Marked enantioselective protein binding in humans of ketorolac in vitro: Elucidation of enantiomer unbound fractions following facile synthesis and direct chiral hplc resolution of tritium‐labelled ketorolac

Peter J. Hayball, Jeffrey W. Holman, Roger L. Nation, Ralph A. Massy‐Westropp, David P.G. Hamon

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The protein binding of the enantiomers of the nonopiate analgesic, ketorolac, was investigated in vitro using human plasma and solutions of human serum albumin (HSA) at physiological pH and temperature. In order to detect the very low levels of unbound enantiomers in protein solutions, tritium‐labelled rac‐ketorolac was synthesised by regiospecific isotopic exchange of the parent drug with tritiated water as the isotope donor. Radio‐chemical purification of this compound by reversed‐phase HPLC followed by direct resolution using a chiral α1‐acid glycoprotein (Chiral‐AGP) HPLC column afforded labelled enantiomers of high specific activity. The in vitro use of (R)‐ and (S)‐[3H4]ketorolac enabled reproducible radiometric detection of enantiomers in protein solution ultrafiltrate. The unbound fractions of (R)‐ and (S)‐ketorolac [fu(R) and fu(S), respectively] were determined when drug was added to various plasma or albumin solutions as either the separate enantiomers or as the racemate. Over an enantiomeric plasma concentration range of 2.0—15.0 μg/ml, fu(S) (mean range: 1.572—1.795%) was more than 2‐fold greater (P < 0.001) than fu(R) (mean range: 0.565—0.674%). Both fu(R) and fu(S) were constant over this concentration range, and each was unaffected by the presence of the corresponding antipode (P > 0.05). At a concentration of 2.0 μg/ml in 40.0 g/liter fatty acid‐free HSA, fu(R) and fu(S) were approximately 0.5 and 1.1%, respectively, and both values declined with increasing concentrations of the long chain fatty acid, oleic acid. We have previously shown that the pharmacokinetics of ketorolac in humans are markedly enantioselective and suggest in this report that these differences are largely the result of substantial differences in the protein binding of ketorolac enantiomers. These findings stress the importance of monitoring the unbound concentrations of the enantiomers of chiral drugs if correct interpretations are to be made of enantioselective pharmacokinetic data. © 1994 Wiley‐Liss, Inc.

Original languageEnglish
Pages (from-to)642-648
Number of pages7
Issue number8
Publication statusPublished - 1 Jan 1994
Externally publishedYes


  • chiral drugs
  • enantiomers
  • enantioselectivity
  • fatty acids
  • human serum albumin
  • ketorolac
  • nonsteroidal antiinflammatory drugs
  • oleic acid
  • pharmacokinetics
  • protein binding

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