Methylphenidate is stereoselectively hydrolyzed by human carboxylesterase CES1A1

Zejin Sun, Daryl J. Murry, Sonal P. Sanghani, Wilhelmina I. Davis, Natalia Y. Kedishvili, Qin Zou, Thomas D. Hurley, William F. Bosron

Research output: Contribution to journalArticlepeer-review

149 Scopus citations


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 (kcat/Km = 7.7 mM-1 min -1) is greater than that of d-methylphenidate (kcat/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.

Original languageEnglish (US)
Pages (from-to)469-476
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Issue number2
StatePublished - Aug 2004
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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