High-affinity stereoselective reduction of the enantiomers of ketotifen and of ketonic nortriptyline metabolites by aldo-keto reductases from human liver

Aldo-keto reductases (AKR) form an enzyme superfamily catalyzing the reduction of carbonyl compounds and in some cases the reverse oxidation of alcohols as well. In particular, a role in drug metabolism has been considered for the AKR1C family, but published data failed to reveal Low K-m drug substrates. Moreover, structure-activity relationships using chemically related substrates have not been established. In the present investigation, a modified procedure was developed for the isolation of AKR1C1, 1C2, and 1C4 (dikydrodiol dehydrogenases 1, 2, and 4) from human liver cytosol along with carbonyl reductase (EC 1.1.1.184), a member of the short-chain alcohol dehydrogenase superfamily. The kinetics of NADPH-dependent reduction by the closely related enzymes AKR1C1 and 1C2 were studied with the structurally similar substrates (R)- and (S)-ketotifen and E- and Z-10-oxonortriptyline by HPLC measurement of the products. K-m Values varied between 2.6 and 53 mu M and V-max values between 5 and 313 mU/mg protein; substrate inhibition with K-i around 30 mu M occurred in the reduction of E- and Z-10-oxonortriptyline by AKR1C1. The reactions were strictly stereospecific with production of one enantiomeric alcohol from each ketotifen enantiomer and of the (+)-enantiomers of E- and Z-10-hydroxynortriptyline. Enzymatic NADP(+)-dependent oxidation of the alcohols mirrored the reduction with regard to stereochemical specificity. All four ketones were no or poor substrates of carbonyl reductase, whereas haloperidol was reduced by this enzyme with low affinity, but high efficiency. (C) 1999 Elsevier Science Inc.