title: Methylphenidate is stereoselectively hydrolyzed by human carboxylesterase CES1A1.
abstract: Methylphenidate is an important stimulant prescribed to treat attention-deficit hyperactivity disorder. It has two chiral centers, but most current commercial formulations consist of the racemic mixture of the threo pair of methylphenidate isomers (d-, l-threo-methylphenidate). The d-isomer is the pharmacologically active component. Numerous studies reported that oral administration of the methylphenidate racemate undergoes first-pass, stereoselective clearance in humans with l-methylphenidate being eliminated faster than d-methylphenidate. Accordingly, the kinetics of hydrolysis of individual enantiomers by purified native and recombinant human liver carboxylesterases CES1A1 and CES2 and a colon isoenzyme CES3 were examined with a liquid chromatography/mass spectrometry assay. The expression of CES1A1, CES2, and CES3 in Sf9 cells and the methods for purification of the three isoenzymes are reported. CES1A1 has a high catalytic efficiency for both d- and l-enantiomers of methylphenidate. No catalytic activity was detected with CES2 and CES3 for either enantiomer. The catalytic efficiency of CES1A1 for l-methylphenidate (k(cat)/K(m) = 7.7 mM(-1) min(-1)) is greater than that of d-methylphenidate (k(cat)/K(m) = 1.3-2.1 mM(-1) min(-1)). Hence, the catalytic efficiency of CES1A1 for methylphenidate enantiomers agrees with stereoselective clearance of methylphenidate reported in human subjects. Both enantiomers of methylphenidate can be fit into the three-dimensional model of CES1A1 to form productive complexes in the active site. We conclude that CES1A1 is the major enzyme responsible for the first-pass, stereoselective metabolism of methylphenidate.
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 1345 W 16th Street, L3-304, Indianapolis, IN 46202-2111, USA.
authors.forename: Zejin
authors.initials: Z
authors.lastname: Sun
authors.forename: Daryl J
authors.initials: DJ
authors.lastname: Murry
authors.forename: Sonal P
authors.initials: SP
authors.lastname: Sanghani
authors.forename: Wilhelmina I
authors.initials: WI
authors.lastname: Davis
authors.forename: Natalia Y
authors.initials: NY
authors.lastname: Kedishvili
authors.forename: Qin
authors.initials: Q
authors.lastname: Zou
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Hurley
authors.forename: William F
authors.initials: WF
authors.lastname: Bosron
mesh_terms: D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D007313:Insecta; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D008099:Liver / Q000201:enzymology; D008774:Methylphenidate / Q000737:chemistry* / Q000378:metabolism*; D013237:Stereoisomerism
chemical_list: D007527:Isoenzymes; D008774:Methylphenidate; D002265:Carboxylic Ester Hydrolases; C474609:CES1 protein, human