Molecular cloning of the baboon UDP-glucuronosyltransferase 2B gene family and their activity in conjugating morphine.

Glucuronidation by UDP-glucuronyltransferase 2B enzymes (UGT2Bs) is a major pathway for the elimination of endobiotics and xenobiotics, including therapeutic drugs. Morphine, a probe drug for UGT2B7, is metabolized to morphine-3-beta-glucuronide (M3G) and morphine-6-beta-glucuronide (M6G) in humans. Morphine has been used in a series of experiments in the baboon to characterize developmental changes in fetal glucuronidation. This study identifies the baboon UGT2B family of enzymes, compares them with that of the human and the monkey (Macaca fascicularis), and measures the activity of the individual baboon UGT2Bs toward morphine. UGT2B cDNAs were cloned from the liver of adult and newborn baboons and expressed in human embryonic kidney 293 cells. The UGT activity toward morphine was assessed by the rate of formation of M3G and M6G by high-performance liquid chromatography. Eight baboon UGT2Bs were cloned and identified: UGT2B41 and UGT2B42, which are 90% homologous to human UGT2B4; UGT2B43, which is 93% homologous to human UGT2B15; and UGT2B39, UGT2B40, UGT2B44, UGT2B45, and UGT2B46, which are 89 to 91% homologous to human UGT2B7. Homology between baboon and monkey UGT2B ranged from 92.6 to 99.1%, with the primary protein structure of UGT2B43 being 99.1% identical to monkey UGT2B20, including a unique R96I substitution. Gene conversion interfered with the phylogenetic signal in the baboon UGT2B7-like and the monkey UGT2B4-like groups and led to concerted evolution of these enzymes. All of the baboon UGT2Bs metabolized morphine to both M3G and M6G. This study lays the foundation for investigating the regulation of UGT2B enzymes during fetal and neonatal development in the baboon.

Placental transfer and fetal elimination of morphine-3-beta-glucuronide in the pregnant baboon.

The glucuronide metabolites of several widely used drugs are detected in fetal plasma after maternal drug administration. However, the disposition of these metabolites is poorly understood and clinical concerns have been raised about accumulation of active metabolites in the fetus. For this reason, morphine-3-beta-glucuronide (M3G), an active metabolite of morphine, was studied to provide quantitative data on disposition. Maternal, fetal, and bidirectional placental clearances of M3G were measured in three pregnant baboons. During maternal infusion of M3G to steady-state, the glucuronide metabolite readily appeared in fetal plasma achieving a mean +/- S.D. fetal-to-maternal concentration ratio of 0.79 +/- 0.04. In paired maternal and fetal infusions, steady-state clearances were 53 +/- 3 (maternal), 1.5 +/- 0.5 (maternal-to-fetal), 2.6 +/- 0.1 (fetal-to-maternal), and -0.70 +/- 0.6 ml x min(-1) (fetal). These clearance values support bidirectional transfer of M3G across the placenta and indicate negligible direct clearance from the fetus. The clearance of M3G across the placenta is more than 20-fold less than that of morphine. Despite this low index of permeability, placental transfer contributes significantly to the glucuronide pool in the fetus. Placental transfer emerges as the major clearance pathway for the glucuronide from the fetus and suggests a component of active efflux. What is more, the results do not support the concept of sequestration in the fetal intestine as a significant route of clearance. Together these results clarify the distribution and clearance of glucuronides in the pregnant primate and facilitate prediction of fetal exposure to active metabolites.

Molecular cloning of the baboon UDP-glucuronosyltransferase 1A gene family: evolution of the primate UGT1 locus and relevance for models of human drug metabolism.

BACKGROUND: Glucuronidation by the UDP glucuronosyltransferase 1A enzymes (UGT1As) is a major pathway for elimination of drugs and endogenous substances, such as bilirubin. OBJECTIVE: To identify the baboon UGT1A gene family, compare it with that of the human, and evaluate the baboon as a model for human glucuronidation. METHODS AND RESULTS: Aligning the human and baboon UGT1 loci identified rearrangements occurring since the divergence of baboons and humans. The baboon UGT1A cDNAs were cloned and shown to have an orthologous relationship with several genes in the human UGT1A family. This indicates that most protein encoding UGT1A first exons were duplicated before the divergence of baboons and humans. Gene conversions interfered with the phylogenetic signal for exons 1A4, 1A5, and 1A10, and led to concerted evolution of exon groups 1A2-1A5 and 1A7-1A13. The activity of the baboon UGT1As resembled those of their human counterparts in glucuronidating endobiotics, such as serotonin, bilirubin, and various xenobiotics. CONCLUSION: These insights demonstrate that the baboon has significant clinical relevance as a model for examining toxicological metabolism in humans.

Fetal morphine metabolism and clearance are constant during late gestation.

Fetal metabolism significantly contributes to the clearance of drugs from the fetus. To understand how the changes in fetal metabolism expected in late gestation alter fetal drug clearance, serial measurements of morphine metabolism were made in the fetal baboon over the latter third of gestation. Clearance and metabolism were evaluated in the context of fetal growth, onset of labor, and the administration of classical enzyme induction agents. Morphine, a probe substrate for the enzyme uridine diphosphate glucuronosyltransferase 2B7 (UGT2B7), was continuously infused to chronically catheterized fetal baboons while measuring morphine, morphine-3-beta-glucuronide, and morphine-6-beta-glucuronide concentrations. In some animals, intermittent infusions of the metabolites provided estimates of metabolite clearance and, hence, the rate of formation of metabolites and metabolic clearance. Overall, metabolic clearance of morphine from the fetus was 27 +/- 9.0 ml x min(-1) or 32% of total clearance. This is similar to the overall clearance in the adult baboon when standardized to weight. No change in any measure of metabolism or clearance of morphine or its glucuronide metabolites was found with gestational age, the presence of labor, or administration of UGT enzyme induction agents. Interpreting these findings using a physiologically based approach suggests that the intrinsic clearance of the fetal liver toward morphine is of sufficient magnitude that fetal hepatic clearance is flow-limited. The implication of a high intrinsic clearance is for significant placento-hepatic first-pass metabolism when drugs are administered to the mother. The previously held view of the "inadequacy of perinatal glucuronidation" needs to be reconsidered.