Purification of a Phenobarbital-Inducible UDP-Glucuronosyltransferase Isoform from Dog Liver Which Catalyzes Morphine and Testosterone Glucuronidation

Oguri, Kazuta; Kurogi, Akiko; Yamabe, Kenji; Tanaka, Mitsuko; Yoshisue, Kunihiro; Ishii, Yuji; Yoshimura, Hidetoshi

doi:10.1006/abbi.1996.0020

Cited by 25 publications

(16 citation statements)

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“…34) Determination of UGT Activity The activity of 3-and 6-glucuronidation of morphine was determined by the published methods. 21,32) Frozen microsomes were thawed and pretreated with a detergent, Brij-58 (0.25 mg/mg protein), on ice for 30 min prior to use. In the assays, the enzyme sources at the protein amounts indicated were used: RLM (15 µg protein), HLM (50 µg protein) and UGT2B7 baculosomes (50 µg protein).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Morphine Glucuronidation in the Liver Microsomes of Rats and Humans by Monoterpenoid Alcohols

Ishii

Iida

Miyauchi

et al. 2012

Biological & Pharmaceutical Bulletin

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Morphine is an important drug used to alleviate moderate to severe pain. This opiate is mainly metabolized by glucuronidation to a non-analgesic metabolite, morphine-3-glucuronide (M-3-G) and an active metabolite morphine-6-glucuronide (M-6-G). To understand the modulation of morphine glucuronidation activity by environmental factors, the effect of endogenous and food-derived compounds on morphine uridine 5′-diphosphate (UDP)-glucuronosyltransferase (UGT) in rat and human microsomes was evaluated examining the 50% inhibitory concentration (IC 50 ). The liver microsomes from Sprague-Dawley rats (RLM) and humans (HLM, 150 donors, pooled microsomes) were used as enzyme sources. Of 27 compounds tested, monoterpenoid alcohols, such as borneol and iso-borneol, exhibited a strong inhibitory effect on morphine glucuronidation in rat liver microsomes (RLM), whereas we failed to detect any inhibitory effect of endogenous substances including amino acids and sugars. The substances which have the ability to inhibit the activity in RLM are also inhibitory toward morphine glucuronidation in HLM and UGT2B7 baculosomes. However, the difference was that while the strongest inhibitory effect was observed for iso-menthol in HLM, borneol was the strongest inhibitor of the activity mediated by RLM. Although zidovudine is a typical substrate of UGT2B7, the inhibition of morphine glucuronidation by zidovudine was far weaker than that of monoterpenoid alcohols. These results demonstrate that dietary and supplementary monoterpenoid alcohols modify the pharmacokinetics and pharmacodynamics of morphine through inhibition of UGT2B7.Key words uridine 5′-diphosphate-glucuronosyltransferase; glucuronidation; morphine; borneol; iso-menthol; monoterpenoid alcohol Morphine is an important analgesic for relieving moderate to severe pain (WHO). The major metabolic pathway for morphine is glucuronidation leading to the formation of morphine-3-glucuronide (M-3-G) and morphine-6-glcuronide (M-6-G).1) While M-3-G is a non-analgesic metabolite, M-6-G is a far more potent analgesic than the parent morphine.2) M-3-G is the major metabolite of morphine in many species. However, the ability to form M-6-G varies from species to species, 3) and relatively higher activity was detected in humans 4,5) and guinea pigs.3) Glucuronidation is catalyzed by uridine 5′-diphosphate (UDP)-glucuronosyltrasferase (UGT) which is expressed mainly in the liver and gastro-intestinal tract.6) UGT is a family of enzymes which transfer the glucuronic acid moiety of the co-substrate, UDP-glucuronic acid (UDPGA), to an acceptor substrate.7) Previous studies have suggested that, among UGT isoforms, UGT2B1 and UGT2B7 contribute to morphine glucuronidation in rats and humans, respectively. 8,9)The accelerated action of narcotics is recommended for curing cancer pain because withdrawal symptoms rarely happen even following the opiate treatment of patients with pain.10) However, a problem associated with the clinical use of narcotics is that the mortality of patients due to overdosing ...

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Section: Methodsmentioning

confidence: 99%

Inhibition of Morphine Glucuronidation in the Liver Microsomes of Rats and Humans by Monoterpenoid Alcohols

Ishii

Iida

Miyauchi

et al. 2012

Biological & Pharmaceutical Bulletin

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“…The animals were fasted for 20 h before sacrifice and then the liver was removed and perfused with physiological saline. The microsomes were prepared and solubilized with Emulgen 911 as described previously (Oguri et al, 1996). The recovered protein in the supernatant amounted to 45 to 55%.…”

Section: Methodsmentioning

confidence: 99%

“…Elution was carried out as described previously (Oguri et al, 1996). Most UGT activity toward the 6-hydroxyl group of morphine was eluted as a single peak at a KCl concentration of 360 mM and coeluted with the activity toward the 3-hydroxyl group of morphine.…”

Section: Methodsmentioning

confidence: 99%

“…Recently, Nagano et al (2000) demonstrated that the formation of M-3-G and M-6-G is comparable when brain homogenate of rat was incubated with an endogenous level of [ 3 H]morphine. UGT isoforms involved in M-3-G formation have been purified from experimental animals (Bock et al, 1979;Mackenzie et al, 1984;Puig and Tephly, 1986;Ishii et al, 1993;Oguri et al, 1996), although the UGT isoform involved in M-6-G formation remains to be purified. On the basis of the substrate specificity of expressed cloned UGT isoforms, human UGT2B7 and monkey UGT2B9 were shown to catalyze the glucuronidation of morphine at the 6-hydroxyl and 3-hydroxyl groups (Coffman et al, 1997;Green et al, 1997).…”

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Simultaneous Expression of Guinea Pig UDP-Glucuronosyltransferase 2B21 and 2B22 in COS-7 Cells enhances UDP-Glucuronosyltransferase 2B21-Catalyzed Morphine-6-Glucuronide Formation

et al. 2001

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Although UDP-glucuronosyltransferases (UGTs) act as an important detoxification system for many endogenous and exogenous compounds, they are also involved in the metabolic activation of morphine to form morphine-6-glucuronide (M-6-G). The cDNAs encoding guinea pig liver UGT2B21 and UGT2B22, which are intimately involved in M-6-G formation, have been cloned and characterized. Although some evidence suggests that UGTs may function as oligomers, it is not known whether hetero-oligomer formation leads to differences in substrate specificity. In this work, evidence for a functional heterooligomer between UGT2B21 and UGT2B22 is provided by studies on the glucuronidation of morphine in transfected COS-7 cells. Cells transfected with UGT2B21 cDNA catalyzed mainly morphine-3-glucuronide formation although M-6-G was also formed to some extent. In contrast, cells transfected with UGT2B22 cDNA did not show any significant activity toward morphine. When UGT2B21 and UGT2B22 were expressed simultaneously in different ratios in COS-7 cells, extensive M-6-G formation was observed. This stimulation of M-6-G formation was not observed, however, when microsomes containing UGT2B21were mixed with those containing UGT2B22 in the presence of detergent. Furthermore, this effect was not very marked when human UGT1A1 and UGT2B21 were coexpressed in COS-7 cells. This is the first report suggesting that UGT hetero-oligomer formation leads to altered substrate specificity.

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“…The reaction was terminated by addition of 0.117 ml of ice-cold 16.34% trichloroacetic acid, and the mixture was chilled in an ice bath for 30 min, followed by centrifugation at 9000 rpm for 8 min. The resulting supernatant was subjected to high-performance liquid chromatography and the formation of M-3-G and M-6-G was determined by a method described elsewhere (Oguri et al, 1996). The kinetic parameters with standard deviations were calculated using a nonlinear least square program MULTI (Yamaoka et al, 1981).…”

Section: Construction Of Expression Plasmids For Ugt2b7 and P450mentioning

confidence: 99%

Modulation of UDP-Glucuronosyltransferase Function by Cytochrome P450: Evidence for the Alteration of UGT2B7-Catalyzed Glucuronidation of Morphine by CYP3A4

Takeda¹,

Ishii²,

Iwanaga³

et al. 2004

Mol Pharmacol

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Modulation of UDP-glucuronosyltransferase 2B7 (UGT2B7)-catalyzed morphine glucuronidation by cytochrome P450 (P450) was studied. The effects of P450 isozymes on the kinetic parameters of UGT2B7-catalyzed glucuronidation of the morphine 3-hydroxyl group were examined by simultaneous expression of UGT2B7 and either CYP3A4, -1A2, or -2C9 in COS-1 cells. Although coexpression of CYP3A4 with UGT2B7 had little effect on V max , the K m was increased by about 9.8-fold compared with the UGT2B7 single expression system. The other P450 isozymes (CYP1A2 and CYP2C9) had some effects on K m and V max values. Immunoprecipitation of UGT from solubilized human liver microsomes resulted in coprecipitation of CYP3A4 with UGT2B7. The protein-protein interaction between CYP3A4 and UGT2B7 was further confirmed by overlay assay using glutathione S-transferase-CYP3A4 fusion protein.Addition of CYP3A4 to untreated COS microsomes expressing UGT2B7 had no or minor effects on morphine glucuronidation. In contrast, the formation of morphine-3-glucuronide by detergent-treated microsomes from COS-1 cells expressing UGT2B7 was reduced by CYP3A4, whereas the formation of the 6-glucuronide was enhanced. These results strongly suggest that 1) the glucuronidation activity of UGT2B7 toward morphine is specifically modulated by interaction with CYP3A4 in microsomal membranes and that 2) CYP3A4 alters UGT2B7 regioselectivity so that the ratio of morphine activation/detoxication is increased. This study provides the first evidence that P450 is not only involved in oxidation of drugs but also modulates the function of UGTs.

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Purification of a Phenobarbital-Inducible UDP-Glucuronosyltransferase Isoform from Dog Liver Which Catalyzes Morphine and Testosterone Glucuronidation

Cited by 25 publications

References 0 publications

Inhibition of Morphine Glucuronidation in the Liver Microsomes of Rats and Humans by Monoterpenoid Alcohols

Inhibition of Morphine Glucuronidation in the Liver Microsomes of Rats and Humans by Monoterpenoid Alcohols

Simultaneous Expression of Guinea Pig UDP-Glucuronosyltransferase 2B21 and 2B22 in COS-7 Cells enhances UDP-Glucuronosyltransferase 2B21-Catalyzed Morphine-6-Glucuronide Formation

Modulation of UDP-Glucuronosyltransferase Function by Cytochrome P450: Evidence for the Alteration of UGT2B7-Catalyzed Glucuronidation of Morphine by CYP3A4

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