Glucuronide and glucoside conjugation of mycophenolic acid by human liver, kidney and intestinal microsomes

Abstract

Mycophenolic acid (MPA) is primarily metabolized to a phenolic glucuronide (MPAG) as well as to two further minor metabolites: an acyl glucuronide (AcMPAG) and a phenolic glucoside (MPAG1s). This study presents investigations of the formation of these metabolites by human liver (HLM), kidney (HKM), and intestinal (HIM) microsomes, as well as by recombinant UDP-glucuronosyltransferases. HLM (n=5), HKM (n=6), HIM (n=5) and recombinant UGTs were incubated in the presence of either UDP-glucuronic acid or UDP-glucose and various concentrations of MPA. Metabolite formation was followed by h.p.l.c. All microsomes investigated formed both MPAG and AcMPAG. Whereas the efficiency of MPAG formation was greater with HKM compared to HLM, AcMPAG formation was greater with HLM than HKM. HIM showed the lowest glucuronidation efficiency and the greatest interindividual variation. The capacity for MPAGls formation was highest in HKM, while no glucoside was detected with HIM. HKM produced a second metabolite when incubated with MPA and UDP-glucose, which was labile to alkaline treatment. Mass spectrometry of this metabolite in the negative ion mode revealed a molecular ion of m/z 481 compatible with an acyl glucoside conjugate of MPA. All recombinant UGTs investigated were able to glucuronidate MPA with K:(M:) values ranging from 115.3 to 275.7 microM l(-1) and V(max) values between 29 and 106 pM min(-1) mg protein(-1). Even though the liver is the most important site of MPA glucuronidation, extrahepatic tissues particularly the kidney may play a significant role in the overall biotransformation of MPA in man. Only kidney microsomes formed a putative acyl glucoside of MPA.

Figure 1

(a) Representative chromatogram of a kidney microsome preparation incubated in the presence of 1 mM MPA and 4 mM UDPG1s. The sample was pre-treated and chromatographed as described in Methods. Compounds eluting from the column were monitored continuously with a diode array detector. This chromatogram illustrates the u.v. absorption at 215 nm. The 7-O glucoside metabolite (MPAG1s) eluted after 4.0 min. A second, as yet unknown metabolite was detected with a retention time of 8.2 min. This metabolite had a very similar u.v. absorption spectrum to that of MPAG1s (shown in the inset). (b) Mass spectra of a kidney microsome preparation incubated in the presence of 1 mM MPA and 4 mM UDPG1s. The sample was pre-treated as described in Methods and introduced into the ion source after h.p.l.c. separation. An extracted ion chromatogram of multiple reaction monitoring (MRM)-transitions of 481/319 amu showed two peaks with retention times of 4.2 and 7.8 min respectively.

Figure 2

Proposed structural formulas of the phenolic and the acyl MPA glucosides.