Genetic predisposition to the metabolism of irinotecan (CPT-11). Role of uridine diphosphate glucuronosyltransferase isoform 1A1 in the glucuronidation of its active metabolite (SN-38) in human liver microsomes.

Irinotecan (CPT-11) is a promising antitumor agent, recently approved for use in patients with metastatic colorectal cancer. Its active metabolite, SN-38, is glucuronidated by hepatic uridine diphosphate glucuronosyltransferases (UGTs). The major dose-limiting toxicity of irinotecan therapy is diarrhea, which is believed to be secondary to the biliary excretion of SN-38, the extent of which is determined by SN-38 glucuronidation. The purpose of this study was to identify the specific isoform of UGT involved in SN-38 glucuronidation. In vitro glucuronidation of SN-38 was screened in hepatic microsomes from normal rats (n = 4), normal humans (n = 25), Gunn rats (n = 3), and patients (n = 4) with Crigler-Najjar type I (CN-I) syndrome. A wide intersubject variability in in vitro SN-38 glucuronide formation rates was found in humans. Gunn rats and CN-I patients lacked SN-38 glucuronidating activity, indicating the role of UGT1 isoform in SN-38 glucuronidation. A significant correlation was observed between SN-38 and bilirubin glucuronidation (r = 0.89; P = 0.001), whereas there was a poor relationship between para-nitrophenol and SN-38 glucuronidation (r = 0.08; P = 0.703). Intact SN-38 glucuronidation was observed only in HK293 cells transfected with the UGT1A1 isozyme. These results demonstrate that UGT1A1 is the isoform responsible for SN-38 glucuronidation. These findings indicate a genetic predisposition to the metabolism of irinotecan, suggesting that patients with low UGT1A1 activity, such as those with Gilbert's syndrome, may be at an increased risk for irinotecan toxicity.

Differential glucuronidation of bile acids, androgens and estrogens by human UGT1A3 and 2B7.

In this work, UDP-glucuronosyltransferases (UGTs), UGT1A3, 2B7(H268) and 2B7(Y268), stably expressed in human embryonic kidney cells (HK293) were used to assess glucuronidation activities with a variety of steroid hormone and bile acid substrates. The rate of synthesis of carboxyl- and hydroxyl-linked glucuronides was determined under optimal reaction conditions. Expressed UGT1A3 catalyzed bile acid glucuronidation at high rates exclusively at the carboxyl moiety for all compounds tested. In contrast, UGT1A4 catalyzed bile acid glucuronidation at very low rates exclusively at the 3alpha-hydroxyl function. Both UGT2B7 allelic variants glucuronidated the bile acid substrates at both carboxyl and hydroxyl moieties, however, the 3alpha-hydroxyl position was preferentially conjugated compared to the carboxyl function. Similarly, androsterone, a 3alpha-hydroxylated androgenic steroid, was glucuronidated at very high rates by expressed UGT2B7. Of the estrogenic compounds tested, UGT2B7 catalyzed the glucuronidation of estriol at rates comparable to those determined for androsterone. Other structural discrimination was found with UGT2B7 which had activity toward estriol and estradiol exclusively at the 17beta-OH position, yielding the cholestatic steroid D-ring glucuronides.

Glucuronidation of catechol estrogens by expressed human UDP-glucuronosyltransferases (UGTs) 1A1, 1A3, and 2B7.

Catechol estrogens are major estrogen metabolites in mammals and are the most potent naturally occurring inhibitors of catecholamine metabolism. These estrogen compounds have been implicated in carcinogenic activity and the 4/2-hydroxyestradiol concentration has been shown to be elevated in neoplastic human mammary tissue compared to normal human breast tissue. Three human liver UDP-glucuronosyltransferases, UGT2B7, UGT1A1, and UGT1A3, have been shown to catalyze the glucuronidation of catechol estrogens and lead to their enhanced elimination via urine or bile. The present study was designed to study the kinetic interaction of expressed human UGT2B7(Y) or (H), UGT1A1, and UGT1A3 toward 2- and 4-hydroxycatechol estrogens. cDNAs encoding UGT2B7(Y) or (H), UGT1A1, and UGT1A3 were expressed in HK293 cells, and cell homogenates or membrane preparations were used to determine their glucuronidation ability. UGT2B7(Y) reacted with higher efficiency toward 4-hydroxyestrogenic catechols, whereas UGT1A1 and UGT1A3 showed higher activities toward 2-hydroxyestrogens. UGT2B7(H) catalyzed estrogen catechol glucuronidation with efficiencies similar to UGT2B7(Y). Flunitrazepam (FNZ), a competitive inhibitor of morphine glucuronidation in hepatic microsomes, competitively inhibited catechol estrogen glucuronidation catalyzed by UGT2B7(Y), UGT1A1, and UGT1A3. Buprenorphine, an opioid substrate that reacts at high efficiency with each of these UGTs, was also studied. FNZ competitively inhibited buprenorphine glucuronidation with UGT1A1 and UGT2B7 but had no inhibitory activity toward UGT1A3. This suggests that buprenorphine and 2-hydroxycatechol estrogens react with separate active sites of UGT1A3. A catecholamine, norepinephrine, did not inhibit UGT2B7(Y)-, UGT1A1-, and UGT1A3-catalyzed glucuronidation of catechol estrogens. These results also suggest that drug-endobiotic interactions are possible in humans and may have implication in carcinogenesis.

Purification and properties of two rat liver phenobarbital-inducible UDP-glucuronosyltransferases that catalyze the glucuronidation of opioids.

Glucuronidation of xenobiotics and endobiotics is catalyzed by a group of intrinsic membrane proteins of the endoplasmic reticulum of cells: the UDP-glucuronosyltransferases. Two isoforms with glucuronidation activity toward opioids have been purified and characterized from liver microsomes obtained from phenobarbital-treated Wistar rats. The proteins have been identified as the gene products of UGT2B1 and UGT1.1r. The purified proteins exhibited the same apparent KM values for morphine glucuronidation (2-3 mM). However, the purified UGT1.1r enzyme exhibited glucuronidation activity toward buprenorphine and bilirubin with high efficiency, but the UGT2B1 protein did not react with these compounds. Both purified enzymes glucuronidated chloramphenicol, 4-hydroxybiphenyl, chrysin, and ibuprofen. Flunitrazepam photoaffinity labeling was demonstrated for both enzymes, and naloxone, the opioid antagonist, antagonized the photoaffinity labeling reactions.

Cloning and stable expression of a cDNA encoding a rat liver UDP-glucuronosyltransferase (UDP-glucuronosyltransferase 1.1) that catalyzes the glucuronidation of opioids and bilirubin.

A chicken anti-rat polyclonal antibody to a purified rat liver UDP-glucuronosyltransferase (UGT) with catalytic activity toward opioid substrates was used to screen a liver cDNA library prepared from phenobarbital-treated Wistar rats. A number of positive clones were obtained, and one of these clones, pM1, was further characterized. Clone pM1 was found to be a full length cDNA coding for a member of the rat UGT1 gene family. Specifically, pM1 represents the full length homologue of the Gunn rat liver pseudo-gene product UGT1.1P and, therefore, has been designated UGT1.1r. The cDNA insert has an open reading frame of 1605 base pairs, which codes for a protein of 535 amino acids and is flanked by 2 and 632 base pairs of 5' and 3' noncoding sequence, respectively. The deduced amino acid sequence of pM1 contains amino acid sequences identical to the amino-terminal and internal peptides of the purified rat liver opioid UGT and to sequences reported for a rat liver bilirubin UGT [FEBS Lett. 299:183-186 (1992)]. Stable expression of UGT1.1r in human embryonic kidney 293 cells showed that a protein with a subunit molecular mass (56 kDa) identical to that of the purified protein was produced. Expressed UGT1.1r protein catalyzed the glucuronidation of buprenorphine and bilirubin at high rates. Other opioids, such as nalorphine and morphine, were also substrates for the expressed UGT1.1r protein. These results show that bilirubin and opioids can be conjugated by the same rat liver UGT.

Characterization of antibodies to a rabbit hepatic UDP-glucuronosyltransferase and the identification of an immunologically similar enzyme in human liver.

An antibody to a UDP-glucuronosyltransferase (UDPGT) isoenzyme which catalyzes the glucuronidation of p-nitrophenol (PNP) in rabbit liver was raised in sheep and used to identify immunologically similar UDPGTs in rabbit and human livers. Immunoblotting experiments showed that the antisera specifically recognized PNP UDPGT but not estrone UDPGT purified from rabbit liver. Sheep anti-rabbit liver PNP UDPGT IgG immunoprecipitated PNP, 1-naphthol, and 4-methylumbelliferone glucuronidation activities in rabbit and human liver microsomal preparations. In rabbit liver microsomes the antibody did not immunoprecipitate estrone or estradiol glucuronidation activities. In human liver microsomes, 4-aminobiphenyl but not estriol glucuronidation activities were immunoprecipitated, suggesting that the antibody recognizes a specific UDPGT (pI 6.2) in human liver microsomes.

Human UGT2B7 catalyzes morphine glucuronidation.

A human UDP-glucuronosyltransferase (UGT) catalyzing the glucuronidation of morphine has been identified. A full length cDNA was isolated from a human liver cDNA library and found to be identical to the UGT2B7 form having a tyrosine at position 288. This cDNA was transfected into HK 293 cells, and stable expression was achieved. Cell homogenates and membrane preparations from HK 293 cells expressing UGT2B7 catalyzed the glucuronidation of morphine and other clinically significant opioid agonists, antagonists, and partial agonists. UGT2B7 catalyzed morphine glucuronidation at the 3- and 6-hydroxy positions and also mediated the formation of codeine-6-glucuronide from codeine. This represents the first demonstration of a UGT capable of catalyzing the glucuronidation of both the 3- and 6-positions of opioids. Since humans excrete morphine-3-glucuronide and morphine-6-glucuronide after morphine administration, it is likely that UGT2B7 is a major isoform in humans responsible for the metabolism of this important drug and its surrogates.

The glucuronidation of opioids, other xenobiotics, and androgens by human UGT2B7Y(268) and UGT2B7H(268).

UGT2B7 has been cloned and expressed previously in COS cells and HK293 cells. Two forms have been identified: one with a tyrosine and one with a histidine at position 268. UGT2B7 has been shown to catalyze NSAIDs, catechol estrogens, and morphine-3- and -6-glucuronidation. cDNAs for UGT2B7Y268 and H268 were cloned and stably expressed in HK 293 cells. Studies were designed to test each form for reactivity toward a number of opioid compounds, xenobiotics such as menthol, oxazepam, and propranolol, and androgens such as androsterone and testosterone using membrane preparations derived from HK 293 cells. Both UGT2B7Y and UGT2B7H are highly reactive with many opioids, menthol, androsterone, and (R)- and (S)-propranolol, and similar kinetic values were observed. UGT2B7Y and UGT2B7H react poorly with oxazepam and no difference in (R)- or (S)-glucuronidation rate ratios was found. Thus, UGT2B7Y and H cannot account for the variability in the plasma or urine concentrations of these glucuronides in human populations. Our data suggest that UGT2B7 is a major isoform responsible for the glucuronidation of androsterone. Neither UGT2B7Y nor H catalyzes the glucuronidation of testosterone although each catalyzes the glucuronidation of epitestosterone. UGT2B7 seems to be a major human isoform responsible for the glucuronidation of opioids of the morphinan and oripavine class and is capable of catalyzing the glucuronidation of both the 3- and 6-hydroxyl moieties on these molecules. Thus, UGT2B7 plays a major role in the conversion of morphine to morphine-6-glucuronide, the potent analgesic metabolite of morphine.

Separation, purification, and characterization of digitoxigenin-monodigitoxoside UDP-glucuronosyltransferase activity.

Glucuronidation of digitoxigenin-monodigitoxoside was investigated in liver microsomes from spironolactone-induced male Wistar rats. Isolation of a specific digitoxigenin-monodigitoxoside UDP-glucuronosyltransferase was possible utilizing chromatofocusing chromatography with a gradient from pH 10.1 to 8.0 after solubilizing the microsomal protein with the nonionic detergent Emulgen 911. The digitoxigenin-monodigitoxoside UDP-glucuronosyltransferase was further purified using UDP-hexanolamine Sepharose 4B affinity chromatography. The highly purified (75-fold) enzyme showed activity toward digitoxigenin-monodigitoxoside and slight activity toward digitoxigenin-bisdigitoxoside, whereas digitoxin and substrates for p-nitrophenol, 17 beta-OH steroid, and 3 alpha-OH steroid UDP-glucuronosyltransferases were not glucuronidated. In addition, bilirubin, morphine, estrone, 4-hydroxybiphenyl, and aromatic amines were not glucuronidated by this protein. These results strongly confirm the presence of a form of UDP-glucuronosyltransferase, which is highly specific for the glucuronidation of digitoxigenin-monodigitoxoside.

Analysis of opioid binding to UDP-glucuronosyltransferase 2B7 fusion proteins using nuclear magnetic resonance spectroscopy.

The UDP-glucuronosyltransferase UGT2B7 is an important human UGT isoform that catalyzes the conjugation of many endogenous and exogenous compounds, among them opioids, resulting in the formation of D-glucuronides. The binding site of the aglycone is located in the N-terminal half of the protein. In this study, we demonstrate that the opioid binding site in UGT2B7 is within the first 119 amino-terminal amino acids. Two maltose binding protein fusion proteins, 2B7F1 and 2B7F2, incorporating the first 157 or 119 amino acids, respectively, of UGT2B7 were expressed in Escherichia coli and purified by affinity chromatography. NMR spectroscopy using one-dimensional spectra, the inversion recovery method, and the transferred nuclear Overhauser effect spectroscopy was used to study the binding properties of opioids to the fusion proteins. Morphine was found to bind at a single site within the first 119 amino acids and to undergo a conformational change upon binding, as demonstrated by transferred nuclear Overhauser effect spectroscopy. Dissociation constants were obtained for morphine, naloxone, buprenorphine, and zidovudine, and the results were confirmed by equilibrium dialysis determinations. Two possible opioid binding sites, based on the nearest neighbors from opioid binding to the micro-receptor and to cytochrome 2D6, are proposed.

Effect of the suicide substrate 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine on the metabolism of xenobiotics and on cytochrome P-450 apoproteins.

Treatment of rats with the cytochrome P-450 suicide substrate, 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP), produced a 95% inhibition of the in vivo demethylation of either aminopyrine or morphine within 2 hr. One-carbon metabolism of formaldehyde or formate to carbon dioxide was not altered. DDEP also produced a time-dependent decrease in total hepatic microsomal cytochrome P-450 but had no effect on either NADPH-cytochrome c reductase or p-nitrophenol glucuronyl-transferase activities up to 24 hr after administration. A rapid decrease in rat liver microsomal aniline hydroxylation and ethoxyresorufin deethylation was observed in vitro following DDEP administration. Although in vitro testosterone metabolism to 16 alpha-, 16 beta-, and 2 alpha-hydroxy metabolites was depressed profoundly by DDEP in microsomes from untreated and 3-methylcholanthrene-treated animals, 7 alpha-hydroxylation of testosterone was much less affected. Immunochemical quantification of various microsomal cytochrome P-450 protein moieties showed that cytochromes P-450 beta NF-B, P-450UT-A, P-450PCN-E, and P-450PB-C were decreased in hepatic microsomes from DDEP-treated rats. However, the protein moiety of cytochrome P-450UT-H was not diminished and the immunoreactive protein for cytochromes P-450UT-F, P-450PB-B, and P-450ISF-G was only slightly decreased. These results show that DDEP treatment leads to marked decreases in holoprotein and apoproteins of many but not all hepatic microsomal cytochrome P-450 isozymes.

The glucuronidation of exogenous and endogenous compounds by stably expressed rat and human UDP-glucuronosyltransferase 1.1.

Rat and human UDP-glucuronosyltransferase (UGT) 1.1 share > 70% identity in their deduced primary amino acid sequences. We have previously shown that rat UGT1.1, stably expressed in human embryonic kidney 293 cells, catalyzes the glucuronidation of bilirubin and the mixed opioid agonist/antagonist buprenorphine with high efficiency. The present study was designed to characterize the reactivity of expressed human UGT1.1 with opioid compounds and compare its substrate specificity for opioids to that of the expressed rat enzyme. The results show that both rat and human UGT1.1 catalyze the glucuronidation of opioids with a relative reactivity of buprenorphine > > nalorphine approximately naltrexone. Comparison of glucuronidation activities in livers from Crigler-Najjar type 1 patients and normal patients indicates that UGT1.1 catalyzes at least 75% of buprenorphine conjugation in normal human liver. In separate studies, the reactivity of expressed rat UGT1.1 was characterized toward various xeno-and endobiotics of various compound classes. It was found that both rat and human UGT1.1 exhibited comparable substrate specificities and efficiencies (Vmax/Km) of glucuronide formation for anthraquinones, coumarins, estrogens, flavonoids, and phenolic compounds. Neither rat nor human UGT1.1 catalyzed the glucuronidation of amines, monoterpenoid alcohols, androgens, or progestins. In general, these data indicate that rat and human UGT1.1 are functionally identical and can be considered orthologous enzymes.

Characterization and primary sequence of a human hepatic microsomal estriol UDPglucuronosyltransferase.

A human liver microsomal UDP glucuronosyltransferase (UDPGT) that demonstrates reactivity with estriol (pI 7.4 UDPGT) has been purified to homogeneity and characterized further. No activity toward morphine, 4-hydroxybiphenyl, bilirubin, or tripelennamine was observed. The estriol UDPGT shows immunoreactivity with antibodies raised against rat hepatic microsomal 3 alpha- and 17 beta-hydroxysteroid UDPGTs but not with antibodies raised against rat hepatic microsomal p-nitrophenol UDPGT. The NH2-terminal sequence of the purified protein was determined and found to correspond to an identical sequence in the deduced amino acid sequence of a cDNA obtained from a human liver library in lambda gt11 (HLUG4). Sequence analysis revealed that HLUG4 is 2094 bp in length and encodes a protein of 523 amino acids which has a 16 amino acid leader sequence, followed by an untranslated 3' region of 525 bp. Three potential N-glycosylation sites were identified in the predicted sequence. The deduced amino acid sequence of estriol UDPGT showed 82% identity with the deduced amino acid sequence of another human hepatic cDNA (HLUG25), which has been expressed as a UDPGT capable of 6 alpha-hydroxyglucuronidation of hyodeoxycholic acid, strongly suggesting that these proteins are members of the same gene subfamily.