highly desirable in cancer treatment, because it is capable of minimizing collateral damage. Herein, we report the synthesis of a nanoplatform, which is composed of a 15 +/- 1 nm diameter core/shell Fe/Fe(3)O(4) magnetic nanoparticles (MNPs) and the topoisomerase I blocker SN38 bound to the surface of the MNPs via a carboxylesterase cleavable linker. This nanoplatform demonstrated high heating ability (SAR = 522 +/- 40 W/g) in an AC-magnetic field. For the purpose of targeted delivery, this nanoplatform was loaded into tumor-homing double-stable RAW264.7 cells (mouse monocyte/macrophage-like cells (Mo/Ma)), which have been engineered to express intracellular carboxylesterase (InCE) upon addition of doxycycline by a Tet-On Advanced system. The nanoplatform was taken up efficiently by these tumor-homing cells. They showed low toxicity even at high nanoplatform concentration. SN38 was released successfully by switching on the Tet-On Advanced system. We have demonstrated that this nanoplatform can be potentially used for thermochemotherapy. We will be able to achieve the following goals: (1) Specifically deliver the SN38 prodrug and magnetic nanoparticles to the cancer site as the payload of tumor-homing double-stable RAW264.7 cells; (2) Release of chemotherapeutic SN38 at the cancer site by means of the self-containing Tet-On Advanced system; (3) Provide localized magnetic hyperthermia to enhance the cancer treatment, both by killing cancer cells through magnetic heating and by activating the immune system.

The safety and efficacy of combination therapy with 7-ethyl-10-[4-[1-piperidino]-1-piperidino]carbonyloxycamptothecin (CPT-11, irinotecan) and S-1 composed of tegafur, a prodrug of 5-fluorouracil, gimeracil, and potassium oxonate, have been confirmed in patients with colorectal cancer. Previously, we showed that p.o. coadministration of S-1 decreased the plasma concentration of both CPT-11 and its metabolites in a patient with advanced colorectal cancer. The aim of this study was to clarify the mechanism of drug interaction using both in vivo and in vitro methods. Rats were administered S-1 p.o. (10 mg/kg) once a day for 7 consecutive days. On day 7, CPT-11 (10 mg/kg) was administered by i.v. injection. Coadministration of S-1 affected the pharmacokinetic behavior of CPT-11 and its metabolites, with a decrease in the C(max) and area under the plasma concentration curve (AUC) of the active metabolite 7-ethyl-10-hydroxycampothecin (SN-38) lactone form. Furthermore, the rate of biliary excretion of the SN-38 carboxylate form increased on coadministration of S-1. In the liver, the level of breast cancer resistance protein (BCRP), but not P-glycoprotein and multidrug resistance-associated protein 2, was elevated after administration of S-1. Enzymatic conversion of CPT-11 to SN-38 by carboxylesterase (CES) was unaffected by the liver microsomes of rats treated with S-1. In addition, components of S-1 did not inhibit the hydrolysis of p-nitrophenylacetate, a substrate of CES, in the S9 fraction of HepG2 cells. Therefore, the mechanism of drug interaction between CPT-11 and S-1 appears to involve up-regulation of BCRP in the liver, resulting in an increased rate of biliary excretion of SN-38 accompanied by a decrease in the C(max) and AUC of SN-38.

PURPOSE: Irinotecan (CPT11) is a prodrug activated in humans mainly by carboxylesterase 2 (CES2) generating the SN38 metabolite responsible for the drug efficacy and toxicity. The interpatients variability in CPT11 activation step could cause unpredictable toxicity. To identify a predictive molecular marker for CPT11 activation in cancer patients, we investigated the CES2 mRNA expression in peripheral blood mononuclear cells (PBMC) and correlated it to CPT11 activation rate, toxic effects, and response. EXPERIMENTAL DESIGN: Forty-five colorectal cancer patients were treated with a CPT11-including regimen (FOLFIRI). CES2 mRNA expression in PBMC was quantified by reverse transcription-PCR in real time. Plasma concentrations of CPT11, SN38, and SN38-glucuronide were determined by high-performance liquid chromatography and the pharmacokinetic variables calculated adopting the noncompartmental model. Toxicity was evaluated by the National Cancer Institute Common Toxicity Criteria scale and response by the WHO criteria.
RESULTS: A high interindividual variability in CES2 mRNA relative expression was observed (median, 1.45; range, 0.01-28.21). CES2 mRNA expression level was significantly associated with CPT11 activation ratio [(AUC(SN38) + AUC(SN38G))/AUC(CPT11)]. Patients with CES2 mRNA expression above the median cutoff value presented an activation ratio higher (median, 0.25; range, 0.15-0.42) than those with CES2 mRNA below the median (median, 0.20; range, 0.10-0.40; P = 0.013). This was associated with a nonsignificant trend of 1.34-fold increase of SN38 AUC in the group of patients with high CES2 mRNA expression (mean, 1.03 +/- 0.62 versus 0.77 +/- 0.32 micromol/L hour). Eight of 23 high CES2 mRNA-expressing patients (34.8%) developed grade 3 to 4 neutropenia or diarrhea compared with 2 of 22 (9.1%) in the low CES2-expressing group (P = 0.071). CONCLUSION: Our data support a predictive power of CES2 mRNA expression in PBMC for the activation rate of CPT11.

Although many drugs have been developed for the treatment of disease, some drugs have complications such as adverse effects, and antitumor agents should target tumors or cells more selectively. It is therefore necessary to develop drug delivery systems, and liposomes are reportedly useful as an effective drug carrier. An antitumor agent, CPT-11, inhibits DNA synthesis by the inhibition of topoisomerase1 and has a strong antitumor activity. SN-38 is converted from CPT-11 as an active metabolite by carboxylesterase in the liver. As SN-38 is insoluble, it has not been applied at the clinical stage as an injection. It is expected that SN-38 liposomalization may increase its usefulness in cancer chemotherapy. Our purpose is to have a clinical application of SN-38 by a novel method of liposomalization to expand the application for the other insolubility drugs. As SN-38 is hydrophobic, SN-38-trapped liposome preparation was attempted using the Bangham method, which is effective for general preparation. However, a high ratio of SN-38 trapped in liposome was not achieved, and this was not improved by the freezing-thawing method or the freeze-drying method. On the other hand, the ratio of SN-38 trapped in liposome by the modified remote loading method was about 4 times that by the Bangham method, and the ratio by the film loading method, novel method of liposomal preparation, reached 2 times and 8 times that by the modified remote loading method and Bangham method, respectively, showing a remarkable increase. In conclusion, it was suggested that the preparation of SN-38 liposome using the film loading method effectively entraps SN-38. Thus, it is expected that SN-38 liposome can be applied as an injection. This preparation method is useful if application is possible in the other insolubility drugs.

PURPOSE: Irinotecan, a drug widely used in the treatment of advanced colorectal cancers, is a prodrug requiring activation to 7-ethyl-10-hydroxycamptothecin (SN-38) by carboxylesterase 2 (hCE2). The existence of functional polymorphisms in the gene encoding this enzyme could explain the individual variability in drug efficacy and toxicity. We have explored this possibility in looking for single nucleotide polymorphisms and their functional consequence.
METHODS: In a series of 115 human deoxyribonucleic acid samples, we have explored the 12 exons of the hCE2 gene, the intron-exon junctions, and the 5'- and 3'-untranslated regions, by denaturing HPLC and sequencing of polymerase chain reaction products. The functionality of the variations identified was studied in 60 human liver samples by measuring hCE2 gene expression by real-time reverse transcriptase-polymerase chain reaction of messenger ribonucleic acid extracts and carboxylesterase activity by use of irinotecan as a substrate.
RESULTS: We have identified a total of 11 single nucleotide polymorphisms, none of them able to alter the amino acid sequence of the protein. They are distributed in 10 distinct genotypes in addition to the wild type. The most frequent variation (localized in IVS10) has an allele frequency of 0.17 and has been identified at the homozygous state in 1 sample. hCE2 gene expression and carboxylesterase activity in the variants identified were not significantly different from those measured in wild-type samples. CONCLUSION: The hCE2 gene presents several polymorphisms, none of which seems to be involved in significant variations in protein activity and, therefore, in irinotecan activation.

Irinotecan is an active cytotoxic agent for various cancers, and is converted to SN-38, its most active metabolite, by carboxylesterase converting enzyme (CCE) in vivo. Although the primary metabolic site is in the liver, ex vivo studies have proven that irinotecan is also converted to SN-38 in intestines, plasma and tumor tissues. The present study attempted to elucidate the in vitro conversion efficiency in human plasma, and to examine possible inter-individual variability and its clinical significance. Plasma samples were taken from 57 patients with lung cancer, 3 patients with benign pulmonary diseases and 9 healthy volunteers. After addition of 157 mM irinotecan to plasma, time courses of SN-38 concentration, measured by high-performance liquid chromatography (HPLC), were investigated. All subjects showed linear increase in SN-38 concentration during the first 60-min period, followed by a plateau. Mean and standard deviation of the conversion rate in the first 60 min were 515.9 +/- 50.1 pmol/ml/h (n = 69), with a coefficient of variation of 0.097. Although most of the subjects showed comparable conversion rates, 3 subjects had significantly higher conversion rates. In conclusion, the results of this study suggest that the enzyme activity of CCE in human plasma may show inter-individual variability.

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.

ONYX-015 has been used successfully in the clinic as a cancer therapeutic in combination with chemotherapy. The combination of ONYX-015 and chemotherapy appears to be more efficacious than either regimen alone. In this study, we try to enhance this combination by "arming" ONYX-015 with a therapeutic transgene, an approach more commonly used with nonreplicating viruses in the context of gene therapy. We chose the prodrug converting enzyme carboxylesterase (CE), which converts the camptothecin derivative CPT-11 (irinotecan) to the much more potent chemotherapeutic SN-38. The transgene was introduced into three distinct positions in the E3 region of the adenovirus genome to allow either early or late expression during the virus life cycle. We demonstrate that each of these ONYX-015-based adenoviruses expresses an active CE enzyme that can efficiently convert CPT-11 to SN-38. Furthermore, the cytotoxicity of CE-expressing viruses, but not control viruses, is enhanced significantly in the presence of the prodrug. Finally, we demonstrate that we can achieve transgene expression and activity in vivo in a human tumor xenograft model, and that treatment with a CE-expressing virus in combination with CPT-11 enhances survival of tumor-bearing mice. These results indicate that the addition of a prodrug converting enzyme may be a feasible approach to additionally enhance the efficacy of replicating adenoviruses as cancer therapeutics.

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.

PURPOSE: To characterize the relationships between human plasma irinotecan carboxylesterase-converting enzyme activity, caboxylesterase-mediated hydrolysis of p-nitrophenyl acetate (pNPA), and the butyrylcholinesterase-mediated hydrolysis of butyrylthiocholine in human plasma and to test the ability of these in vitro tests to predict the variability in SN-38 pharmacokinetics in adult patients during a prolonged infusion of irinotecan.
METHODS: Individual plasma-converting enzyme activity was measured in 20 adult cancer patients participating in a pharmacokinetic and phase I clinical trial of a prolonged 96-h intravenous infusion of irinotecan. The pNPA and butyrylthiocholine hydrolysis in patient plasma was also assayed.
RESULTS: The irinotecan carboxylesterase-converting enzyme in human plasma had a Vmax of 89.9 +/- 22.7 pmol/h per ml plasma and a Km of 207 +/- 56 microM (mean +/- SD, n = 3). The mean value of the specific activity of this enzyme in 20 adult cancer patients was 10.08 +/- 2.96 pmol/h per ml plasma ranging from 5.43 to 15.39 pmol/h per ml. The area-under-the-concentration-versus time curve (AUC) ratio of SN-38 to irinotecan (AUCSN-38/AUCCPT-11) was used to assess the relative SN-38 exposure to the active metabolite in individual patients. Pharmacokinetic variations in the relative exposure to SN-38 did not correlate with the measured carboxylesterase-converting enzyme activity nor with plasma butyrylcholinesterase activity in our patient population. However, it did correlate with the measured pNPA hydrolysis activity in patient plasma (r2 = 0.350, P = 0.0124, n = 18).
CONCLUSIONS: Determination of patient plasma pNPA hydrolysis activity may have utility in predicting SN-38 pharmacokinetics during prolonged infusions of irinotecan.

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.

Irinotecan, or CPT-11 (7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecine++ +), is a water-soluble derivative of camptothecine with promising activity against several types of malignancies. In addition to 7-ethyl-10-hydroxycamptothecine (SN-38), its active metabolite, we were able to identify several metabolites in the plasma of patients treated with this drug, especially an oxidative metabolite, 7-ethyl-10[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxy-camptothecine. During our study of the biosynthesis of 7-ethyl-10[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxy-camptothecine from CPT-11 by human liver microsomes, we were able to detect another quantitatively important polar metabolite, which was also present in the plasma and urine of patients treated with CPT-11. On the basis of preliminary experiments, the structure of this compound was postulated to be 7-ethyl-10-(4-amino-1-piperidino)carbonyloxycamptothecine, and this structure was synthesized by Rhone-Poulenc Rorer. Urine samples and human liver microsomal extracts were studied by high-performance liquid chromatography/atmospheric pressure chemical ionization/tandem mass spectrometry to identify its structure formally. The identification of the metabolite was supported by identical retention time, mass-to-charge ratio and tandem mass spectrometry fragmentation as a synthetic standard. Like irinotecan, 7-ethyl-10-(4-amino-1-piperidino) carbonyloxycamptothecine was a weak inhibitor of cell growth of P388 cells in culture (IC50 = 3.4 micrograms/ml vs. 2.8 micrograms/ml for irinotecan and 0.001 microgram/ml for SN-38). It was also a poor inducer of topoisomerase I-DNA cleavable complexes (100-fold less potent than SN-38). However, unlike 7-ethyl-10[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxy-camptothecine, this new metabolite could be hydrolyzed to SN-38 by human liver microsomes and purified human liver carboxylesterase, though to a lesser extent than irinotecan. This compound can therefore contribute to the activity and toxicity profile of irinotecan in vivo.

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) 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.

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.

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.