Methylphenidate is stereoselectively hydrolyzed by human carboxylesterase CES1A1

J Pharmacol Exp Ther. 2004 Aug;310(2):469-76. doi: 10.1124/jpet.104.067116. Epub 2004 Apr 13.


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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Carboxylic Ester Hydrolases / biosynthesis*
  • Carboxylic Ester Hydrolases / genetics
  • Cell Line
  • Gene Expression Regulation, Enzymologic / drug effects
  • Gene Expression Regulation, Enzymologic / physiology
  • Humans
  • Hydrolysis / drug effects
  • Insecta
  • Isoenzymes / biosynthesis
  • Isoenzymes / genetics
  • Liver / enzymology
  • Methylphenidate / chemistry*
  • Methylphenidate / metabolism*
  • Stereoisomerism


  • Isoenzymes
  • Methylphenidate
  • Carboxylic Ester Hydrolases
  • CES1 protein, human