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The purpose of the present study was to identify the structures of cannabidiol (CBD) metabolites during CO formation by human liver microsomes and human recombinant cytochrome P450 (CYP) enzymes. CBD was NADPH-dependently metabolized by human liver microsomes and human recombinant CYP enzymes. Less-polar metabolites were analyzed by gas chromatography–mass spectrometry monitoring, and their estimated molecular ions were m/z 286, 358 and 481 after non-derivatization, trimethylsilylation and pentafluorobenzyl oxime formation, respectively. We tentatively identified novel decarbonylated metabolites of CBD as keto-enol tautomers. Among eight major recombinant human CYP enzymes, only CYP3A4 catalyzed the formation of decarbonylated metabolites. CBD was biotransformed to two decarbonylated metabolites, an enol-form (cyclopentadienol structure), and a keto-form (cyclopentenone structure) by human liver microsomes and CYP3A4.

Hydrophilic nano-carbon particle solution has been prepared up to 1.2 wt%-C by anodic electro-oxidation of graphite in water. It is composed of mellitic acid and graphite. A new application to metal ion trapping has been tested using a mineral water. Ca ions have been well trapped by nano-carbon particles, however, Mg ions did not been so well.

Many oxidative metabolites of tetrahydrocannabinols (THCs), active components of Cannabis sativa L. (Cannabinaceae), were pharmacologically potent, and 11‐hydroxy‐THCs, 11‐oxo‐Δ8‐THC, 7‐oxo‐Δ8‐THC, 8β,9β‐epoxyhexahydrocannabinol (EHHC), 9α,10α‐EHHC and 3'‐hydroxy‐Δ9‐THC were more active than THC in pharmacological effects such as catalepsy, hypothermia and barbiturate synergism in mice, indicating that these metabolites are active metabolites of THCs. Cannabidiol (CBD), another major component, was biotransfomred to two novel metabolites, 6‐hydroxymethyl‐Δ9‐THC and 3‐pentyl‐6, 7, 7a, 8, 9, 11a‐hexahydro‐1, 7‐dihydroxy‐7,10‐dimethyldibenzo[b,d]oxepin (PHDO) through 8R,9‐epoxy‐CBD and 8S, 9‐epoxy‐CBD as intermediates, respectively, identified by us. Both metabolites have some pharmacological effects comparable to Δ9‐THC. Cannabinol (CBN), the other major component, was mainly metabolized to 11‐hydroxy‐CBN by hepatic microsomes of animals including humans. The pharmacological effects of the metabolite were h...

Six halogenated derivatives of cannabidiol (CBD, 1) substituted on the aromatic ring at the 3' and/or 5' position, 3'-chloro- (2), 3',5'-dichloro- (3), 3'-bromo- (4), 3',5'-dibromo- (5), 3'-iodo- (6) and 3',5'-diiodo-CBD (7) were synthesized and their pharmacological effects of barbiturate-induced sleep prolongation, anticonvulsant effects and locomotor activity were evaluated by intravenous (i.v.) injection in mice. 2 (10 mg/kg, i.v., 69 +/- 10 min) significantly prolonged pentobarbital-induced sleeping time by 3.1-fold, compared to control (22 +/- 2 min), although other 1 derivatives used did not significantly affect the sleeping time. 2, 4 and 6 (10 mg/kg, i.v.) significantly prolonged hexobarbital-induced sleeping time by 2.0-, 2.0- and 2.3-fold, respectively, compared with control (52 +/- 5 min). On the other hand, 1 and all halogenated derivatives did not significantly prolong barbital-induced sleeping time. The monohalogenated derivatives, 2, 4 and 6 were able to prolong pentobarbital and hexobarbital-induced sleeping time, although the dihalogenated derivatives, 3, 5 and 7 did not exhibit a prolongation of the sleeping time. All halogenated derivatives of 1 except for brominated derivatives (2, 3, 6, 7) tended to prolong tonic seizure latency induced by pentylenetetrazol. 1 and its halogenated derivatives did not exhibit any prolongation of seizure latency induced by picrotoxin or strychnine. Maximal electroshock test demonstrated that 1 and 4 exhibited almost the same potency in their anticonvulsant effects, although other 1 derivatives 2, 3, 5, 6 and 7 did not show significant effect up to a dose of 63 mg/kg, i.v. The ED50 values (mg/kg, i.v.) of 1 and 4 were 38 and 44, respectively. 1 and 4 also showed anticonvulsant effect in minimal and maximal electroshock-threshold tests. 2, 4 and 6 tended to decrease the total distance (horizontal activity) and number of rearings (vertical activity) of mice, whereas 3, 5 and 7 tended to increase the number of rearings. However, the effects of all derivatives were not statistically significant from the control. 2 and 4 were the most potent derivatives on pharmacological activities among the synthetic cannabinoids examined in the present study. These results indicate that monohalogenation of 1 leads to some modification of the pharmacological profile of CBD.

The metabolism of 2,3′,4′,5-tetrachlorobiphenyl (TCB) was compared using liver microsomes and six isoforms of cytochrome P450 purified from rats, guinea pigs and hamsters. In microsomal study, the following species differences were observed: 1) Untreated guinea pigs and hamsters but not rats can metabolize this TCB to 3-hydroxy- or 4-hydroxy-2,3′,4′,5-TCB, 2) Guinea pig microsomes showed only 3-hydroxylatiog activity, whereas hamster microsomes showed higher activity of 4-hydroxylation than that of 3-hydroxylation. In common with three species, the 3-hydroxylation was accelerated by phenobarbital. The 4-hydroxylation in rats and hamsters was increased by pretreatment with 3-methylcholanthrene and 3,3′,4,4′,5-pentachlorobiphenyl. The hydroxylation activities of liver microsomes from the three species could be explained by an involvement of different isoforms of cytochrome P450. In addition, it is apparent that hamster CYP1A2 as well as hamster CYP2A8 is involved in the 4-hydroxylation of 2,3′,4′,5-TCB although it has no activity for 2,2′,5,5′-TCB.

2,5,2',5'-Tetrachlorobiphenyl (TCB) is one of the major components of PCB preparation, Kanechlor 400, which caused a mass food poisoning called Yusho in Japan in 1968 (Kuratsune 1996) and has phenobarbital (PB)-type inducing ability of rat liver microsomal enzymes (Yoshimura et al. 1979). The main metabolic pathway of this PCB isomer is 3-hydroxylation of aromatic ring catalyzed by cytochrome P450 (P450). So far, two groups of P450 superfamily, namely CYP1A and CYP2B subfamily, have been known to be involved in the hydroxylation of PCBs in mammals (Koga and Yoshimura 1996). In particular, the 3-hydroxylation of 2,5,2',5'-TCB is catalyzed by some P450 isoforms belonging to CYP2B subfamily, for example, CYP2B1 and 2B2 in rat liver and P450HPB-1 in hamster liver (Ishida et al. 1990; Koga et al. 1995a).

Anandamide (N-Arachidonoylethanolamine) amidohydrolase catalyzing hydrolysis of anandamide was characterized in mice. The enzymatic activity was highest in the liver, followed by the brain and testis. Negligible activity was found in heart, lung and spleen. The activity in brain and liver was mainly localized in the microsomal fractions. Kinetic experiments demonstrated that Km (μM) and Vmax (nmol/min/mg protein) for the brain microsomes were 9.3 and 2.58, respectively, while those for the hepatic microsomes were 180 and 18.9, respectively. The activity in the microsomes from the liver and brain was markedly inhibited by Cu2+, Hg2+, Se4+, phenylmethylsulfonylfluoride and sodium dodecylsulfate. Brain but not hepatic microsomal enzyme activity was inhibited by Δ9-tetrahydrocannabinol, cannabidiol and cannabinol. Kinetic parameters demonstrated that the inhibition by the cannabinoids was competitive in nature. Relatively high distribution of the enzyme activity in brain suggests an importance of the enzyme in the central nervous system to regulate the neuromodulatory fatty-acid amides.

delta 9-Tetrahydrocannabinol (delta 9-THC), cannabinol, cannabidiol and cannabichromene were detected in commercially available cannabis seeds by silica gel TLC and gas chromatography. These cannabinoids existed in rather high content (0.10-2.02 mg/100 g of seeds) in the feed for birds especially bracts (82.3-441 mg/100 g). When the suspension prepared from the benzene washing solution of cannabis seeds, BenW, was administered at a dose of 3 mg/kg corresponding to delta 9-THC into a mouse, i.v., BenW caused hypothermia, catalepsy, pentobarbital-induced sleep prolongation and suppression of locomotor activity. These pharmacological activities of BenW were significantly higher than those of delta 9-THC (3 mg/kg, i.v.). These results may indicate the necessity to reconsider the present regulations on marihuana.

Guinea pig hepatic enzyme, microsomal alcohol oxygenase, was able to oxidize both 7 alpha- and 7 beta-hydroxy-delta 8-tetrahydrocannabinol (7 alpha- and 7 beta-hydroxy-delta 8-THC) to 7-oxo-delta 8-THC. A cytochrome P450, named P450GPF-B, which mediates this oxidative metabolism was purified from hepatic microsomes of untreated female guinea pigs. The purified enzyme showed a single protein band of molecular mass 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal amino acid sequence of P450GPF-B is highly homologous with those of several cytochrome P450s belonging to the CYP3A subfamily. 18O derived from atmospheric oxygen was incorporated into 31 and 6%, respectively, of 7-oxo-delta 8-THC formed from 7 alpha- and 7 beta-hydroxy-delta 8-THC when the substrates were incubated with P450GPF-B under 18O2. The antibody against P450GPF-B significantly suppressed the oxidative activities of 7 alpha- and 7 beta-hydroxy-delta 8-THC to 7-oxo-delta 8-THC in hepatic microsomes of guinea pig. These results indicate that P450GPF-B is a major enzyme responsible for the hepatic microsomal alcohol oxygenase activities in the guinea pig.

ES46.5K, a novel esterase, was found in mouse hepatic microsomes. The enzyme catalyzed hydrolysis of 2-acetylaminofluorene and cannabinoid esters. In latter case, the enzyme regioselectively hydrolyzed acetates of 11-hydroxy-delta8-tetrahydrocannabinol. Western immunoblotting analysis demonstrated that none of immuno-reactive proteins against ES46.5K were present in hepatic microsomes from rats, guinea-pigs, monkeys and humans. Rabbit hepatic microsomes contained an immuno-reactive protein, although molecular weight of the protein was rather high (50 kDa) by SDS-PAGE. Esterase activity stained after PAGE demonstrated that ES46.5K retained at origin. Hepatic microsomes of above animal species contained several activity bands on the PAGE, while only mouse hepatic microsomes exhibited significant activity at origin. In mice, liver was only an organ containing ES46.5K by analyzing Western immunoblotting. These results indicate that distribution of ES46.5K is quite different from known carboxylesterases, and suggest that the enzyme has some role in the biotransformation of xenobiotic amide and esters.

Deamination or oxidative cleavage of the carbon-nitrogen bond in various phenylisopropylamines was examined in liver microsomes from rabbits and rats, and in reconstituted systems containing CYP2C subfamily isozymes. Kinetic studies of phenylacetone formation from six amphetamine (AP) derivatives, catalyzed by rabbit liver microsomes, indicated that AP had the highest apparent affinity (lowest K(m)) and increasing the size of the substituent on the nitrogen atom decreased the affinity. The values of maximal velocity increased with increasing size of the substituent. Experiments with purified CYP2C3 from rabbit liver gave similar results: this enzyme showed the highest activity for phenylacetone formation from benzphetamine (BZP) and showed lower activities with compounds having smaller nitrogen substituents. Based on these results, we conclude that among a series of AP derivatives, the parent phenylisopropylamine has the highest affinity for rabbit liver deaminase, where as BZP has the highest turnover. However, the intrinsic clearance (Vmax/K(m)) values for the individual reactions tended to be comparable. The rates of BZP and deprenyl N-demethylation by rat CYP2C11 and 2C13 were far greater than those of the reactions at other N-alpha-positions. This result indicated that rat CYP2C enzymes have a more rigid regioselectivity than rabbit CYP2C3 for the deamination/N-dealkylation of phenylisopropylamines.

1. The in vitro and in vivo metabolism of 2,4,5,2',4',5'-hexachlorobiphenyl (PCB153) in guinea pig has been studied. 2. Seven metabolites were detected in the faeces of PCB153-treated animals and three were identical to those produced by dog liver microsomes. The detection of a metabolite where a chlorine atom was shifted from the 2- to 3-position strongly suggested the involvement of 2,3-arene oxide intermediate, and evidence for the concomitant formation of a 3,4-arene oxide intermediate was provided by identifying other two minor metabolites which were dechlorinated at the 4-position. 3. In vitro studies using liver microsomes from guinea pigs revealed that the 2,3-arene oxide and 3-hydroxylation pathways are the predominant metabolic routes compared with the 3,4-arene oxide pathway. Although the guinea pig is an another species that can metabolize PCB153 mainly to the 2,3-arene oxide intermediate, the rate of formation was only about one-tenth of the dog. 4. These results indicate that the ability to form this unusual 2,3-arene oxide intermediate may not be responsible for high excretion rate of this congener. Our data also suggest that the cytochrome P450-catalysed metabolism of PCB153 in the guinea pig and dog are similar, whereas for post-cytochrome P450 metabolism, the guinea pig resembles the rabbit.

Metabolism of 2,5,2′,5′-tetrachlorobiphenyl (TCB) was studied using liver microsomes of hamsters and two hamster P450 isoforms, CYP1A2 and 2A8. CYP2A8 catalyzed selectively 4-hydroxylation of 2,5,2′,5-TCB at a rate of 21.7 pmol/min/nmol P450. In contrast, CYP1A2 showed no activity for hydroxylation of 2,5,2′,5′-TCB. Immunological study revealed that rabbit antiserum against CYP2A8 almost completely inhibited the microsomal 4-hydroxylation but that against CYP1A2 did not. It was also shown that the induction pattern of CYP2A8 protein by P450 inducer was similar to that of the 4-hydroxylase activity in hamster liver microsomes. These results suggest that CYP2A8 plays a major role in the 4-hydroxylation of 2,5,2′,5′-TCB in hamster liver.

The metabolic formation of an oxepin derivative, 3-pentyl-6, 7, 7a, 8, 9, 11a-hexahydro-1, 7-dihydroxy-7, 10-dimethyldibenzo-[b,d]-oxepin, from cannabidiol was studied in-vitro using guinea-pig hepatic microsomes. The hepatic microsomes catalysed the formation of the metabolite from cannabidiol and 8S, 9-epoxycannabidiol in the presence of an NADPH-generating system and 3, 3, 3-trichloropropene-1, 2-oxide. 8S, 9-Epoxycannabidiol was thought to be an intermediate in the formation of the metabolite, which was identified by gas chromatography-mass spectrometry. The metabolite synthesized from 8S, 9-epoxycannabidiol diacetate exhibited catalepsy, hypothermia and pentobarbitone-induced sleep prolongation in mice, although the pharmacological effect was less potent than that of Δ9-tetrahydrocannabinol.

This review describes recent advances in the metabolism of cannabinoids. Cannabidiol was metabolized to cannabielsoin, 6 beta-hydroxymethyl-delta 9-tetrahydrocannabinol and an oxepine derivative through epoxide intermediates by hepatic microsomal enzymes containing cytochrome P450 of animals. Cannabidiol inactivated cytochrome P450 UT-2 (CYP2C11) not equal to in male rats and a member of 3A subfamily in mouse liver. These inactivations may be very important because serious drug-drug interactions will occur in the case that cannabidiol is co-administered with drugs which are metabolized mainly by the enzyme system containing these P450 isozymes. A member of cytochrome P450 belonging to 2C subfamily was the major isozymes responsible for the cannabinoid metabolism in many experimental animals and that of 3A subfamily made some contribution to the metabolism of cannabinoids by human hepatic microsomes. Microsomal aldehyde oxygenase, a particular isozyme of cytochrome P450 catalyzing the oxidation of 11-oxo-tetrahydrocannabinol to tetrahydrocannabinol-11-oic acid, was found for the first time by the authors. Cytochrome P450 MUT-2 (CYP2C29) is the major isozyme responsible for the microsomal aldehyde oxygenase activity in mouse hepatic microsomes.

Metabolism of 2,4,5,2′,4′,5′-hexachlorobiphenyl was studied with cDNA-expressed human P450 2B isoform, CYP2B6. 3-Hydroxy-2,4,5,2′,4′,5′-hexachlorobiphenyl was identified as a major metabolite, and the formation activity was compared with that of dog CYP2B11 and guinea pig P450GP-1. The activity of 3-hydroxylation was comparable with that of P450GP-1, but one-tenth of CYP2B11. These results indicate that P450 2B in humans as well as other animal species can metabolize 2,4,5,2′,4′,5′-hexachlorobiphenyl, and the reason why this PCB congener remained most abundantly in human bodies is discussed.

The most abundant metabolite of delta 9-tetrahydrocannabinol (delta 9-THC) formed with hepatic microsomes of monkeys was 11-OH-delta 9-THC, followed by 8 alpha-OH-, 8 beta-OH- and 3'-OH-delta 9-THCs. Two cytochrome P450 isozymes, P450RM-A and P450JM-C, were purified from monkey hepatic microsomes and found to have a molecular weight of 51,000. In the reconstituted system, the activities of P450RM-A towards formation of 11-OH-, 8 alpha-OH-, 8 beta-OH- and 3'-OH-delta 9-THCs were 19-, 40-, 22- and 10-fold higher, respectively, than the corresponding activities of the hepatic microsomes. The activity of P450JM-C towards formation of 3'-OH-delta 9-THC was 10-fold higher than that of P450RM-A, while the activities of both isozymes for 11- and 8 alpha-hydroxylation were not so much different and the 8 beta-hydroxylation activity was 14-fold higher in P450RM-A than in P450JM-C. Antibodies against P450RM-A and P450JM-C markedly inhibited the microsomal formation of 11-OH- and 8 alpha-OH-delta 9-THCs, and 3'-OH-delta 9-THC, respectively. These results suggest that P450RM-A and P450JM-C are major isozymes responsible for the formation of 11-OH- and 8 alpha-OH-delta 9-THCs, and 3'-OH-delta 9-THC, respectively, in monkeys.

In the hamster liver, 2,5,2',5'-tetrachlorobiphenyl (TCB) is metabolized to 3-hydroxy- and 4-hydroxy-2,5,2',5'-TCB to a similar extent, and formation of the former metabolite is stimulated by phenobarbital pretreatment of the animals, while that of the latter metabolite is stimulated by 3-methylcholanthrene pretreatment. In the present study, we identified a new isoform (designated P450HPB-1) of cytochrome P450 which proved to be phenobarbital-inducible and responsible for 3-hydroxylation of this TCB isomer. This isoform was purified from liver microsomes of phenobarbital-treated hamsters and characterized. P450HPB-1 has a molecular mass of 50 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the absorption maxima of the oxidized form at 417 nm and of the reduced CO-complex form at 450 nm. The sequence of 28 amino acids of P450HPB-1 at the amino-terminal has a 68% similarity with those of rat P450 2B1 and mouse P450 2b10, 57% similarity with that of guinea pig P450GP-1, and 54% similarity with that of guinea pig P450GP-1, and 54% similarity with those of rabbit P450 2B4 and dog P450 2B11. P450HPB-1 in the reconstituted system catalyzed the 3- but not 4-hydroxylation of 2,5,2',5'-TCB, at a rate of 19.0 pmol/min/nmol P450. The isoform also has high catalytic activity for 17-oxidation of testosterone but low activity for the N-demethylation of benzphetamine and 16 alpha- and 16 beta-hydroxylations of testosterone. In microsomal metabolism of 2,5,2',5'-TCB, rabbit antiserum against P450HPB-1 almost completely inhibited 3- but not 4-hydroxylation. Immunoblot analysis of hamster liver microsomes revealed that P450HPB-1 was constitutive and phenobarbital-inducible but was decreased by pretreatment with 3-methylcholanthrene or 3,4,5,3',4'-pentachlorobiphenyl. These results suggest that P450HPB-1 belongs in the P450 2B subfamily and apparently plays a major role in the 3-hydroxylation of 2,5,2',5'-TCB, in hamster liver.

3,4,5-Trimethoxyphenylacetaldehyde, an intermediate metabolite of mescaline, was oxidized to 3,4,5-trimethoxyphenylacetic acid by mouse hepatic microsomes. The reaction was NADPH-dependent, and inhibited by SKF 525-A, metyrapone and disulfiram. A P450 isozyme in mouse hepatic microsomes, P450 MUT-2 (CYP2C29), catalyzed the reaction (0.96 nmol/min/nmol P450) in which NADPH and NADPH-cytochrome c reductase were essential for the catalytic activity. The reaction was confirmed to be an oxygenation since molecular oxygen was incorporated into the carboxylic acid metabolite formed under oxygen-18 gas by GC-MS analysis. By addition of antibody against CYP2C29 to the microsomes (3.2 mg/mg microsomal protein) the MALDO activity was inhibited by 35% of the control value with preimmune serum, suggesting that CYP2C29 or an immunologically-related isozyme(s) plays a major role in the NADPH-dependent oxidation of 3,4,5-trimethoxyphenylacetaldehyde to 3,4,5-trimethoxyphenylacetic acid by mouse hepatic microsomes. Pharmacological experiments on mescaline and its deaminated metabolites using mice indicated that the metabolites were much less active or were inactive in cataleptogenic effect and pentobarbital-induced sleep prolongation as compared with the parent compound.

Cytochrome P450 isozymes involvement in the metabolic activation of tetrahydrocannabinol (THC) have been investigated. CYP2C29 was a major isozyme responsible for 11-hydroxylation as well as 7α- and 8 α-hydroxylation of THC in the mouse liver. In rats, CYP2C11 and CYP2C6 were major isozymes involvement in the 11-hydroxylation of THC in the male and female rat liver, respectively. P450G-A belonging to CYP2B subfamily was a major isozyme for metabolizing THC in the guinea pig liver. In monkey liver, P450RM-A (CYP2A) and P450JM-D (CYP2C), and P45OJM-C (CYP2B) were responsible for the 11-hydroxylation and 3'-hydroxylation of TUC, respectively. the 11-hydroxylation of TUC In human liver was also catalyzed by P450 isozyme belonging to CYP2C, weheras 8 β-hydroxylation of Δ9-TIC and 7 α-hydroxylation of Δ8-THC were catalyzed by CYP3A. The present results indicate that CYP2C Is mainly responsible for the metabolic activation of 1HC in most of animals including humans, while CYP2A has some role in the metabolic activation of 1W in the monkey liver.

The hepatic microsomal metabolism of cannabinoids was studied using the liver from an old woman. delta 8-Tetrahydrocannabinol, delta 9-tetrahydrocannabinol and cannabinol were biotransformed to their respective 11-hydroxy metabolites by a microsomal fraction with specific activities (pmol/min/mg protein) of 29.1, 47.1 and 27.9, respectively. In addition, both 11-oxo-delta 8-tetrahydrocannabinol and 11-oxo-delta 9-tetrahydrocannabinol were metabolized to the corresponding carboxylic acids with the microsomes. An antibody against mouse CYP2C29 almost completely inhibited 11-hydroxylation of the cannabinoids and microsomal aldehyde oxygenase (MALDO) activity for 11-oxo-delta 8-tetrahydrocannabinol and 11-oxo-delta 9-tetrahydrocannabinol, used as substrates, whereas an antibody against rat CYP3A2 conversely stimulated the 11-hydroxylation of delta 8-tetrahydrocannabinol and MALDO activity for 11-oxo-delta 8-tetrahydrocannabinol. The results indicate that a member of CYP2C is primarily responsible for the metabolism of the above cannabinoids in the human hepatic microsomes.

The ability of animals to metabolize xenobiotics has been acquired over a period of more than 2 thousand million years during the process of adaptation to the environment. The acquired character has been accumulated and conserved in the genome as genetic information. Thus, the ability of animals to metabolize the large number of chemicals produced by modem chemical industries must be an inheritance of enforced contact with natural xenobiot­ ics. In early studies with liver microsomes, the substrate specificity of cytochrome P450 (P450) was thought to be very general, but investigators now believe that its broad specificity can be explained in terms of an integrated system with numerous P450 isoenzymes. Regioand stereoselec­ tive aspects of drug metabolism by P450 are important concerns in studies of the pharmacokinetics of racemates or in the extrapolation of metabolic profiles of drugs from laboratory animals to humans. In this review, we summarize current knowledge concerning the regioselectivity and stereo­ selectivity of the major P450 isozymes.

Induction of hepatic microsomal bilirubin UDP_glucuronyltransferase and CYP4A1 P450 by 3,4,3′,4′-tetrachlorobiphenyl (TCB) and 3,4,5,3′,4′-pentachlorobiphenyl (PenCB) was studied in guinea pigs and rats. Both enzyme activities in guinea pigs were increased 4-fold or more over the control by PenCB treatment, while by treatment with a less toxic congener, TCB, these were increased to a lesser extent. On the contrary, the rat enzymes were significantly decreased by treatment with these co-planar polychlorinated biphenyls. These results suggest that the alterations of both bilirubin UDP-glucuronyltransferase and CYP4A P450 are the characteristic indices for the toxicites of polychlorinated hydrocarbons.

1. Enzymatic dehydration of atropine and scopolamine was studied in guinea pig. 2. The incubation of these alkaloids with guinea pig liver cytosol in the absence of cofactors gave no dehydrated metabolite. However, when atropine and scopolamine were incubated with cytosol supplemented with ATP and sodium sulphate, dehydrated metabolites, apoatropine and aposcopolamine were formed. The formation of these metabolites was confirmed by gas chromatography-mass spectrometry. 3. The reaction required ATP as well as cytosol as the obligatory factors. Deletion of sodium sulphate from the reaction mixture also resulted in a decrease of the activities, although this treatment showed limited effect when the low concentration of atropine was used. Furthermore, dehydroepiandrosterone, an excellent substrate for hydroxysteroid-sulphotransferase, effectively inhibited the in vitro activity of atropine dehydration. 4. Administration of dehydroepiandrosterone to guinea pig followed by atropine treatment caused decreased urinary excretion of apoatropine. 5. These results strongly suggested that the dehydration of atropine and scopolamine takes place via the sulphate conjugate intermediates produced from the sulphotransferase-catalysed reaction. The present finding is the first example of the sulphotransferase-dependent dehydration of a drug, and its generality in drug metabolism is discussed.

Imaging mass spectrometry based on matrix-assisted laser desorption/ionization gives distributions of multiple biomolecules and identification of analytes in a tissue section. We are developing a stigmatic imaging mass spectrometer using a multi-turn time-of-flight mass spectrometer, MULTUM-IMG. A mesh pattern image was obtained with spatial resolution of 3 μm. Separation of stigmatic ion images according to the mass-to-charge ratio was also successfully demonstrated with the micro-dot pattern made with crystal violet and methylene blue. An ion image of a micro-dot pattern sample was observed after ten cycles in the multi-turn circuit of MULTUM-IMG, and the pattern of the sample was maintained. Then, stigmatic ion images of the ions of crystal violet and methylene blue produced from the stained section of a brain were observed. An ion image of whole part of a hippocampus (3.25 mm × 1.5 mm) in the brain section can be obtained within 13 minutes.

Effect of a co-planar PCB, 3,4,5,3′,4′-pentachlorobiphenyl (PenCB) on gluconeogenesis in guinea pigs was compared with that in rats. The key enzyme of gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK), in liver cytosol of rats was reduced by PenCB treatment to about 50% of control level. Hypoglycemia was also seen in the rat. On the contrary, the liver cytosol PEPCK activity in guinea pigs was slightly increased by PenCB treatment. Plasma levels of alanine and tryptophan were increased in the rat treated with PenCB, but the level of glycogenic amino acid, alanine, was not raised significantly in the guinea pig. Thus the modification of the gluconeogenesis was not involved in the PenCB toxicity in highly sensitive guinea pigs.

A morphine UDP-glucuronyltransferase was purified from liver microsomes of untreated Sprague-Dawley rats. A new gel, omega(beta-carboxypropionylamino)octyl Sepharose 4B, was prepared by coupling monomethylsuccinate with omega-aminooctyl Sepharose 4B and this was used as an efficient tool for the separation of microsomal enzymes. Emulgen 911 solubilized microsomes were applied to a column packed with the gel and eluted at pH 7.4 while increasing KCl concentration in a stepwise manner. An isoform was further purified with UDP-hexanolamine Sepharose 4B gel. The purified UDP-glucuronyltransferase (morphine UGT of untreated rat, morphine UGTUT) exhibited a molecular weight of 52000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and was capable of glucuronidating the 3-hydroxyl group of morphine. The isoform catalyzed to a small extent the glucuronidation of 4-hydroxybiphenyl; however, no glucuronidation activity towards androsterone, testosterone, bilirubin, 4-nitrophenol and the 6-hydroxyl group of morphine was observed. The difference in properties, compared with morphine UGT (molecular weight 56000) which was purified previously from phenobarbital-treated rats, is discussed.

A novel membrane-bound esterase was purified from hepatic microsomes of male ddN mice using ω-aminooctyl Sepharose-4B, DEAE-5PW, CM-Sephadex C-50 and/or hydroxylapatite column chromatographies. The purified enzyme (ES46.5K) showed a single band on a sodium dodecylsulfate-polyacrylamide gel with a mimimum molecular weight of 46.5 Kd. The N-terminal amino acid sequence of the enzyme had no homology to those of known carboxylesterases. ES46.5K exhibited esterase activities toward 11-acetoxy derivatives of tetrahydrocannabinol (THC), whereas the enzyme did not hydrolyze phenolic acetates of cannabinoids (Δ8-THC, Δ9-THC and cannabidiol) except for 1-acetyl-cannabinol. ES 46.5K also possessed esterase activity toward p-nitrophenylacetate, 1-naphtylacetate and 2-acetylaminofluorene. Emulgen 911 and deoxycholate affected differently on the esterase activity of ES46.5K and carboxylesterase purified from porcine liver. Immuno-reactive protein to antibody against ES46.5K was present in hepatic microsomes from mice and rabbits, but not those from rats, guinea pigs and monkeys. The present study demonstrates that mouse hepatic microsomes contain a novel type of esterase having molecular weight of 46.5 Kd and high catalytic activity toward 11-hydroxy-esters of cannabinoids.

A stigmatic and microscopic imaging mass spectrometer has been developed using matrix-assisted laser desorption/ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG. Ion images of rhodamine 6G masked by fine grids with pitches of 63.5, 25.4, and 12.7 µm were clearly observed in the linear mode. Separation of stigmatic ion images according to the time-of-flight, i.e., the mass-to-charge ratio of ions was also successfully demonstrated with a micro-dot pattern ma de with crystal violet and methylene blue. Stigmatic ion images of a micro-dot pattern made with crystal violet was also observed and the ion image of the pattern was maintained after 10 cycles in MULTUM-IMG. A section of a mouse brain stained with crysta l violet and methylene blue was observed in the linear mode, and the stigmatic total ion image of crystal violet and methylene blue was in good agreement with the optical microphotograph of the hippocampus in the section. Keywords: imaging mass spectrometry, matrix-assisted laser desorption/ionization, multi-turn time-of-flight mass spectrometer, delay-line detector, mid-infrared tunable laser

Six halogenated derivatives of cannabidiol (CBD, 1) substituted on the aromatic ring at the 3' and/or 5' position, 3'-chloro- (2), 3',5'-dichloro- (3), 3'-bromo- (4), 3',5'-dibromo- (5), 3'-iodo- (6) and 3',5'-diiodo-CBD (7) were synthesized and their pharmacological effects of barbiturate-induced sleep prolongation, anticonvulsant effects and locomotor activity were evaluated by intravenous (i.v.) injection in mice. 2 (10 mg/kg, i.v., 69 +/- 10 min) significantly prolonged pentobarbital-induced sleeping time by 3.1-fold, compared to control (22 +/- 2 min), although other 1 derivatives used did not significantly affect the sleeping time. 2, 4 and 6 (10 mg/kg, i.v.) significantly prolonged hexobarbital-induced sleeping time by 2.0-, 2.0- and 2.3-fold, respectively, compared with control (52 +/- 5 min). On the other hand, 1 and all halogenated derivatives did not significantly prolong barbital-induced sleeping time. The monohalogenated derivatives, 2, 4 and 6 were able to prolong pentobarbital and hexobarbital-induced sleeping time, although the dihalogenated derivatives, 3, 5 and 7 did not exhibit a prolongation of the sleeping time. All halogenated derivatives of 1 except for brominated derivatives (2, 3, 6, 7) tended to prolong tonic seizure latency induced by pentylenetetrazol. 1 and its halogenated derivatives did not exhibit any prolongation of seizure latency induced by picrotoxin or strychnine. Maximal electroshock test demonstrated that 1 and 4 exhibited almost the same potency in their anticonvulsant effects, although other 1 derivatives 2, 3, 5, 6 and 7 did not show significant effect up to a dose of 63 mg/kg, i.v. The ED50 values (mg/kg, i.v.) of 1 and 4 were 38 and 44, respectively. 1 and 4 also showed anticonvulsant effect in minimal and maximal electroshock-threshold tests. 2, 4 and 6 tended to decrease the total distance (horizontal activity) and number of rearings (vertical activity) of mice, whereas 3, 5 and 7 tended to increase the number of rearings. However, the effects of all derivatives were not statistically significant from the control. 2 and 4 were the most potent derivatives on pharmacological activities among the synthetic cannabinoids examined in the present study. These results indicate that monohalogenation of 1 leads to some modification of the pharmacological profile of CBD.

The tissue-specific expression and induction of P450 GP-1 , a constitutive form of cytochrome P450 of the guinea pig classified into the CYP2B subfamily, were studied. Prior to these studies, a P450 form (P450 GP-1 PB ) was purified from phenobarbital-treated guinea pigs and the properties were compared with those of the P450 GP-1 . This form was judged to be the same as P450 GP-1 existing in untreated animals by comparisons of their N-terminal amino acid sequences, peptide maps, and affinities toward anti-P450 GP-1 antibody. Immunostaining of P450 GP-1 revealed that the lung and small intestine as well as the liver of untreated guinea pigs contain P450 GP-1 , while none or only small amounts of this P450 form were observed in the kidney, heart, spleen, urinary bladder, and testis. The amount of liver P450 GP-1 protein expressed in untreated guinea pigs was estimated to be 19.4% of the total cytochrome P450 and this form was increased 1.7-fold by phenobarbital treatment. Similarly, intestinal P450 GP-1 was increased by phenobarbital treatment. However, lung P450 GP-1 was not increased by the treatment. It was also observed that the liver P450 GP-1 is induced with SKF-525A to the same extent as with phenobarbital. On the other hand, dexamethasone, p,p′ -dichlorodiphenyltrichloroethane, and isosafrole showed no or only a weak ability to increase the liver P450 GP-1 content. The drug-metabolizing activities in the liver microsomes of SKF-525A-pretreated guinea pigs were lower than those in phenobarbital-treated animals, although the P450 GP-1 protein was induced equally by these treatments. The low activities of SKF-525A-treated animals in the drug metabolisms were attributed to the formation of the metabolic-intermediate complex between P450 GP-1 and SKF-525A metabolite. These results permitted us to conclude that the tissue specificity in the expression of guinea pig P450 belonging to the CYP2B subfamily and the inducibility with chemicals are similar to those of rat CYP2B1, although the constitutive expression of guinea pig liver P450 GP-1 is much higher than that of CYP2B1.

An effective therapy which assists the elimination of the residual chlorinated compounds in Yusho patients has still not been established. To determine the behavior of polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PCDFs) in patients 20 years since the occurrence of Yusho disease, the concentrations of PCBs and PCDFs in the blood, subcutaneous adipose tissue and stools were investigated in six patients with typical symptoms of Yusho disease. The daily excretion levels of 2,3,4,7,8‐penta‐chlorodibenzofuran (PnCDF), and 1,2,3,4,7,8‐ and 1,2,3,6,7,8‐hexachlorodibenzofurans (HxCDFs) into the stools of Yusho patients were 200 to 1380pg, and 190 to 1590pg, respectively. Similarly, PCBs were excreted at levels of 320 to 1370 ng per day, into the stools. The amounts of PnCDF and HxCDFs excreted into the stool per day was correlated to the levels of those chemicals in the blood and subcutaneous adipose tissue. The daily levels of PnCDF and HxCDFs excreted into the stool were comparable with th...

A protein (designated as protein-B) was purified from liver microsomes of adult male guinea pigs by an affinity chromatography with omega-aminooctyl Sepharose 4B, followed by HPLC using DEAE-5PW and hydroxyapatite columns which had been used to purify a cytochrome P450 (P450) isozyme (P450-A) from the same subcellular fraction (Narimatsu et al., Biochem Biophys Res Commun 172: 607-613, 1990). Protein-B had a molecular mass of 49 kDa in SDS-PAGE, but did not show absorbance at 417 nm for heme. Further, it did not show any oxidative activities towards aniline (AN), d-benzphetamine (d-BP), p-nitroanisole (p-NA) or delta 9-tetrahydrocannabinol (delta 9-THC) in a reconstituted system including dilauroylphosphatidylcholine, NADPH-P450 reductase, and cytochrome b5. However, antiserum against protein-B raised in rabbits suppressed liver microsomal oxidative activities towards d-BP and p-NA dose-dependently. The antibody decreased delta 9-THC oxidative activity most effectively, but did not decrease AN hydroxylation activity. Antiserum against P450-A suppressed all the activities towards these four substrates, especially towards delta 9-THC, in liver microsomes of male guinea pigs. Moreover, reconstitution with hemin made it possible for protein-B to produce some oxidative activity toward delta 9-THC. These results suggest that protein-B is also a cytochrome P450 isozyme which has lost a heme moiety during purification steps. Both P450-A and protein-B could have a role as cytochrome P450 isozymes in the oxidative metabolism of drugs, especially that of delta 9-THC by the liver microsomes of adult male guinea pigs.

A mouse hepatic microsomal enzyme that catalyzes the oxidation of cannabinol (CBN) to 11-hydroxycannabinol (11-OH-CBN) was characterized. 11-OH-CBN formed was determined by HPLC. The reaction required NADPH as a cofactor and the optimal pH for the reaction was at 7.8. The Km and Vmax values for the reaction were 122 μM and 8.82 nmol/min/mg protein, respectively. The reaction was inhibited by inhibitors of cytochrome P450 such as SKF 525-A and by cannabidiol. The pretreatment of mice with phenobarbital significantly increased the enzyme activity but not with 3-methylcholanthrene, whereas that with cobaltous chloride significantly decreased the activity. These results indicate that cytochrome P450 plays a major role in the formation of 11-OH-CBN in the mouse hepatic microsomes.

Our previous study suggested that squalane would be a good candidate for an antidote to reduce the toxicity of drug ingested accidentally at a high dose by enhancing the drug elimination from the body. In the present study, we investigated whether squalane given orally could enhance the elimination of theophylline, phenobarbital and strychnine which were administered parenterally to rats or mice. Squalane increased the fecal excretion of theophylline and reduced the serum level of the drug in rats. Squalane accelerated the fecal excretion of strychnine in mice. These results suggest that squalane may stimulate more the elimination of neutral (theophylline) or basic (strychnine) drugs which should be present in unionized form in intestinal lumen, than that of acidic drugs.

Cytochrome P450 isozymes purified from rat hepatic microsomes were able to catalyse the oxidation of 11-oxo-delta 8-tetrahydrocannabinol (11-oxo-delta 8-THC) to delta 8-THC-11-oic acid in the presence of NADPH, cytochrome P450 reductase and dilauroylphosphatidylcholine. The catalytic activities (nmol/min/nmol P450) of cytochrome P450s, UT-2 (IIC11), UT-4 (IIA2), UT-5 (IIC13), PB-1, PB-2 (IIC6), PB-4 (IIB1), MC-1 (IA2), MC-5 (IA1) and IF-3 (IIA1), were 0.69, 0.08, 0.07, 0.23, 0.46, 0.02, 0.06, 0.07 and 0.34, respectively, whereas the activities of cytochrome P450s, PB-5 (IIB2) and DM (IIE1), were less than 0.02 nmol/min/nmol P450. Cytochrome P450 IIC11 showed the highest catalytic activity of the cytochromes examined. The mechanism for the oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid by cytochrome P450 IIC11 was established as being an oxygenation since one atom of oxygen-18 was exclusively incorporated into the carboxylic acid formed under 18O2. The antibody raised to cytochrome P450 IIC11 inhibited by 60% the hepatic microsomal oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid in male rats. These results indicate that cytochrome P450 IIC11 is a major form of the cytochrome to catalyse the oxidation of 11-oxo-delta 8-THC to delta 8-THC-11-oic acid in the hepatic microsomes of male rats and that the oxidation of aldehyde to carboxylic acid is a catalytic activity common to most isozymes of P450.

In order to provide evidence that a cytochrome P450 belonging to the IIB subfamily is expressed as a constitutive form in the guinea pig, we tried to purify an isozyme from liver microsomes of untreated guinea pigs by assessing its reactivity with anti-P450b antibody in the present study. One form of cytochrome P450, named P450GP-1, was obtained. The minimum molecular weight of this isozyme was estimated to be 52,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino terminal sequence up to the 33rd amino acid of P450GP-1 was determined. As expected, comparison of the amino acid sequence with those of cytochrome P450 isozymes from other species reported so far indicated that P450GP-1 was highly homologous to P450s categorized in the IIB subfamily; that is, 67% similarity to rat P450b, 82% to rabbit LM2, 76% to dog PBD-2, 70% to mouse pf 3 46 , and 73% to human IIB1. On the other hand, P450GP-1 showed only low similarity, less than 41%, to other cytochrome P450s of the II subfamily and those of the I, III, and IV families. Affinity of P450GP-1 to anti-P450b immunoglobulin G was confirmed to be comparable with that of a principal antigen, P450b. Immunoblot analysis revealed that P450GP-1 in the guinea pig liver microsomes was induced by phenobarbital treatment, but the increase was not as large as in the rat. P450GP-1 efficiently catalyzed benzphetamine N-demethylation, strychnine 2-hydroxylation, and testosterone 16β-hydroxylation, all of which are also catalyzed by P450b. Based on these results, it was strongly suggested that the IIB-type of cytochrome P450 in guinea pigs, at least one of them, is a constitutive form which is moderately induced by phenobarbital.

Metabolism of two polychlorinated biphenyls, 3, 4, 3', 4'- and 2, 5, 2', 5'-tetrachlorobiphenyl (TCB), was studied using rat liver microsomes and the four forms of cytochrome P-450 (P-450), P-450b, P-450e, P-450c and P-450d. At first, effects of various inducers of P-450 such as phenobarbital (PB), 3-methylcholanthrene (MC), isosafrole (ISF) and pregnenolone 16α-carbonitrile on the formation of metabolites of these TCBs by liver microsomes were compared. 3, 4, 3', 4'-TCB was significantly metabolized by liver microsomes from MC-treated rats to form two previously reported metabolites, 4-hydroxy-3, 5, 3', 4'-TCB and 5-hydroxy-3, 4, 3', 4'-TCB with a relative ratio of 2.5 : 1. Incubation with microsomes from untreated or PB-treated rats produced none of the metabolites. On the other hand, 2, 5, 2', 5'-TCB was metabolized to 3-hydroxy-2, 5, 2', 5'-TCB most easily by liver microsomes from PB-treated rats and at a moderate rate by liver microsomes from ISF-treated rats. Activities of microsomes from untreated or MC-treated rats to hydroxylate 2, 5, 2', 5'-TCB were low or undetectable. When these TCB hydroxylase activities were examined with a reconstituted system consisting of each P-450, reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase, dilauroylphosphatidylcholine and NADPH-generating system, only P-450c catalyzed both the 4-and 5-hydroxylations of 3, 4, 3', 4'-TCB at a ratio of 2.2 : 1. On the contrary, the hydroxylation of 2, 5, 2', 5'-TCB proceeded efficiently with P-450b and P-450e, being more efficient with the former. P-450d did not show any catalytic activity toward 3, 4, 3', 4'-TCB and 2, 5, 2', 5'-TCB. These results indicated that P-450c and P-450b play a major role in the metabolism of 3, 4, 3', 4'-TCB in MC-treated microsomes and of 2, 5, 2', 5'-TCB in PB-treated microsomes, respectively. The inhibition studies using antibodies against P-450c and P-450b further supported the above conclusions.

Cannabidiol hydroxy-quinone (CBDHQ) was identified as an air oxidation product of cannabidiol (CBD). The in vitro incubation of mouse hepatic microsomes with CBDHQ resulted in a decrease of cytochrome P-450 content. CBDHQ inhibited the hepatic microsomal drug-metabolizing enzymes of mice. This inhibitory effect was stronger than that of CBD. CBDHQ (150 microM) inhibited aniline hydroxylase, p-nitroanisole O-demethylase and aminopyrine N-demethylase in the microsomes by 70, 52 and 77%, respectively, whereas the same concentration of CBD caused the inhibition by 39, 30 and 26%, respectively. CBDHQ (91.5 microM) significantly decreased total heme content by 21% and free SH groups by 11% in the microsomes. The results indicate that CBDHQ, which is an oxidation product of CBD, inhibits the hepatic microsomal drug-metabolizing enzymes through the decrease of cytochrome P-450 content.

1. An unknown metabolite of strychnine termed M-5 was isolated, from an incubation mixture using guinea pig liver microsomes, by repeated preparative t.l.c. 2. The structure of M-5 was shown to be 22-hydroxystrychnine by mass, n.m.r. and i.r. spectrometry. 3. 22-Hydroxystrychnine is a stable enol, a unique metabolite possessing a hydroxyl group on the double bond of the alicylic moiety.

Two forms of cytochrome P-450 (P-450) from liver microsomes of hamsters treated with 2,3,4,7,8-pentachlorodibenzofuran (PenCDF), which possesses the potent acute toxicity and 3-methylcholanthrene (MC)-type inducing ability of liver microsomal monooxygenases in animals, were purified and characterized. These P-450 forms, designated as hamster P-450H and hamster P-450L, had the molecular masses of 52 and 50 kDa, respectively, and showed the absorption maximum of CO-reduced difference spectra at 446 nm. The absolute spectra of their oxidized forms indicated that hamster P-450H was in high-spin state and hamster P-450L was in low-spin state. A part of PenCDF injected into hamster was tightly bound to purified hamster P-450H at a ratio of 0.107 nmol PenCDF/nmol P-450. In a reconstituted system, both hamster P-450H and hamster P-450L showed relatively low catalytic activities for 3-hydroxylation of benzo[a]pyrene and O-deethylations of both 7-ethoxyresorufin and 7-ethoxycoumarin, while they both catalyzed 7 alpha- and 2 alpha-hydroxylations of testosterone effectively to a similar extent. Addition of cytochrome b5-to a reconstituted system accelerated the formation of 7 alpha-hydroxytestosterone 5.3-fold with hamster P-450L and 2.2-fold with hamster P-450H. In addition, hamster P-450H catalyzed estradiol 2-hydroxylation at a high rate but hamster P-450L did not.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatic microsomal oxygenation of aldehydes to carboxylic acids was investigated. Aldehydes (veratrum aldehyde, cinnamic aldehyde, myrtenal, cuminaldehyde, 3-phenylpropionaldehyde, perillaldehyde and 9-anthraldehyde) were incubated with hepatic microsomes of mice in the presence of an NADPH-generating system under 18O2 (97 atom%). The incorporation of oxygen-18 into carboxylic acids formed was determined by gas chromatography-mass spectrometry. Oxygen-18 was incorporated into the carboxylic acids formed from all aldehyde substrates examined. Hepatic microsomal formation of 3,4-dimethoxybenzoic acid and cumic acid from veratrum aldehyde and cuminaldehyde, respectively, was inhibited by CO and SKF 525-A. These results indicate that the oxygenation of aldehydes which may be catalyzed by cytochrome P450 is a common reaction in the biotransformation of xenobiotic aldehydes.

The GC-MS and TLC methods for the determination and identification of Δ9-tetrahydrocannabinol-11-oic acid, a major urinary metabolite of Δ9-tetrahydrocannabinol which is one of main constituents of marihuana, were established. Δ9-Tetrahydrocannabinol-11-oic acid was detected on TLC by using Fast Blue BB salt as a color reagent. The metabolite was determined as trimethylsilyl derivative of its methyl ester by using electron impact ionization GC-MS and 5'-nor-Δ8-tetrahydrocannabinol-4'-oic acid as an internal standard. The assay provides excellent sensitivity, reproducibility and specificity with detection limit of 1 ng/ml human urine.

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.

The metabolism of strychnine was studied with hepatic microsomes of rats, mice, guinea pigs, rabbits and dogs, using high-performance liquid chromatography. Eight metabolites were found by the incubation with rabbit liver microsomes. Five of them were identified as strychnine N-oxide (St N-oxide), 2-hydroxystrychnine (2-OH St), strychnine 21,22-epoxide, 16-hydroxystrychnine (16-OH St) and 18-oxostrychnine by comparing the chromatographic and spectral data to those of authentic samples. Significant differences in metabolic profiles of strychnine were observed among the above species. The main metabolite in rats and mice was 16-OH St, while in guinea pigs and rabbits, and in dogs, it was 2-OH St, and St N-oxide, respectively. The metabolic activity in guinea pig liver microsomes was much higher than those of other species. There seems to be a fairly good inverse correlation between the metabolic activity and strychnine toxicity in guinea pigs and other animal species.

Interactions of anandamide (N-arachidonylethanolamide), an endogenous compound for cannabinoid receptors, with the receptors for 5-hydroxytryptamine (5-HT), benzodiazepine, and gamma-aminobutyric acid(A) (GABA[A]) receptors in bovine synaptic membrane were examined. Anandamide decreased the 5-HT receptor bindings at concentrations of 1-100 microM, although it did not cause any change in benzodiazepine or GABA(A) receptor bindings. A high concentration of anandamide, 100 microM, significantly decrease both [3H]5-HT and [3H]ketanserin bindings. The present study revealed that the pharmacological activity of anandamide might be partially mediated through the 5-HT receptor.

A cDNA clone encoding a cytochrome P-450 (P-450) isozyme was isolated from a mouse liver cDNA library. This P-450, designated CYP2C29, the first member of the mouse CYP2C subfamily to be reported, contained the complete coding region of 490 amino acid residues. The deduced amino acid sequence of CYP2C29 exhibited 83% identity with that of the rat CYP2C7 and had an N-terminal sequence identical to that of P-450 MUT-2, a microsomal aldehyde oxygenase. Two peptides, derived from BrCN cleavage of P-450 MUT-2, were also identical to the cDNA deduced protein of CYP2C29. These results indicate that CYP2C29 cDNA encodes P-450 MUT-2.

The hepatic microsomal metabolism of cocaine and the pharmacological effects of some metabolites were studied in experimental animals. Hepatic microsomes from mice, rats and guinea pigs catalyzed the oxidation of cocaine to m- and p-hydroxycocaines. Only trace amounts of m-hydroxycocaine were detected in the extract of the incubation mixture with rabbit hepatic microsomes. The total amount of the two hydroxycocaines was less than 12% that of norcocaine which was the most predominant microsomal metabolite in all the animals species examined. The administration of p-hydroxycocaine (20 mg/kg i.p.) to mice significantly increased locomotor activity (total distance and number of rearing movements). The effect of p-hydroxycocaine was more active or comparable with that of cocaine, indicating that this metabolite is an active metabolite of cocaine.