Kazuta Oguri

Biochemical basis for analgesic activity of morphine-6-glucuronide. I. Penetration of morphine-6-glucuronide in the brain of rats.

Abstract To understand the potent analgesic action of morphine-6-glucuronide (M-6-G), which was reported previously to be a minor metabolite of morphine in several mammalian species, the penetration of this conjugate into the brain was investigated using 14 C-labeled compound. A similar study was also conducted with 14 C-morphine. These studies presented evidence that, although M-6-G was a highly polar conjugate, it can penetrate the blood brain barrier and react with the receptor of analgesic action without prior hydrolysis of the glucuronide linkage. It was further suggested that the lack of analgesic activity produced with morphine-3-glucuronide (M-3-G), a major metabolite of morphine, was attributable to its inability to react with the receptor, because it penetrates the brain as well as M-6-G.

Enhanced binding of morphine and nalorphine to opioid delta receptor by glucuronate and sulfate conjugations at the 6-position.

Effect of the modification of morphine and nalorphine by glucuronate and sulfate conjugations at the 3- and 6-positions on the binding to opioid receptors was examined in a particulate fraction of rat brain. Competing potencies of both drugs against [3H]morphine and [3H]leucine enkephalin bindings were extremely decreased by either glucuronate or sulfate conjugation at the 3-position. On the other hand, the potencies of morphine and nalorphine against [3H]leucine enkephalin binding were considerably enhanced by the conjugations at the 6-position, whereas the potencies against [3H]morphine binding were decreased. These altered interactions of the conjugates at the 6-position with the two ligands were attributed to their enhanced binding to delta-receptor and reduced binding to mu-receptor by Hill plot and modified Scatchard analysis. Resulted comparable and simultaneous interactions with mu- and delta- receptors were assumed to be a cause of the enhanced mu-receptor-directed analgesia of morphine and elevated same receptor-directed antagonistic effect of nalorphine, which have been found previously in our laboratory.

Chemical synthesis and analgesic effect of morphine ethereal sulfates

Abstract Chemical synthesis of morphine-3- and -6-ethereal sulfate was accomplished, utilizing chlorosulfonic acid as sulfonating reagent. The analgesic effect of morphine-6-sulfate in mice was about same order of magnitude as that of morphine-6-glucuronide, being much higher in potency and longer in duration than that of morphine. On the other hand, no effect was observed with morphine-3-sulfate (20 mg/kg, s. c.). Morphine-6-sulfate, an active conjugate, could not be detected in the urine of cats injected with morphine by careful examination with thin-layer chromatography.

INHIBITION OF UDP-GLUCURONOSYLTRANSFERASE 2B7-CATALYZED MORPHINE GLUCURONIDATION BY KETOCONAZOLE: DUAL MECHANISMS INVOLVING A NOVEL NONCOMPETITIVE MODE

Glucuronidation of morphine in humans is predominantly catalyzed by UDP-glucuronosyltransferase 2B7 (UGT2B7). Since our recent research suggested that cytochrome P450s (P450s) interact with UGT2B7 to affect its function [Takeda S et al. (2005) Mol Pharmacol 67:665–672], P450 inhibitors are expected to modulate UGT2B7-catalyzed activity. To address this issue, we investigated the effects of P450 inhibitors (cimetidine, sulfaphenazole, erythromycin, nifedipine, and ketoconazole) on the UGT2B7-catalyzed formation of morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G). Among the inhibitors tested, ketoconazole was the most potent inhibitor of both M-3-G and M-6-G formation by human liver microsomes. The others were less effective except that nifedipine exhibited an inhibitory effect on M-6-G formation comparable to that by ketoconazole. Neither addition of NADPH nor solubilization of liver microsomes affected the ability of ketoconazole to inhibit morphine glucuronidation. In addition, ketoconazole had an ability to inhibit morphine UGT activity of recombinant UGT2B7 freed from P450. Kinetic analysis suggested that the ketoconazole-produced inhibition of morphine glucuronidation involves a mixed-type mechanism. Codeine potentiated inhibition of morphine glucuronidation by ketoconazole. In contrast, addition of another substrate, testosterone, showed no or a minor effect on ketoconazole-produced inhibition of morphine UGT. These results suggest that 1) metabolism of ketoconazole by P450 is not required for inhibition of UGT2B7-catalyzed morphine glucuronidation; and 2) this drug exerts its inhibitory effect on morphine UGT by novel mechanisms involving competitive and noncompetitive inhibition.

Species differences in metabolism of codeine: urinary excretion of codeine glucuronide, morphine-3-glucuronide and morphine-6-glucuronide in mice, rats, guinea pigs and rabbits.

1. Metabolites of codeine were determined by use of h.p.l.c. in urine of male mice, rats, guinea pigs and rabbits injected with 10 mg codeine/kg subcutaneously. 2. In 24 h urines of these species, unchanged codeine, codeine glucuronide, free morphine, and morphine-3-glucuronide were as follows: mice, 6.8, 1.6, 0.8 and 7.6% dose; rats, 1.6, 0.2, 4.3 and 23.9% dose; guinea pigs, 1.6, 39.8, 0.2 and 1.6% dose; rabbits, 2.2, 24.5, 1.3 and 17.9% dose. Urinary excretion of morphine-6-glucuronide was 0.7% dose in guinea pigs, 1.9% in rabbits, and not detectable in mice and rats. Norcodeine was found only in the urine of mice. 3. These results indicate that codeine is metabolized in all four species by glucuronidation and by oxidative N- and O-demethylation, but the quantitative excretions of metabolites were quite different in different species.

Participation of cytochrome P-450 isozymes in N-demethylation, N-hydroxylation and aromatic hydroxylation of methamphetamine

1. Five isozymes of cytochrome P-450 were purified from liver microsomes of phenobarbital-pretreated (P-450-SD-I and -II), 3-methylcholanthrene-pretreated (P-450-SD-III) and untreated rats (P-450-SD-IV and -V) to determine their catalytic activities in metabolic reactions of methamphetamine. 2. All the isozymes except P-450-SD-III showed considerably high N-hydroxylating activity of methamphetamine. The cytochromes P-450 initiate N-demethylation of this drug by two metabolic pathways, C-hydroxylation and N-hydroxylation. 3. Both N-demethylation and N-hydroxylation of methamphetamine were efficiently catalysed by the phenobarbital-inducible forms P-450-SD-I and -II and constitutive forms P-450-SD-IV and -V. 4. The constitutive forms P-450-SD-IV and -V revealed high catalytic activities of p-hydroxylation of methamphetamine, but phenobarbital- and 3-methylcholanthrene-inducible isozymes showed only low activities. 5. The present results indicate that the different extents of the metabolic intermediate complex formation with cytochrome P-450 (455 nm complex) in the microsomes from phenobarbital-, 3-methylcholanthrene-pretreated, and untreated rats is not attributable to the activities of the respective isozymes of cytochrome P-450 to form the precursor of the complex, N-hydroxymethamphetamine.

Comparison in mice of pharmacological effects of Δ8-tetrahydrocannabinol and its metabolites oxidized at 11-position

Abstract Pharmacological effects of Δ 8 -tetrahydrocannabinol ( Δ 8 -THC) and its metabolites, 11-hydroxy- Δ 8 -THC, 11-oxo- Δ 8 -THC and Δ 8 -THC-11-oic acid were compared using mice. The cataleptogenic effect of the 11-hydroxy and 11-oxo metabolites was 5 and 1.5 times greater respectively, than that of the parent compound. The hypothermic effect of Δ 8 -THC, 11-hydroxy- Δ 8 -THC and 11- oxo - Δ 8 -THC was almost equivalent in both potency and duration at a dose of 10 mg/kg i.v., but the metabolites exhibited a somewhat higher potency and longer duration that the parent compound at a dose of 5 mg/kg i.v. In addition, 11-hydroxy- and 11-oxo- Δ 8 -THC were more active to prolong pentobarbital-induced sleeping time than Δ 8 -THC. In spite of the loss of cataleptogenic and hypothermic effects, Δ 8 THC-11-oic acid slightly prolonged pentobarbital-induced sleeping time at a dose of 10 mg/kg i.v. The LD 50 s (i.v.) with their 95% confidence limits of Δ 8 -THC, 11-hydroxy- Δ 8 -THC and 11-oxo- Δ 8 -THC were estimated to be 27.5 (23.1–32.7), 110.0 (79.1–152.9) and 63.0 (54.5–72.8) mg/kg (P Δ 8 -THC-11-oic acid. These results indicate that both 11-hydroxy- and 11-oxo- Δ 8 -THC can be categorized as active metabolites of Δ 8 -THC. Further studies are necessary, however, to clarify whether or not these metabolites contribute, at least in part, to the effect of Δ 8 -THC on biological systems in vivo.

Interaction of Cytochrome P450 3A4 and UDP-Glucuronosyltransferase 2B7: Evidence for Protein-Protein Association and Possible Involvement of CYP3A4 J-Helix in the Interaction

We have reported that the protein-protein interaction between UDP-glucuronosyltransferase (UGT) 2B7 and cytochrome P450 3A4 (CYP3A4) alters UGT2B7 function. However, the domain(s) involved in the interaction are largely unknown. To address this issue, we examined in more detail the CYP3A4-UGT2B7 association by means of immunoprecipitation, overlay assay, and cross-linking involving 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Purified CYP3A4 or glutathione transferase (GST)-tagged CYP3A4 was cross-linked to UGT2B7 in solubilized baculosomes. The formation of the cross-linked complex was detected by immunoblotting using both antibodies against CYP3A4 and UGTs. Although the GST-tagged CYP3A4 containing the region ranging from Tyr25 to Ala503 was cross-linked to UGT2B7, the same did not occur when another construct containing Met145 to His267 was used. This observation was consistent with the result of the overlay assay indicating that CYP3A4 lacking the N-terminal hydrophobic segment retains the ability to associate with UGT2B7, whereas the Met145-to-His267 region loses this capacity. Although the Met145-to-His267 peptide was recognized by one anti-CYP3A4 antibody that has the ability to coimmunoprecipitate UGT2B7, it was not recognized by another antibody incapable of coimmunoprecipitating UGT2B7. The epitope of the latter antibody was mapped to the Leu331-to-Lys342 region, which is located on the J-helix of CYP3A4. Taken together, the results obtained suggest that 1) CYP3A4 and UGT2B7 are a pair of enzymes in proximity to each other and 2) either the Leu331-to-Lys342 domain or the surrounding region plays a role in the interaction with UGT2B7, whereas the hydrophobic Met145-to-His267 region does not contribute to this interaction.

Induction of the hepatic cytochrome P450 2B subfamily by xenobiotics : Research history, evolutionary aspect, relation to tumorigenesis, and mechanism

The cytochrome P450 belonging to the CYP2B subfamily has long been of great interest because it can be induced by xenobiotics. While a well known diagnostic ligand-receptor theory explains the induction of the CYP1A subfamily, the mechanism by which xenobiotics induce the CYP2B subfamily is not fully understood. Although the constitutive androstane receptor (CAR) undoubtedly plays a crucial role in the induction, many questions regarding the mechanism of CAR activation by xenobiotics have not yet been answered. It is a puzzle that many structurally-unrelated chemicals can increase the expression of the CYP2B subfamily. This may support a mechanism(s) distinct from the signaling induced by ligand-receptor binding. Indeed, phenobarbital, a typical inducer, cannot associate with CAR. Thus, no one is able to answer a fundamental question: what is the initial target of xenobiotics to produce induced expression of CYP2B enzymes? In this review, we survey the research history of CYP2B induction, list the inducers reported so far, and discuss the mechanism of induction including the target site of inducers.

Characteristic glucuronidation pattern of physiologic concentration of morphine in rat brain.

Formation of conjugated metabolites from morphine at a very low level in brain was studied in vitro in rats. Incubation of a low concentration of 3H-morphine with brain homogenate followed by two successive high-performance liquid chromatographic analyses showed that endogenous morphine is converted by brain enzymes to its 3- and 6-glucuronides (M-3-G and M-6-G), and codeine glucuronide (Cod-G). However, the formation of morphine-6-sulfate was likely to be low if it was produced at all. All of the cerebral hemisphere, brain stem and cerebellum were capable of producing M-3-G, M-6-G and Cod-G, although there were differences in selectivity. The capacity of the brain for glucuronide formation was far less than that of the liver, but UDP-glucuronosyltransferase in brain was much more selective in forming M-6-G and Cod-G than liver enzymes.