T. Inaba

Human UDP‐glucuronosyltransferase (UGT)1A3 enzyme conjugates chenodeoxycholic acid in the liver

Chenodeoxycholic acid (CDCA) is a liver‐formed detergent and plays an important role in the control of cholesterol homeostasis. During cholestasis, toxic bile acids (BA) accumulate in hepatocytes causing damage and consequent impairment of their function. Glucuronidation, a conjugation reaction catalyzed by UDP‐glucuronosyltransferase (UGT) enzymes, is considered an important metabolic pathway for hepatic BA. This study identifies the human UGT1A3 enzyme as the major enzyme responsible for the hepatic formation of the acyl CDCA‐24glucuronide (CDCA‐24G). Kinetic analyses revealed that human liver and UGT1A3 catalyze the formation of CDCA‐24G with similar Km values of 10.6 to 18.6 μmol/L, respectively. In addition, electrophoretic mobility shift assays and transient transfection experiments revealed that glucuronidation reduces the ability of CDCA to act as an activator of the nuclear farnesoid X‐receptor (FXR). Finally, we observed that treatment of human hepatocytes with fibrates increases the expression and activity of UGT1A3, whereas CDCA has no effect. In conclusion, UGT1A3 is the main UGT enzyme for the hepatic formation of CDCA‐24G and glucuronidation inhibits the ability of CDCA to act as an FXR activator. In vitro data also suggest that fibrates may favor the formation of bile acid glucuronides in cholestatic patients. (HEPATOLOGY 2006;44:1158–1170.)

The Human UGT1A3 Enzyme Conjugates Norursodeoxycholic Acid into a C23-ester Glucuronide in the Liver

Norursodeoxycholic acid (norUDCA) exhibits efficient anti-cholestatic properties in an animal model of sclerosing cholangitis. norUDCA is eliminated as a C23-ester glucuronide (norUDCA-23G) in humans. The present study aimed at identifying the human UDP-glucuronosyltransferase (UGT) enzyme(s) involved in hepatic norUDCA glucuronidation and at evaluating the consequences of single nucleotide polymorphisms in the coding region of UGT genes on norUDCA-23G formation. The effects of norUDCA on the formation of the cholestatic lithocholic acid-glucuronide derivative and of rifampicin on hepatic norUDCA glucuronidation were also explored. In vitro glucuronidation assays were performed with microsomes from human tissues (liver and intestine) and HEK293 cells expressing human UGT enzymes and variant allozymes. UGT1A3 was identified as the major hepatic UGT enzyme catalyzing the formation of norUDCA-23G. Correlation studies using samples from a human liver bank (n = 16) indicated that the level of UGT1A3 protein is a strong determinant of in vitro norUDCA glucuronidation. Analyses of the norUDCA-conjugating activity by 11 UGT1A3 variant allozymes identified three phenotypes with high, low, and intermediate capacity. norUDCA is also identified as a competitive inhibitor for the hepatic formation of the pro-cholestatic lithocholic acid-glucuronide derivative, whereas norUDCA glucuronidation is weakly stimulated by rifampicin. This study identifies human UGT1A3 as the major enzyme for the hepatic norUDCA glucuronidation and supports that some coding polymorphisms affecting the conjugating activity of UGT1A3 in vitro may alter the pharmacokinetic properties of norUDCA in cholestasis treatment.

Oxazepam as a probe of hepatic metabolism in patients with Alzheimer's disease

1. Hepatic metabolism of oxazepam in Alzheimers disease (AD) was assessed by measurement of urinary metabolites in a group of hospitalized patients with AD, a hospitalized schizophrenic control group and a normal community based group. 2. A subgroup of six AD patients showed marked elevations of the hydroxylated metabolite. The median excretion of conjugated oxazepam in the AD and schizophrenic patients was almost one third that in normal controls (p less than .005). 3. A relationship between decline in level of conjugated metabolite and increase in the mental confusion score on the London Psychiatric Rating Scale (r = -.5253, p less than .05) was found in the AD patients. 4. Changes in hepatic metabolism in AD may be relevant not only for drug metabolism and the development of side effects, but also for the pathogenesis of AD.

A Human Liver Model of Metabolism as a Tool in the Identification of Potential PET Radiotracers

The potent 5-HT1A antagonist WAY 100635 is metabolized in vivo by human liver. The metabolite, WAY 100634, readily crosses the blood-brain barrier and may confound positron emission tomography (PET) studies of [11C]WAY 100635. WAY 100635 and three analogs were studied by an in vitro model of human liver metabolism using HPLC to analyze the reaction mixtures. The objective of the study was to determine whether the analogs were likely to suffer the same metabolic fate as WAY 100635 in vivo, an outcome that would limit their potential as PET radiotracers for imaging 5-HT1A receptors. In human liver cytosolic media, WAY 100635 was quantitatively metabolized to WAY 100634 (amide hydrolysis), but none of the three analogs were affected. In human liver microsomal media, WAY 100635 was again metabolized predominantly to WAY 100634 with some more polar products as well. All three analogs were also metabolized to some extent in the microsomal preparations, but none followed the major pathway of WAY 100635, i.e., no metabolite from simple amide hydrolysis was detected. The metabolic products from microsomal incubation were all more polar (by reverse-phase HPLC) than the anticipated product of amide hydrolysis. In vitro screening of potential PET ligands using human liver preparations can provide useful information in helping decide which ligands merit further study.The potent 5-HT 1A antagonist WAY 100635 is metabolized in vivo by human liver. The metabolite, WAY 100634, readily crosses the blood-brain barrier and may confound positron emission tomography (PET) studies of [ 11 C]WAY 100635. WAY 100635 and three analogs were studied by an in vitro model of human liver metabolism using HPLC to analyze the reaction mixtures. The objective of the study was to determine whether the analogs were likely to suffer the same metabolic fate as WAY 100635 in vivo, an outcome that would limit their potential as PET radiotracers for imaging 5-HT 1A receptors. In human liver cytosolic media, WAY 100635 was quantitatively metabolized to WAY 100634 (amide hydrolysis), but none of the three analogs were affected. In human liver microsomal media, WAY 100635 was again metabolized predominantly to WAY 100634 with some more polar products as well. All three analogs were also metabolized to some extent in the microsomal preparations, but none followed the major pathway of WAY 100635, i.e., no metabolite from simple amide hydrolysis was detected. The metabolic products from microsomal incubation were all more polar (by reverse-phase HPLC) than the anticipated product of amide hydrolysis. In vitro screening of potential PET ligands using human liver preparations can provide useful information in helping decide which ligands merit further study.