Review Human Carboxylesterase Isozymes: Catalytic Properties and Rational Drug Design
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Drug Metab. Pharmacokinet. 21 (3): 173–185 (2006). Review Human Carboxylesterase Isozymes: Catalytic Properties and Rational Drug Design Teruko IMAI Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk Summary: Human carboxylesterase 1 (hCE-1, CES1A1, HU1) and carboxylesterase 2 (hCE-2, hiCE, HU3)areaserineesteraseinvolvedinbothdrugmetabolismandactivation.AlthoughbothhCE-1and hCE-2 are present in several organs, the hydrolase activity of liver and small intestine is predominantly attributed to hCE-1 and hCE-2, respectively. The substrate speciˆcity of hCE-1 and hCE-2 is signiˆcant- ly diŠerent. hCE-1 mainly hydrolyzes a substrate with a small alcohol group and large acyl group, but its wide active pocket sometimes allows it to act on structurally distinct compounds of either large or small alcohol moiety. In contrast, hCE-2 recognizes a substrate with a large alcohol group and small acyl group, and its substrate speciˆcity may be restricted by a capability of acyl-hCE-2 conjugate formation due to the presence of conformational interference in the active pocket. Furthermore, hCE-1 shows high transesteriˆcation activity, especially with hydrophobic alcohol, but negligible for hCE-2. Transesteriˆ- cation may be a reason for the substrate speciˆcity of hCE-1 that hardly hydrolyzes a substrate with hydrophobic alcohol group, because transesteriˆcation can progress at the same time when a compound is hydrolyzed by hCE-1. From the standpoint of drug absorption, the intestinal hydrolysis by CES during drug absorption is evaluated in rat intestine and Caco2-cell line. The rat in situ single-pass perfusion shows markedly exten- sive hydrolysis in the intestinal mucosa. Since the hydrolyzed products are present at higher concentra- tion in the epithelial cells rather than blood vessels and intestinal lumen, hydrolysates are transported by a speciˆc eŒux transporter and passive diŠusion according to pH-partition. The expression pattern of CES in Caco-2 cell monolayer, a useful in vitro model for rapid screening of human intestinal drug absorption, is completely diŠerent from that in human small intestine but very similar to human liver that expresses a much higher level of hCE-1 and lower level of hCE-2. Therefore, the prediction of human intestinal absorption using Caco-2 cell monolayers should be carefully monitored in the case of ester and amide-containing drugs such as prodrugs. Further experimentation for an understanding of detailed substrate speciˆcity for CES and develop- ment of in vitro evaluation systems for absorption of prodrug and its hydrolysates will help us to design the ideal prodrug. Key words: carboxylesterase; prodrug; hydrolysis; substrate speciˆcity; intestinal absorption requirements because they play an important role in Introduction biotransformation of a variety of ester-containing drugs The hydrolase activity within several tissues is increas- and prodrugs4–6) such as an angiotensin-converting en- ingly used as the basis for drug design, particularly on zyme inhibitor (temocapril, cilazapril, quinapril and im- prodrugs and softdrugs containing functional groups idapril),7) anti-tumor drugs (CPT-11 and capecitabin)8,9) such as carboxylic acid ester.1–3) Introduction of an ester and narcotics (cocain, heroin and meperidine).10,11) linkage generally improves the bioavailability of ther- CESs are members of the aWb hydrolase fold family apeutic agents due to increased passive transport and show ubiquitous tissue expression proˆles with high following oral administration. Carboxylesterases levels in the liver, small intestine and lung.12,13) The (CESs, EC.3.1.1.1) are essential to achieve these mammalian CESs comprise a multigene family, and the Received; March 14, 2006, Accepted; June 6, 2006 To whom correspondence should be addressed: Teruko IMAI, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe- honmachi, Kumamoto 862-0973, Kumamoto, Japan. Tel. & Fax. ＋81-96-371-4626, E-mail: iteruko＠gpo.kumamoto-u.ac.jp 173 174 Teruko IMAI Table 1. MolecularpropertyofhCE-1andhCE-2 hCE-1 ref hCE-2 ref Molecular weight 180 kDa 18, 32 60 kDa 10 Subunit weight 60 kDa Isoelectric point 5.6–5.8 18, 32 4.8–5.0 10 Glycosylation site Asn-X-Thr 10 Asn-X-SerWThr 10 Asn79 Asn103, Asn267 Optimal pH 6.5 18 7.5–8.0 18 isozymes are classiˆed into four main CES groups (CES1-CES4) and several subgroups according to the homology of the amino acid sequence.10,12) The majority of CESs has been segregated into the CES1 and CES2 families. Recent studies have shown some diŠerences between these CES1 and CES2 families in terms of sub- strate speciˆcity, tissue distribution, immunological properties and gene regulation. Fig. 1. Contribution of carboxylesterase in the hydrolase activity of This review focuses on the molecular characteristics humanliverandsmallintestine. of human CES and the contribution of CES isozymes in Each column represents a relative activity for hydrolysis of p- the hydrolase activity of the human liver and small nitrophenylacetate as percent of control hydrolase activity in human intestine. In addition, it explains the extensive intestinal liver and small intestine. ``Hydrolysis by CES'' is represented as an inhibited hydrolase activity after inhibition by bis-p-nitrophenyl ˆrst-pass hydrolysis and the di‹culty of prediction of phosphate. Black and hatched column represent a relative hydrolase human intestinal absorption of ester-containing drugs activity of hCE-1 and hCE-2, respectively. using Caco-2 cells. Tissue Distribution of Human Carboxylesterase is detected in the blood of humans.22–24) Recent molecular biology techniques have identiˆed Contribution of Carboxylesterase on Hydrolase the two major human CESs, hCE-1 (CES1A1, HU1), a Activity in the Liver and Small Intestine human CES1 family isozyme, and hCE-2 (hiCE, HU3), a human CES2 family isozyme. hCE-1 is highly The hydrolase activity in the liver and small intestine expressedintheliverandalsoobservedinmacrophages, in mammals is attributable to several esterase molecules. human lung epithelia,14) heart, testis and other tissues.13) However, when native PAGE gel of microsomes is hCE-1 expression is markedly low in the gastrointestinal stained by esterase activity using 1-naphthylacetate, tract. hCE-2 is present in the small intestine, colon, predominant bands are corresponding CES isozymes.25) kidney, liver, heart, brain and testis. hCE-2 expression Also, 80–95z andmorethan90z of hydrolase activity is essentially absent in all other organs.15,16) hCE-1 and is inhibited by bis-p-nitrophenyl phosphate, a speciˆc hCE-2 share 48z amino acid sequence identity and are CES inhibitor,26,27) in rat liver and small intestine,28) predicted to be glycoproteins of 60kDa.17,18) Some respectively. The content of CES in rat liver is found to molecular properties of hCE1 and hCE2 are listed in be about 1 mg per g of fresh tissue while microsomal Table 1. A third, brain-speciˆc CES was isolated in fraction contains about 30 mg of CES per g of 199919) andtermedhBr3(hCE3).However,relatively microsomal protein.29) Thus, CES activity is signiˆcant- little characterization of this CES has been reported. ly high in comparison with other esterases. Although the hBr3 shares 77z and 49z sequence identity with hCE-1 liver and small intestine of mammals contains CES1 and and hCE-2, respectively. Furthermore, a new class of CES2 enzymes, no CES isozyme is present in the small human CES, CES3, was isolated in 2004.20) CES3 has intestine of the beagle dog. Therefore, the beagle dog about 40z identity with both hCE-1 and hCE-2, and is shows a diŠerent absorption behavior of some drugs expressed in the liver and gastrointestinal tract at an containing esters such as prodrugs in comparison with extremely low level in comparison with hCE-1 and other animals. hCE-2.20,21) In humans, the native PAGE gel of tissue microsomes Typically, expression of CES is maximal in the shows only one band (hCE-2) for human small intestine epithelia of most organs, suggesting that these enzymes but two bands (hCE-1, upper strong band; hCE-2, play a protective role against xenobiotics. In addition, lower weak band) for human liver after visualization of although high levels of CES activity can be detected in esterase activity with staining by 1-naphthylacetate, the blood of the majority of mammals, no such activity indicating the predominant esterase is CES isoforms in Hydrolysis of Prodrug by Carboxylesterase 175 Fig. 2. The two-step catalytic mechanism of mammalian carboxylesterase. Bold arrow shows the typical route of hydrolysis. In the ˆrst step (Step 1), the active site serine hydroxyl group attacks the carboxylester linkage in the substrate to generate the alcohol product and the covalent acyl-enzyme intermediate. The second step (Step 2), the second substrate, water, attacks the covalent acyl-enzyme intermediate to produce acyl product, and serine residue changes to original state. However, when an alcohol presents in abundance, the enzyme can facilitate a transesteriˆcation reaction to generate a new ester product. The alcohol produced in ``Step1'' is possible to react with acyl-enzyme intermediate to generate an original ester compound. human liver and small intestine.30) The hydrolysis much higher than hCE2 even by considering the pattern for several substrates in the human small intes- variation of expression level. tine microsomes is nearly the same as those of recom- Carboxylesterase Reactions binant hCE-2, as expected by result from the native PAGE.30) Although human liver microsomes express Catalysis of ester cleavage by CESs is base-mediated, both hCE-1 and hCE-2, their substrate speciˆcity requiring water as a co-reactant. This reaction is closely resembles recombinant hCE-1. Furthermore, achieved via a triad of catalytic amino acids (Ser, His anti-hCE-1 antibody inhibited 80–95z of the hepatic and Glu) that are all essential for enzymatic activity.12) hydrolysis.