Hydrolysis of Ester-Type Drugs by the Purified Esterase From Human Intestinal Mucosa

Abstract

Abstract-Esterase from human intestinal mucosa was purified 210 fold by solubilization with Triton X-100, chromatography on DEAF-cellulose, Sephadex G-100 and hydroxy lapatite, and isoelectric focusing. The purified esterase showed a single band by polyacrylamide gel electrophoresis.The molecular weight of the purified esterase was estimated to be about 55,000 by gel filtration on Sephadex G-150, and the isoelectric point was 5.02. The purified esterase was strongly inhibited by diethyl p-nitrophenyl phosphate (E-600) and diisopropyl fluorophosphate (DFP), and was not inhibited by eserine sulfate and p-chloromercuribenzoate.The purified esterase from human intestinal mucosa was found to be one of the carboxylesterases.The purified esterase hydrolyzed ester-type drugs, i.e., aspirin, clofibrate, indanyl carbenicillin and procaine, but did not hydrolyze amide-type drugs and choline-type drugs.Carboxylesterase hydrolyzing carboxyl esters, thiol esters and aromatic amides, are found in most animal tissues (1). The esterases have been purified from human liver and brain (2-3). There are however, only a few reports on intestinal esterases (4-5) and there are apparently few reports regarding these drug-metabolizing activities and detailed properties.We reported elsewhere that the intestinal estrase was present in the absorption site of the intestine, and the intestinal esterase differed characteristically from hepatic esterase (6).Furthermore, there was a difference in intestinal esterase activities among several species of animals (6). Human intestinal esterase hydrolyzes ester-type drugs to a large extent during absorption. Recently, a variety of estertype drugs is being used in many ways as prodrugs (7). Reported herein is the purification of esterase from human intestinal mucosa and examination of the enzyme properties. MATERIALS AND METHODS Crude EnzymeThe small intestine (jejunum) from humans was obtained at autopsy 12 hr after death.It was excised, shown to be non-pathologic, frozen and kept at -40'C until use . The intestinal mucosa was collected by scraping and was homogenized in Tris-mannitol medium;Tris-HC1 buffer (pH 7.4) containing 0.278 M mannitol at O 'C. Assay of Esterase ActivityEsterase and lipase activities were determined according to the methods described previously using /3-naphthyl derivatives, phenyl acetate (PA), tributyrin and olive oil as substrates (8-9). The esterase activities using acetylsalicylic acid (aspirin), ethylchloro phenoxyisobutyrate (clofibrate, CPIB) and indanyl carbenicillin (I-CBPC) as substrates were determined according to the methods described previously (6). Procaine and procainamide hydrolyzing activity was assayed at 37'C for 30 min by incubating with 1 mM procaine, procainamide as substrates and the product formed was determined according to the method of Tobin et al. (10). Acetanilide and phenacetin-hydrolyzing activity was determined by measuring the formation of aniline by the method of Krisch (11). Lidocaine-hydrolyzing activity was determined according to the met...

Carboxylesterases: Structure, Function and Polymorphism

“…For example, Inoue et al (Inoue et al, 1979a) showed that esterase activity in the dog intestine is very weak and produced no appreciable active band in a disk electrophoresis coupled with staining of esterase activity. On the other hand, esterase activities were observed in the intestines of other species (human, rat, mouse, guinea pig and rabbit) (Inoue et al, 1979b;Prueksaritanont et al, 1996;Mansbach and Nevin, 1998;Imai et al, 2006;Satoh and Hosokawa, 2006) and found to produce a few active bands in an electrophoretic assay.…”

Bioactivation of Capecitabine in Human Liver: Involvement of the Cytosolic Enzyme on 5′-Deoxy-5-Fluorocytidine Formation

“…1): carboxylesterase (CES), cytidine deaminase (CDA), and thymidine phosphorylase (TP). In humans, CES is mainly expressed in the liver and intestine (Junge et al, 1974;Inoue et al, 1979). CDA is found in most tissues, including the liver and in tumor tissues (Camiener and Smith, 1965).…”

Comparative study of human intestinal and hepatic esterases as related to enzymatic properties and hydrolizing activity for ester-type drugs.

“…There are, however, only a few reports on the purification and properties of human intestinal esterase and hepatic esterase (3). We have already reported the purification of esterase from human intestinal mucosa (4,5). When esterase from intestinal mucosa has been examined for its action in the metabolism of ester-type drugs in the body, esterase from liver must also be considered.…”