Comparative N-glucuronidation kinetics of ketotifen and amitriptyline by expressed human UDP-glucuronosyltransferases and liver microsomes

Like other basic amphiphilic drugs, the (S)-enantiomer of the antiallergic drug ketotifen exhibited biphasic kinetics when it was converted to two iso meric quaternary ammonium-linked glucuronides in human liver microsomes. Fo r (R)-ketotifen this applied when incubations were carried out in the absen ce of a detergent. Two UDP-glucuronosyltransferases (UGTs) present in human liver, UGT1A4 and UGT1A3, were previously shown to catalyze tertiary amine N-glucuronidation when expressed in HK293 cells. Therefore, the conjugatio n kinetics of (R)- and (S)-ketotifen were investigated with the two express ed proteins. When homogenates of HK293 cells expressing UGT1A4 were incubat ed without detergent, N-glucuronidation kinetics were monophasic with K-M v alues of 59 +/- 5 mu M for (R)- and 86 +/- 26 mu M for (S)-ketotifen. In ex periments with membranes containing expressed UGT1A3, somewhat higher K-M v alues were obtained. These values correspond to the high rather than to the low K-M components of ketotifen glucuronidation in liver microsomes, the l atter exhibiting K-M values around 2 and 1 mu M, respectively, with (R)- an d (S)-ketotifen. With amitriptyline as the substrate, N-glucuronidation kin etics in the absence of detergent were biphasic in human liver microsomes a nd monophasic with a high K-M value in cell homogenates containing UGT1A4. The results suggest that UGT1A4 and UGT1A3 catalyze high-K-M N-glucuronidat ion of tertiary amine drugs, whereas the low-K-M reaction requires either a n alternative enzyme or a special conformation of UGT1A4 or UGT1A3 that can be attained in liver microsomes, but not in HK293 cell membranes.