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Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan (T.I., M.T., M.S.); Faculty of Pharmaceutical Sciences, Chiba Institute of Sciences, Chiba, Japan (M.H.); and Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan (K.C.)
(Received January 19, 2006; Accepted July 11, 2006)
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Abstract |
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; Buchwald and Bodor, 2002). Introduction of an ester linkage generally improves the bioavailability of therapeutic agents, because of increased passive transport following oral administration. The requirements for a better oral prodrug are that it is stable to hydrolytic breakdown in its absorptive stage and that it is easily hydrolyzed to an active drug once it enters the systemic circulation (Beaumont et al., 2003). Carboxylesterases (CESs; EC.3.1.1.1) play an important role in biotransformation of a variety of ester-containing drugs and prodrugs such as angiotensin-converting enzyme inhibitors (e.g., temocapril, cilazapril, quinapril, and imidapril; Takai et al., 1997), antitumor drugs (CPT-11 and capecitabin; Humerickhouse et al., 2000; Tabata et al., 2004), and narcotics (cocaine, heroin, and meperidine; Pindel et al., 1997; Zhang et al., 1999).
CESs are members of the 
/ß-hydrolase family and show ubiquitous tissue expression profiles with high levels in the liver, small intestine, and lung (Satoh and Hosokawa, 1998; Satoh et al., 2002). CESs use a catalytic triad (Ser-His-Glu) for catalysis, which is located at the base of a deep catalytic gorge (Bencharit et al., 2002, 2003a,b). CES cleaves esters via a two-step process that involves the formation and degradation of an acyl-enzyme intermediate. First, the acyl carbonyl group of the substrate binds to the hydroxyl group of serine to produce an acyl-enzyme complex while the alcohol moiety is released. Then the acid component is released by an attack of histidine-activated water on this acyl-enzyme complex. The microenvironment surrounding the catalytic triad is important, not only for binding of substrate but also for the ease with which alcohol and/or acyl components can be released.
Mammalian CESs comprise a multigene family, in which the isozymes are classified into four main groups and several subgroups according to the homology of the amino acid sequence (Satoh and Hosokawa, 1998; Satoh et al., 2002). The majority of CESs belong to the CES1 and CES2 families, and are differentiated on the basis of substrate specificity, tissue distribution, immunological properties, and gene regulation. For example, hCE-1 (CES1A1, HU1), a human CES1 family isozyme, is widely distributed in many tissues but is only found at very low levels in the intestine, whereas hCE-2 (CES2A1, hiCE), a human CES2 family isozyme, is widely distributed in the intestine, liver, and kidney (Xu et al., 2002). Whereas hCE-1 preferentially catalyzes the hydrolysis of compounds esterified with a small alcohol group, hCE-2 hydrolyzes compounds with a relatively small acyl group and large alcohol group (Pindel et al., 1997; Takai et al., 1997; Satoh et al., 2002). In the case of cocaine, hCE-1 catalyzes the hydrolysis of the methyl ester of cocaine, producing benzoylecgonine and methanol, whereas hCE-2 catalyzes the hydrolysis of the benzoyl ester (Pindel et al., 1997). hCE-1 has also been found to catalyze the transesterification of cocaine with ethanol to generate cocaethylene (Brzezinski et al., 1994). Furthermore, hCE-1 has been reported to possess acyl coenzyme A:cholesterol acyltransferase activity, which generates cholesterol esters from fatty-acyl coenzyme A and free cholesterol (Becker et al., 1994). Therefore, bioconversion of orally administered ester-containing drugs is affected by the expression level of hCE-1 and hCE-2 in the liver and small intestine. Some reports have described greater expression of hCE-1 than hCE-2 in human liver and little expression of hCE-1 in human small intestine (Satoh et al., 2002; Xie et al., 2002). However, the extent to which the overall hydrolase activity of the liver and small intestine is dependent upon hCE-1 and hCE-2 activities has not been reported, and differences of substrate specificity between hCE-1 and hCE-2 have not been systematically demonstrated using structurally related compounds.
The purpose of the present study is to demonstrate the relative contributions of CES isozymes in the hydrolase activity of microsomes from human liver and small intestine, and to investigate differences in substrate specificity between hCE-1 and hCE-2 expressed in V79 and Sf9 cells using several structurally related compounds. In addition, we examine whether or not the transesterification activity of enzymes via acyl-CES intermediates affects substrate specificity for hCE-1 and hCE-2.
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Materials and Methods |
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). Flurbiprofen derivatives were synthesized from the appropriate alcohol and 2-(2-fluoro-4-biphenylyl)-propionyl chloride, which had been synthesized by flurbiprofen and SOCl2, according to a previously reported method (Imai et al., 1993). The identity and purity of the propranolol derivatives and the flurbiprofen derivatives were confirmed by IR, NMR, atomic analysis, and HPLC. Temocapril and temocaprilat were kindly provided by Sankyo Co., Ltd. (Tokyo, Japan). Betamethasone valerate, betamethasone, and 1-naphthylbutyrate were purchased from Wako Pure Chemical Industries (Tokyo, Japan). Benzoic acid derivatives were purchased from Tokyo Kasei Kogyo Co. Ltd. (Tokyo, Japan). Fast Red TR was purchased from Sigma (St. Louis, MO). Pooled human liver microsomes from 10 subjects (5 males and 5 females, 21–61 years old, of mixed Caucasian, Hispanic, and African-American races) and pooled small intestine microsomes from 10 subjects (4 males and 6 females, 24–63 years old, of mixed Caucasian, Hispanic, and African-American races) were obtained from BD Gentest (Woburn, MA). All other chemicals and reagents were of analytical grade.
Expression of hCE-1 and hCE-2. Expression of hCE-1 (GenInfo Identifier: 34740321) and hCE-2 (GenInfo Identifier: 37622885) in V79 cells was carried out as described previously (Mori et al., 1999; Hosokawa et al., 2001). The parental V79 cells [V79-4; American Type Cell Culture Collection (CL93), Manassas, VA] were cultured at cell densities of 1 x 106 cells/100-mm plate with D-MEM (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), penicillin/streptomycin (Invitrogen), and L-glutamine (Invitrogen) in an atmosphere of 5% CO2 and 95% air at 37°C. The next day, 10 µg of vector DNA (CES/pTARGET or pTARGET) was transfected into the cells using Lipofectamine Transfection Reagent (Invitrogen) with OPTI-MEM medium (Invitrogen). On day 3, the V79 cells were spread over 100-mm plates with media containing the selective agent, G418 (Invitrogen), at 400 to 800 µg/ml. The medium was changed every 2 to 3 days, and the cells were maintained for at least 3 weeks to obtain stable expression colonies. Ten colonies were identified and homogenized in SET buffer (0.25 M sucrose, 1 mM EDTA, 100 mM Tris-HCl buffer, pH 7.4), and the expression of hCE-1 or hCE-2 was determined by measurement of hydrolase activity, immunoblot analysis, and the molecular weight of a subunit. The background esterase activity in the mock V79 cells (V79 cells transfected by pTARGET), which were used as a control, was extremely low, comparable with the hydrolysis activity in buffer solution. These G418-resistant colonies could be stored in liquid N2 for several months without loss of expression level.
Recombinant hCE-1 was expressed in Sf9 cells using a BAC-TO-BAC Baculovirus Expression System (Invitrogen) according to a previously described procedure (Furihata et al., 2003). The cDNA in the cloning vector was subcloned into the pFAST BAC1 vector using EcoRI and alkaline phosphatase. The pFAST BAC1 vector containing hCE-1 was transformed into DH10Bac cells, and this was followed by transposition of the inserts into bacmid DNA. Likewise, nonrecombinant bacmid DNA (mock) was also prepared. The recombinant and mock bacmid DNAs were separately transfected into Sf9 cells with CELL FECTIN Reagent (Invitrogen), and the virus was harvested 72 h later. The cells were centrifuged at 1700g for 10 min to separate cells and virus. The supernatant containing the virus was stored at 4°C in the dark with 5% fetal bovine serum until required for use. Cells were routinely infected with the virus and were harvested 72 h after infection, washed twice with phosphate-buffered saline, and stored at –80°C until used. Lysates were prepared by disrupting the cells with a sonicator until the cells were completely lysed as determined by microscopy. Cytosol from Sf9 cells expressing hCE-1 and from Sf9 cells infected with mock virus were prepared by subjecting the cell lysate to centrifugation (105,000g for 60 min at 4°C). The expression of hCE-1 was identified by molecular weight of a subunit, immunoblot analysis, and hydrolase activity. The background esterase activity in the mock cells was extremely low, comparable to hydrolysis activity in buffer solution.
The recombinant hCE-1 and hCE-2 showed the same substrate specificity as purified human hCE-1 and hCE-2 in the preliminary experiment (data not shown), indicating expression of actual CES isozyme. Although the recombinant hCE-1 expressed in V79 cells showed the same pattern as hydrolysis of propranolol derivatives by hCE-1 expressed in Sf9 cells, the activity of the hCE-1 expressed in V79 cells was very low because of its low expression. Therefore, the activity of recombinant hCE-1 was evaluated using the recombinant hCE-1 in Sf9 cells.
Hydrolysis Experiments. Hydrolysis experiments were performed using tissue microsomes and the homogenates or cytosol of cells expressing CES. The initial hydrolytic activity was measured under reaction conditions such that less than 25% of substrate was hydrolyzed. Enzyme solutions were diluted to the appropriate concentration with HEPES buffer (50 mM, pH 7.4). The hydrolysis reaction was initiated by the addition of test compounds dissolved in dimethyl sulfoxide after preincubation of each subcellular fraction (200 µl) for 5 min. The final concentration of dimethyl sulfoxide was less than 1%, which has no effect on hydrolase activity. The reaction of benzoic acid derivatives was terminated by the addition of acetonitrile. After centrifugation, the supernatant was injected onto an HPLC column. For betamethasone valerate, propranolol derivatives, and flurbiprofen derivatives, the reaction was terminated by the addition of 5 ml of ethyl acetate. Saturated NaCl (0.5 ml), adjusted to pH 1 by phosphoric acid, was added to reaction samples of flurbiprofen derivatives, whereas 0.5 ml of saturated NaCl, adjusted to pH 4 by phosphate buffer, was added to the reaction samples of propranolol derivatives. After each sample had been shaken for 10 min, the isolated organic phase was evaporated and the residue was redissolved in HPLC mobile phase and injected onto an HPLC column. The rates of hydrolysis of aspirin and methyl salicylate were determined by the increase in fluorescence intensity of salicylic acid at ex 296 nm and em 405 nm over 7 min (Hitachi F-4500; Hitachi, Tokyo, Japan). The rate of hydrolysis of p-nitrophenylacetate was determined by the initial linear increase in absorbance of p-nitrophenol at 405 nm over 3 min (JASCO V530; JASCO Co., Tokyo, Japan). The rate of hydrolysis was determined by subtraction of the hydrolysis rate in buffer from that in tissue microsomes, and by subtraction of the hydrolysis rate in mock/cell preparations from that in CES/cell preparations. Hydrolytic activity was represented as the concentration of hydrolysis product per milligram of protein. Protein content was determined using the method described by Bradford (1976), with bovine serum albumin as standard.
HPLC Analysis. Concentrations of betamethasone, flurbiprofen, propranolol, and benzoic acid derivatives were determined by HPLC. The HPLC system comprised a JASCO PU-980 pump, a JASCO 980-UV detector, a JASCO AS950 autosampler, a JASCO CO-965 column oven, and a JASCO FP-1520S fluorescence detector, and a Shimadzu Chromatopac C-R7A plus (Shimadzu Co., Ltd., Kyoto, Japan). The column and composition of the mobile phase used for each assay are listed in Table 1. All substrates and hydrolysates were clearly separated. Each hydrolysate was measured in a quantitatively linear range.
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Polyacrylamide Gel Electrophoresis. Polyacrylamide gel electrophoresis (PAGE) was performed as described by Mentlein et al. (1980). Polyacrylamide gels (7.5% w/w) containing 1% w/v Nonidet P-40 for solubilization of proteins were used for the separation of native enzymes. After electrophoresis of the microsomes and S9 samples (5–15 µg of protein), the gels were stained for esterase activity with 1-naphthylbutyrate, through coupling of the liberated 1-naphthol with Fast Red TR-salt.
Inhibition Experiments for Hydrolysis of Valeryl-Propranolol Using Anti-hCE-1 Polyclonal Antibody. Anti-hCE-1 polyclonal IgG (Yamada et al., 1994) was purified from anti-hCE-1 rabbit serum using a Protein D column (Funakoshi, Tokyo, Japan). Human liver microsomes (1 mg protein/ml in pH7.4 HEPES buffer) were incubated with 0.5% cholic acid for 30 min on ice. After centrifugation at 10,000g for 30 min at 4°C, the supernatant was diluted to 25 µg protein/ml by 50 mM HEPES buffer (pH7.4) and then incubated with anti-hCE-1 IgG for 30 min at 37°C. The mixtures (200 µl) were allowed to stand for 24 h at 4°C before adding 30 µl of Protein A Sepharose 4 Fast Flow (GE Healthcare, Little Chalfont, Buckinghamshire, UK). After centrifugation at 20,000g for 5 min, the resulting supernatant was used to assay hydrolase activity for valeryl-propranolol. The control activity was measured using control rabbit IgG (Sigma).
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), was found to inhibit the hydrolysis of a variety of compounds tested in this study by more than 90% (data not shown). In comparison with the liver microsomes, the small intestine microsomes showed lower activity for substrates with a large acyl group, for example, temocapril, p-nitrobenzoic acid methylester, and methyl salicylate. In contrast, betamethasone valerate and aspirin, substrates with a small acyl group, were hydrolyzed to either the same or a greater extent in the small intestine microsomes than in the liver microsomes. Furthermore, the substrate with the largest alcohol group, betamethasone valerate, was preferentially hydrolyzed in the small intestine microsomes.
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Hydrolysis of Flurbiprofen Derivatives in Human Liver and Small Intestine Microsomes. Flurbiprofen ethylene glycol ester and trimethylene glycol ester were used as model compounds with large acyl groups. As shown in Fig. 3, both flurbiprofen derivatives were barely hydrolyzed in the small intestine microsomes, while being rapidly hydrolyzed in the liver microsomes. Moreover, the R-isomers were preferentially hydrolyzed in the liver microsomes. The recombinant hCE-1 showed a hydrolysis pattern similar to that of the human liver microsomes, although the latter expressed both hCE-1 and hCE-2. In common with the small intestine microsomes, the recombinant hCE-2 showed only low activity. A significant difference in the enantioselectivity of hydrolysis was not observed in recombinant hCE-2 or the small intestine microsomes due to the low activity observed.
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The major CES isozyme in human liver microsomes was identified using immunoinhibition experiments (Fig. 6). Racemic valeryl propranolol (4) was used as the substrate. Inhibition by anti-hCE-1 IgG (Yamada et al., 1994) was dose-dependent, and hydrolysis of R- and S-isomers was inhibited by 95% and 80%, respectively, at 500 µg/ml anti-hCE-1 IgG. The residual hydrolysis rates were nearly the same for R- and S-isomers, indicating that the residual activity was responsible for hCE-2. These data suggest that the major component of human liver hydrolase activity is hCE-1.
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Hydrolytic activity as a function of affinity for hCE-2 is a normal property of the enzyme reaction. However, hCE-1 showed a nearly identical Km value and increasing Vmax value with alcohol chain length. Since CES catalyzes ester hydrolysis in two steps, the Vmax value might depend on the binding velocity of the acyl group with the serine residue of CES and the releasing velocity of the acyl group from the acyl-enzyme intermediate upon attack of surrounding water. The acyl group for p-aminobenzoic acid derivatives is p-aminobenzoyl in all these substrates. The essential velocity for binding of the p-aminobenzoyl group with the serine residue of hCE-1 might be similar for the three ester compounds, given the invariance of the Km values and their similar structures. However, the velocity of the release of the p-aminobenzoyl group from the acyl-enzyme intermediate might be affected by alcohol released in the first step of the reaction. That is, interference may occur if the released alcohol can attack the acyl-enzyme intermediate. Therefore, p-aminobenzoic acid propyl ester was selected as a substrate, ethanol and butanol were added to the reaction mixture, and production of p-aminobenzoic acid and its ethyl or butyl ester was measured. The results are given in Table 3. Ethanol and butanol inhibited the production of p-aminobenzoic acid in reactions with both recombinant hCE-1 and hCE-2. The inhibition ratio was greater for hCE-1. The formation of ethyl ester on the addition of ethanol was nearly the same for hCE-1 and hCE-2. Interestingly, when the more hydrophobic alcohol, butanol, was added, p-aminobenzoic acid butyl ester was easily formed by hCE-1, but barely by hCE-2. These data suggest that hCE-1 catalyzes transesterification with hydrophobic rather than hydrophilic alcohols, and that hCE-2 possesses low transesterification ability.
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). These findings indicate the predominant contribution of hCE-1 to human hepatic hydrolase activity. Individual levels of both human liver CESs, hCE-1 and hCE-2, are highly variable. Xu et al. (2002) reported a 3-fold range variance for hCE-2 among 13 human liver microsomes, whereas Hosokawa et al. (1995) reported a more than 8-fold range variance of CES1 protein levels among 12 human liver microsomes. Therefore, in the present study, we used pooled human liver microsomes from 10 subjects whose expression levels of hCE-1 and hCE-2 showed interindividual differences. Our results nevertheless indicate that hCE-1 is the main component in human liver.
hCE-1 and hCE-2 exhibit 48% sequence identity, and differing substrate specificities have been proposed. That is, hCE-1 preferentially recognizes a substrate with large acyl and small alcohol moieties, whereas hCE-2 prefers substrates with smaller acyl and bulky alcohol moieties (Bosron and Hurley, 2002; Satoh et al., 2002). Our results regarding hydrolysis by hCE-2 concurred with this proposal; a bulky acyl moiety, such as flurbiprofen, was barely recognized by hCE-2, whereas propranolol derivatives with a small acyl moiety and bulky alcohol group were good substrates for hCE-2. These findings suggested the presence of sterically distorted regions, which affect the formation of an acyl-enzyme intermediate, in the active site of hCE-2. Interestingly, propranolol derivatives 9 and 11 (see Fig. 5) were scarcely hydrolyzed by hCE-2 or human small intestine microsomes. These compounds have a branched acyl moiety with a methyl group at the 3-position. In contrast, compounds 8 and 10 were easily hydrolyzed at almost the same rate as the corresponding straight acyl derivatives, although they possessed methyl groups at the 2-position. In general, the chemical hydrolysis of ester bonds is affected by substitution of the methyl group at the 2-position but not the 3-position, due to steric hindrance. The reduction in the hydrolysis rate resulting from substitution of the methyl group at the 3-position also suggests the presence of conformational interference in the active site of hCE-2.
In contrast to hCE-2, hCE-1 recognized substrates with either large or small acyl moieties. Flurbiprofen derivatives (a bulky acyl moiety) and R-propranolol derivatives (a small acyl moiety and bulky alcohol group) were recognized by hCE-1, although S-propranolol derivatives were poor substrates for hCE-1. Furthermore, acetyl derivatives such as p-nitrophenyl acetate and aspirin were hydrolyzed by both liver and small intestine microsomes (Fig. 1). The crystal structure of hCE-1 has been defined by Bencharit et al. (2003a,b). They reported that the substrate-binding site of hCE-1 was composed of a "small, rigid" pocket and a "large, flexible" pocket; the small, rigid pocket being selective, and the large, flexible pocket being promiscuous with regard to substrate specificity. These pockets allow hCE-1 to act on structurally distinct compounds containing either large or small acyl moieties. Therefore, R-propranolol derivatives might be easily recognized by hCE-1. In preliminary studies, we determined the Km and Vmax values for R- and S-butyryl-propranolol in human liver microsomes. The Km value (22.1 ± 0.8 µM) for the S-isomer was 10-fold smaller than that for the R-isomer (251 ± 18.8 µM), indicating the higher affinity of the S-enantiomer for hCE-1, despite the lower hydrolysis rate of the S-isomer. The Vmax value for S-butyryl-propranolol (95.7 ± 23.4 nmol/min/mg protein) was much smaller than that obtained for the R-enantiomer (1580 ± 4.0 nmol/min/mg protein). These preliminary findings suggest that both the R- and S-enantiomers are recognized by hCE-1, although their reactivity may be greatly affected by the conformational orientation upon incorporation into the active site pocket of hCE-1. It has been reported that S-cocaine and cis-cypermethrin analogs are poor substrates for hCE-1, in contrast to R-cocaine and trans-cypermethrin analogs (Brzezinski et al., 1997; Huang et al., 2005). The differences in hydrolysis rate between these enantiomers have been explained by steric clashes with the loop containing Gly142 and Gly143 in the rigid pocket (Bencharit et al., 2003b; Huang et al., 2005), where Gly residues form the oxianion hole (140–144) to stabilize the transition state of substrate via their amide nitrogen. Bencharit et al. (2003b) also determined the Z-site surface ligand-binding site for an inactive substrate. This Z-site is formed by interdigitation of the 
1 and 2 loops adjacent to the active site of hCE-1. The enantioselective hydrolysis of propranolol derivatives is possibly explained by enantiomerically distinct active site orientations in hCE-1 due to structural clashes. Structural analysis will be required to establish the detailed nature of such enantioselectivity.
It was interesting that the substrate requirements for hydrolysis of various benzoate derivatives by hCE-1 and hCE-2 were quite different. Such substrate specificity might be explained by conformational fitting of substrates in the active sites. However, we propose that transesterification via hCE-1 may be due to different hydrolytic mechanisms in hCE-1 and hCE-2. CES-catalyzed hydrolysis proceeds in two steps. For benzoate derivatives, the first step is the formation of a covalent benzoyl-CES intermediate. At the same time, the released alcohol moiety is present in the active site gorge. Subsequently, the benzoyl-CES intermediate is attacked by histidine-activated water, and benzoic acid is released from the enzyme. Bencharit et al. (2002) reported the crystal structure of rabbit liver CES, in which 4-piperidino piperidine, a product of CPT-11 activation, was bound between the first N-acetylglucosamine of the Asn389 glycosylation site and the Trp550 side-chain of the C-terminal helix. There are two N-linked glycosylation sites in rabbit liver CES at Asn residues 79 and 389; Asn389 is modified via a long carbohydrate chain and plays a role as a novel exit pore for the release of small products from the active site of the enzyme. hCE-2 contains glycosylation sites at two positions (residues 103 and 267). Although there are no crystallographic data for hCE-2, one of these two glycosylation sites might function as an exit pore for the release of alcohol.
In contrast to hCE-2, hCE-1 maintains a glycosylation site at Asn79 but not at residue 389. Asn79 is modified by two N-acetylglucosamine groups and appears to be involved in the stabilization of the hCE-1 trimer by packing into the adjacent monomer (Bencharit et al., 2003a,b). hCE-1 is also capable of transesterifying cocaine in the presence of ethanol to cocaethylene (Brzezinski et al., 1994). During the two-step hydrolysis of cocaine, hCE-1 forms a covalent acyl-enzyme intermediate at the carboxylic methyl ester position of cocaine, which is then attacked by ethanol to create cocaethylene. Bencharit et al. (2003b) proposed the following mechanism based on their X-ray crystalline analysis. Ethanol enters the active site of hCE-1 through the side-door secondary pore adjacent to the large, flexible substrate-binding pocket. The entrance to the side-door secondary pore on the surface of hCE-1 is lined by structurally flexible residues including both ß-strands (ß14 and ß15) and random coils. This flexibility may facilitate the passage of small molecules through the side-door. The alcohol produced may also be released from this side-door. The presence of the side-door secondary pore adjacent to the large, flexible substrate-binding pocket in hCE-1 might prolong the presence of the alcohol molecule so that the alcohol can attack the benzoyl-hCE-1 intermediate to reproduce the original substrate. Furthermore, the transition time of alcohol in hCE-1 might be prolonged with increasing hydrophobicity due to interaction of alcohol with the flexible site of hCE-1. This may be another explanation for the apparently slow hydrolytic rate of substrates with large alcohol moieties by hCE-1. Conversely, if hCE-2 can release an alcohol from the glycosylation site, its transesterification activity may be extremely low. This would agree with the observation of the low transesterification ability of hCE-2.
Although our proposal is a speculative mechanism of hydrolysis of hCE-1 and hCE-2, the fact that hCE-1 possesses transesterification activity suggests that, in addition to the binding structure of the substrate, this property should be considered in a complete analysis of the hydrolytic mechanism.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: CES, carboxylesterase; CPT-11, camptothecin-11 (irinotecan); hCE-1, human carboxylesterase-1; hCE-2, human carboxylesterase-2; HPLC, high-performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.
Address correspondence to: Dr. Teruko Imai, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-Honmachi, Kumamoto 862-0973, Japan. E-mail: iteruko{at}gpo.kumamoto-u.ac.jp
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