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Objectives. In recent years, video-assisted thoracoscopic surgery (VATS) has increasingly become the preferred technique for thoracic surgery. However, the inherent characteristics of the lungs as large, soft, slippery, and delicate creates difficulties for pulmonary surgery. In this article, we outline the development and assessment of a balloon-based organ retractor for VATS via collaboration between medical and engineering personnel. Methods. A dry lab trial and accompanying questionnaire assessment were performed by a group of thoracic surgeons. Objective pressure measurements were obtained, and animal experiment on pigs was performed. Results. In the dry lab trial, use of the developed organ retractor required significantly less time and resulted in fewer difficulties than using a Cherry Dissector. The measured pressure per mm2 of the developed retractor was clearly lower than that for the Cherry Dissector. The questionnaire completed by the surgeons following the dry lab and animal experiments showed that most of the surgeons (7 surgeons out of 9) were satisfied with the quality of the balloon-based retractor based on a score of 3.13 ± 0.28 (mean ± standard deviation) out of 4.0. During the animal experiment, the balloon-based retractor provided stable and clear viewing with minimal need for adjustment. Conclusion. This balloon-based retractor could contribute to increased safety and less-invasive VATS.

Although isolation and identification of bacteria in a clinical specimen constitute essential steps for the diagnosis of bacterial infection, positive results of the bacterial culture are not always attained, despite observing the bacteria by Gram staining . As bacteria phagocytosed by the leukocytes are considered as the causative agents of infectious diseases, this study aims to introduce a new approach for the collection of only bacteria phagocytosed by the neutrophils in an animal model using laser capture microdissection (LCM) followed by the DNA identification using polymerase chain reaction (PCR). We inoculated representative bacteria ( Escherichia coli and Staphylococcus aureus ) into the abdominal cavities of specific pathogen-free C57BL/6 J mice. After 6 h inoculation , we collected the fluid samples from the peritoneal cavities of mice and demonstrated peritonitis by the increase of neutrophils. Then, we smeared the neutrophils on the membrane slides and collected single-cell phagocytosing bacteria by LCM. The supernatant of the cell lysate was supplied for the PCR reaction to amplify the 16S rRNA gene , and we validated the DNA sequences specific for the inoculated bacteria. In addition, PCR using specific primers for E. coli and S. aureus identified each species of bacteria. Hence, this study suggests that the combination of LCM and PCR could be a novel approach to determine bacteria in infectious diseases. Nevertheless, further investigation is warranted to test various additional bacterial taxa to demonstrate the general applicability of this method to clinical samples.

We previously demonstrated that cannabidiol (CBD) was a potent mechanism-based inhibitor of human cytochrome P450 1A1 (CYP1A1). However, the moiety of CBD that contributes to the potent mechanism-based inhibition of human CYP1A1 remains unknown. Thus, the effects of compounds structurally related to CBD on CYP1A1 activity were examined with recombinant human CYP1A1 in order to characterize the structural requirements for potent inactivation by CBD. When preincubated in the presence of NADPH for 20 min, olivetol, which corresponds to the pentylresorcinol moiety of CBD, enhanced the inhibition of the 7-ethoxyresorufin O-deethylase activity of CYP1A1. In contrast, d-limonene, which corresponds to the terpene moiety of CBD, failed to inhibit CYP1A1 activity in a metabolism-dependent manner. Pentylbenzene, which lacks two free phenolic hydroxyl groups, also did not enhance CYP1A1 inhibition. On the other hand, preincubation of the CBD-2′-monomethyl ether (CBDM) and CBD-2′,6′-dimethyl ether (CBDD) enhanced the inhibition of CYP1A1 activity. Inhibition by cannabidivarin (CBDV), which possessed a propyl side chain, was strongly potentiated by its preincubation. Orcinol, which has a methyl group, augmented CYP1A1 inhibition, whereas its derivative without an alkyl side chain, resorcinol, did not exhibit any metabolism-dependent inhibition. The preincubation of CBD-hydroxyquinone did not markedly enhance CYP1A1 inhibition. We further confirmed that olivetol, CBDM, CBDD, CBDV, and orcinol, as well as CBD (kinact = 0.215 min−1), inactivated CYP1A1 activity; their kinact values were 0.154, 0.0638, 0.0643, 0.226, and 0.0353 min−1, respectively. These results suggest that the methylresorcinol structure in CBD may have structurally important roles in the inactivation of CYP1A1., Article, CHEMICO-BIOLOGICAL INTERACTIONS.215:62-68(2014)

The inhibitory effect of nordihydroguaiaretic acid (NDGA) (a nonselective lipoxygenase (LOX) inhibitor)-mediated 15-LOX inhibition has been reported to be affected by modification of its catechol ring, such as methylation of the hydroxyl group. Cannabidiol (CBD), one of the major components of marijuana, is known to inhibit LOX activity. Based on the phenomenon observed in NDGA, we investigated whether or not methylation of CBD affects its inhibitory potential against 15-LOX, because CBD contains a resorcinol ring, which is an isomer of catechol. Although CBD inhibited 15-LOX activity with an IC(50) value (50% inhibition concentration) of 2.56 microM, its monomethylated and dimethylated derivatives, CBD-2'-monomethyl ether and CBD-2',6'-dimethyl ether (CBDD), inhibited 15-LOX activity more strongly than CBD. The number of methyl groups in the resorcinol moiety of CBD (as a prototype) appears to be a key determinant for potency and selectivity in inhibition of 15-LOX. The IC(50) value of 15-LOX inhibition by CBDD is 0.28 microM, and the inhibition selectivity for 15-LOX (i.e., the 5-LOX/15-LOX ratio of IC(50) values) is more than 700. Among LOX isoforms, 15-LOX is known to be able to oxygenate cholesterol esters in the low-density lipoprotein (LDL) particle (i.e., the formation of oxidized LDL). Thus, 15-LOX is suggested to be involved in development of atherosclerosis, and CBDD may be a useful prototype for producing medicines for atherosclerosis.

The transmission of human T-lymphotropic virus Type 1 (HTLV-1) occurs mainly via breast-feeding, sexual intercourse and blood transfusions. After transmission, the HTLV-1 infection is predominantly maintained by cell-to-cell infection and clonal expansion; however, the details have not yet been clarified. To investigate how HTLV-1 infected cells act in an environment without an effective immune reaction, peripheral blood mononuclear cells (PBMCs) from asymptomatic HTLV-1 carriers were inoculated into nonobese diabetic/severe combined immunodeficient (NOD/SCID)/γcnull (NOG) mice, which have immunological dysfunctions of T- and B-lymphocytes and NK cells. Human mononuclear cells including both CD4+ and CD8+ T cells were found to have infiltrated into various organs, including the liver, kidney, spleen and lung, when the mice were sacrificed 1 month after inoculation. The copy numbers of HTLV-1 provirus detected in the tissue-infiltrating human cells were much higher than those in the original PBMCs from the carriers. The expression of HTLV-1 mRNA was demonstrated in the tissue-infiltrating cells by reverse transcriptase-polymerase chain reaction. Inverse-long polymerase chain reaction showed that the pattern of HTLV-1 proviral integration was different from that of the original carrier and that it varied among NOG mice inoculated with PBMCs from the same carrier. These results suggest the selective proliferation of particular clones of HTLV-1 infected cells in NOG mice. Alternatively, transmission and new integration of HTLV-1 from infected cells to noninfected cells might have occurred in an environment without an effective immune reaction. The NOG mouse is considered a good animal model for the patho-physiological study of HTLV-1 infection with immunodeficiency. © 2007 Wiley-Liss, Inc.

In the area of manufacturing surgical instruments, the ability to rapidly design, prototype and test surgical instruments is critical. This paper provides a simple case study of the rapid development of two bio-mechanism based surgical instruments which are ergonomic, aesthetic and were successfully designed, prototyped and conceptually tested in a very short period of time.

Metabolism of nafamostat, a clinically used serine protease inhibitor, was investigated with human blood and liver enzyme sources. All the enzyme sources examined (whole blood, erythrocytes, plasma and liver microsomes) showed nafamostat hydrolytic activity. V(max) and K(m) values for the nafamostat hydrolysis in erythrocytes were 278 nmol/min/mL blood fraction and 628 microM; those in plasma were 160 nmol/min/mL blood fraction and 8890 microM, respectively. Human liver microsomes exhibited a V(max) value of 26.9 nmol/min/mg protein and a K(m) value of 1790 microM. Hydrolytic activity of the erythrocytes and plasma was inhibited by 5, 5'-dithiobis(2-nitrobenzoic acid), an arylesterase inhibitor, in a concentration-dependent manner. In contrast, little or no suppression of these activities was seen with phenylmethylsulfonyl fluoride (PMSF), diisopropyl fluorophosphate (DFP), bis(p-nitrophenyl)phosphate (BNPP), BW284C51 and ethopropazine. The liver microsomal activity was markedly inhibited by PMSF, DFP and BNPP, indicating that carboxylesterase was involved in the nafamostat hydrolysis. Human carboxylesterase 2 expressed in COS-1 cells was capable of hydrolyzing nafamostat at 10 and 100 microM, whereas recombinant carboxylesterase 1 showed significant activity only at a higher substrate concentration (100 microM). The nafamostat hydrolysis in 18 human liver microsomes correlated with aspirin hydrolytic activity specific for carboxylesterase 2 (r=0.815, p

The properties of ES46.5K, an esterase from mouse hepatic microsomes, were compared with those of carboxylesterases from rabbit and porcine liver. The inhibitory profile with a serine hydrolase inhibitor (bis-p-nitrophenylphosphate) and detergents (sodium dodecylsulfate, Emulgen 911) was different between ES46.5K and the carboxylesterases. Bis-p-nitrophenylphosphate (0.1 mM) markedly inhibited the catalytic activity of the carboxylesterases but not that of ES46.5K. Emulgen 911 (0.05-0.25%) inhibited the catalytic activity of the carboxylesterases, whereas the detergent conversely stimulated that of ES46.5K by 150%. The two carboxylesterases catalyzed the hydrolysis of acetate esters of tetrahydrocannabinol (THC) analogues with different side chain lengths (C1-C5), although ES46.5K showed marginal activity only against the acetate of Delta8-tetrahydrocannabiorcol, a methyl side chain derivative of Delta8-THC. ES46.5K hydrolyzed cannabinoid esters stereospecifically and regioselectively. The esterase hydrolyzed 8alpha-acetoxy-Delta9-tetrahydrocannabinol (8alpha-acetoxy-Delta9-THC, 5.62 nmol/min/mg protein), while the enzyme did not hydrolyze 8beta-acetoxy-Delta9-THC, 7alpha-acetoxy-, and 7beta-acetoxy-Delta8-THC at all. In contrast, the carboxylesterases from rabbit and porcine liver hydrolyzed 8beta-acetoxy-Delta9-THC efficiently but not 8alpha-acetoxy-Delta9-THC. ES46.5K hydrolyzed side chain acetoxy derivatives of Delta8-THC at the 3'- and 4'-positions, and a methyl ester of 5'-nor-Delta8-THC-4'-oic acid. The enzyme, however, could not hydrolyze methyl esters of Delta8- and Delta9-THC-11-oic acid, while both carboxylesterases hydrolyzed side chain acetoxy derivatives of Delta8-THC and three methyl esters of THC-oic acids. These differences in stereospecificity and regioselectivity between ES46.5K and carboxylesterases suggest that the configurations of important amino acids for the catalytic activities of these enzymes are different from each other.

Thirty-six allyl substituted oxopyrimidine analogues such as barbituric acid (BA), barbiturates, uracil, thymine, and related derivatives including 13 new compounds were synthesized and their pharmacologic effects ([hypnotic activity, anticonvulsant activity against pentylentetrazol (PTZ)-induced seizures, and LD(50)]) and interactions with the barbiturates were evaluated in mice and rats. The results are briefly and parially summarized as follows. BA prolonged pentobarbital (PB)-induced sleep and had some central depressant effects. N,5,5-triallyl-BA exhibited some hypnotic and anticonvulsant activities, although the other 5,N-allyl-compounds did not show any activity except for allobarbital (AlloB). N-allyl-BA, 5-allyl-BA, N(1),N(3),5-triallyl-BA, N,5,5-triallyl-BA, and N(1),N(3),5,5-tetraallyl-BA also prolonged PB-induced sleep. Interestingly, N,5,5-triallyl-BA was the most potent in the interaction with AlloB, phenobarbital (PheB), amobarbital (AB), PB, and thiopental (TP) but not barbital (B). N(1),N(3),5,5-tetraallyl-BA prolonged AlloB-, PB-, and AB-induced sleep but not B-, PheB-, and TP-induced sleep. N(1),N(3),5-triallyl-B prolonged only PB- and TP-induced sleep. 5,5-diallyl-BA prolonged PheB- and TP-induced sleep. N,5-diallyl-BA prolonged only TP-induced sleep. In contrast, BA and N(1),N(3),5-triallyl-AB tended to antagonize AlloB, AB, and B. N(1),N(3),5,5-tetraallyl-BA also slightly antagonized B, PheB, and TP. 5,5-diallyl-BA antagonized only AB. The prolonging effects of BA, N,5,5-triallyl-BA, and N(1),N(3),5,5-tetraallyl-BA on PB-induced sleep were dose dependent. These results indicate that the position and number of allyl groups substituted on the structure of BA play an important role in their depressant activities. This review deals with the structure-activity relationship of allyl-substituted oxopyrimidines as part of our search for antagonists and agonists of barbiturates as well as their mechanisms of action.

The progesterone 17alpha-hydroxylase activity, which is one of the steroidogenic enzymes in rat testis microsomes, was significantly inhibited by crude marijuana extracts from Delta(9)-tetrahydrocannabinolic acid (THCA)- and cannabidiolic acid (CBDA)-strains. Delta(9)-Tetrahydrocannabinol, cannabidiol and cannabinol also inhibited the enzymatic activity with relatively higher concentration (100-1000 microM). Testosterone 6beta- and 16alpha-hydroxylase activities together with androstenedione formation from testosterone in rat liver microsomes were also significantly inhibited by the crude marijuana extracts and the cannabinoids. Crude marijuana extracts (1 and 10 microg/ml) of THCA strain stimulated the proliferation of MCF-7 cells, although the purified cannabinoids (THC, CBD and CBN) did not show significant effects, such as the extract at the concentration of 0.01-1000 nM. These results indicate that there are some metabolic interactions between cannabinoid and steroid metabolism and that the constituents showing estrogen-like activity exist in marijuana.

The NADH-dependent activity by hepatic microsomes of Japanese monkeys for 7-oxo-Delta(8)-tetrahydrocannabinol (7-oxo-Delta(8)-THC) formation from 7beta-hydroxy-Delta(8)-THC exhibited about 70% of the NADPH-dependent activity (100%) at the substrate concentration of 72.7 microM, although NADPH was an obligatory cofactor for maximal activity. Both NADH- and NADPH-dependent activities were significantly inhibited by the typical P450 inhibitors, such as SKF525-A and metyrapone. Both activities were almost completely inhibited by the NADPH-P450 reductase inhibitor diphenyliodonium chloride. The ratio of NADH- and NADPH-dependent activities varied significantly according to the substrate concentration. Interestingly, the NADH-dependent activity was higher than that of NADPH at low substrate concentrations of 13-50 microM. The ratio was also affected by the cofactor concentration. In the reconstituted system of CYP3A8 purified from hepatic microsomes of Japanese monkeys as a major enzyme responsible for the NADPH-dependent oxidation, NADH as well as NADPH could sustain the oxidation of 7beta-hydroxy-Delta(8)-THC to the corresponding ketone. The NADH-dependent oxidation of 7beta-hydroxy-Delta(8)-THC by monkey livers is mainly catalyzed by CYP3A8 as well as the NADPH-dependent oxidation. These results indicate that NADH as a cofactor may be also useful for the oxidation of 7beta-hydroxy-Delta(8)-THC, and that the cofactor requirement for the reaction is varied by the concentrations of substrate and/or cofactor.

Microsomal alcohol oxygenase catalyzes the stereoselective oxidation of 7alpha- and 7beta-hydroxy-delta8-tetrahydrocannabinol (7alpha- and 7beta-hydroxy-delta8-THC) to 7-oxo-delta8-THC in monkey liver, and the activity for 7beta-hydroxy-delta8-THC is relatively higher than that for 7alpha-hydroxy-delta8-THC. We previously reported that purified P450JM-E, assumed to be CYP3A8, is a major enzyme responsible for the oxidation of 7-hydroxy-delta8-THC to 7-oxo-delta8-THC in monkey liver and is capable of catalyzing the oxidative reaction by NADH as well as NADPH. In the present study, we demonstrated that some steroids such as testosterone and progesterone stimulated both the NADH- and NADPH-dependent conversions of 7beta-hydroxy-delta8-THC to 7-oxo-delta8-THC in monkey liver microsomes. Kinetic analyses revealed that both the NADH- and NADPH-dependent 7-oxo-delta8-THC formation showed sigmoid kinetics. Testosterone caused a decrease in S50 and an increase in V(max) for the NADH-dependent activity, and resulted in a decrease in S50 without changing the V(max) for the NADPH-dependent activity. On the other hand, NADH-dependent testosterone 6beta-hydroxylation activity showed Michaelis-Menten kinetics and was also inhibited by 7beta-hydroxy-delta8-THC, resulting in a decrease in V(max) with no effect on the K(m). NADPH-dependent testosterone 6beta-hydrozylation activity was also inhibited by 7beta-hydroxy-delta8-THC, resulting in a decrease in both S50 and V(max). In order to explain the metabolic interaction between 7beta-hydroxy-delta8-THC and testosterone, we propose a kinetic model involving at least three binding sites, for the mechanism of activation by testosterone.

OBJECTIVE: Thyroid peroxidase (TPO) deficiency is one of the causes of thyroid dyshormonogenesis, because TPO plays a key role in thyroid hormone biosynthesis. To determine the frequency and pattern of TPO abnormalities, we have been screening TPO genes of patients with congenital goitrous hypothyroidism. SUBJECTS AND METHODS: TPO genes of a patient with congenital goitrous hypothyroidism and her parents were directly sequenced, and two novel missense mutations (R665W and G771R) were found. The former was derived from her father and the latter from her mother. R665 and G771 were well conserved in the peroxidase superfamily. When mRNAs containing each of the mutations were transfected into CHO-K1 cells, each cell showed faint TPO enzyme activity. However, immunofluorescence and immunoelectron microscopic analyses revealed that neither of the mutated TPOs reached the plasma membrane. CONCLUSIONS: Two novel missense mutations in the TPO gene were found. TPO proteins encoded by these mutated alleles showed abnormal cellular localization; namely, localization on the plasma membrane was disturbed. The loss of plasma membrane localization in mutated TPOs brought about the iodide organification defect, which was diagnosed as congenital hypothyroidism.

The formation of 7-oxo-delta8-tetrahydrocannabinol (7-Oxo-delta8-THC) from 7alpha- or 7beta-hydroxy-delta8-THC (7alpha- or 7beta-OH-delta8-THC) was found in hepatic microsomes of monkeys. The activity in 7beta-OH-delta8-THC was stereoselectively 2.5- to 4.6-fold higher than that from 7alpha-OH-delta8-THC. The oxidative activities of 7alpha- and 7beta-OH-delta8-THC to 7-Oxo-delta8-THC were inhibited to 35% and 10%, respectively, of the control value by the antibody against P450GPF-B (CYP3A), a major enzyme responsible for the formation of 7-Oxo-delta8-THC in guinea pigs. In the Lineweaver-Burk double-reciprocal plot analysis, testosterone 6beta-hydroxylase activity was competitively inhibited by 7beta-OH-delta8-THC. Two cytochrome P450 enzymes, called P450JM-D and P450JM-E, were purified from hepatic microsomes of Japanese monkeys. P450JM-E, assumed to be CYP3A8, immunologically reacted with the antibody against P450GPF-B and showed high forming activity of 7-Oxo-delta8-THC from 7-OH-delta8-THC. On the other hand, 7-Oxo-delta8-THC forming activity of P450JM-D, assumed to be CYP2C, was less than 10% of that of P450JM-E (CYP3A8). Oxygen-18 (18O) derived from atmospheric oxygen was incorporated into about 40% of the corresponding ketone formed from 7alpha-OH-delta8-THC or 8beta-OH-delta9-THC by P450JM-E (CYP3A8), although the incorporation of the stable isotope into the oxidized metabolite from 7beta-OH-delta8-THC or 8alpha-OH-delta9-THC was negligible. These results indicate that P450JM-E (CYP3A8) is a major enzyme of the oxidation of 7-OH-delta8-THC in monkey hepatic microsomes. The oxidation mechanism may proceed as follows: the alpha- and beta-epimers of 7-OH-delta8-THC or 8-OH-delta9-THC may be converted to ketone through dehydration of an enzyme-bound gem-diol by P450JM-E (CYP3A8), although this stereoselective dehydration differentiates between two epimers.

This review deals with the concept of sleep mechanism based on our uridine receptor theory. It is well established that uridine is one of the sleep-promoting substances, we have, therefore, synthesized new types of hypnotic compounds from oxopyrimidine nucleosides. Their mechanism of action in CNS depressant effects is elucidated based on the receptor theory. In this study, structure-activity relationship for CNS depressant properties, sleep-promoting effects, interaction with certain CNS receptors, and receptor binding assay of uridine derivatives as oxopyrimidine nucleoside were investigated. In the studies of structure-activity relationship of N3-substituted uridine, we found for the first time that both N3-benzyluridine and N3-phenacyluridine synthesized exhibited potent hypnotic activity (loss of righting reflex) by intracerebroventicular injection in mice. Moreover, certain derivatives of these compounds possessed synergistic effects with barbiturate and benzodiazepine, and decreased in spontaneous activity, motor incoordination, and antianxiety effects in mice. Especially, N3-phenacyluridine markedly enhanced pentobarbital- and diazepam-induced sleep by 6- and 70-fold, respectively. However, N3-benzyluracil and N3-phenacyluracil that have no ribose moiety did not possess any hypnotic activity, indicating specific effects of nucleoside derivatives. Effects of N3-benzyluridine on natural sleep in rats were thus examined. N3-Benzyluridine also possessed the sleep promoting effect assessed by electrocorticogram at the dose of 10 pmol. For elucidating the mechanism of action of N3-phenacyluridine, the interactions of this compound with benzodiazepine, GABA, 5-HT, or adenosine receptors were also investigated. Although the pharmacological activity of N3-phenacyluridine was high, the affinities to benzodiazepine, GABA, 5-HT, and adenosine receptors were quite low. [3H]N3-Phenacyluridine concentration-dependently bound to synaptic membrane prepared from the bovine brain. The Scatchard analysis revealed a single component of the binding site. This binding site is proposed here as a novel receptor called "uridine receptor" for hypnotic activity of the uridine derivatives. The rank order of the distribution of these specific binding sites was found to be striatum > thalamus > cerebral cortex > cerebellum > mid brain > medulla oblongata in the rat brain. In the metabolic study of N3-phenacyluridine, we found that this compound was exclusively metabolized to N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine, but not the (R)- form, in mice. N3-(S)-(+)-alpha-Hydroxy-beta-phenylethyluridine possessed not only strong hypnotic activity but also a high affinity to the uridine receptor of synaptic membranes, while the (R)-isomer was low in both activities. Racemic mixture was shown to be intermediate for pharmacological effects of the compounds. These studies which used (R)- or (S)-isomer indicate that uridine binding site or uridine receptor, exists in the CNS and plays some role in sleep regulation in mammals as one of the triggering steps in inducing hypnotic activity. It is suggested that uridine is released from steps of nucleic acid-nucleic protein biosynthesis (catabolism), and reaches the binding sites in the areas of the brain which regulate natural sleep. The uridine dissociated from the receptor is then utilized for the synthesis of nucleic acid (anabolism). We propose here that the induction of sleep may be mediated by uridine through uridine receptor in the CNS, although the structure of uridine receptor is not yet elucidated.

Since few previous studies have examined the effects of urocortin on physiological fed and fasted gastrointestinal motility, we administered urocortin intracerebroventricularly (icv) or intravenously (iv) in freely moving conscious rats and examined the changes in antral and duodenal motility. Icv and iv injection of urocortin disrupted fasted motor patterns of gastroduodenal motility, which were replaced by fed-like motor patterns. When urocortin was given icv and iv in the fed state, the motor activity remained like the fed patterns but % motor index (%MI) was decreased in the antrum and increased in the duodenum. Increase in the %MI in the duodenum induced by urocortin was shown as a nonpropagated event, since the transit of nonnutrient contents in the duodenum was decreased by icv and iv injection of urocortin. Changes in the gastroduodenal motility induced by icv injection of urocortin were abolished in animals with truncal vagotomy but not altered in animals with mechanical sympathectomy, suggesting that the vagal pathway may mediate the central action of urocortin. Neither urocortin antiserum nor α-helical CRF-(9–41) affected fed and fasted gastroduodenal motility, suggesting that endogenous urocortin is not involved in regulation of basal gastroduodenal motility.

The formation of 7-oxo-delta8-tetrahydrocannabinol (7-oxo-delta8-THC) from 7beta-hydroxy-delta8-THC was found in hepatic microsomes of rats. The activity was stereoselective and about 3-fold higher than that from 7alpha-hydroxy-delta8-THC. The oxidative activity of 7alpha- and 7beta-hydroxy-delta8-THC to 7-oxo-delta8-THC was significantly higher in male than in female, and significantly enhanced by both dexamethasone and phenobarbital, and then inhibited up to about 20% of the control value by antibody against P450GPF-B, presumably a member of the 3A subfamily, a major enzyme responsible for the formation of 7-oxo-delta8-THC in guinea pigs. This antibody also inhibited the formation of 7alpha- and 7beta-hydroxy-delta8-THC, and 7-oxo-delta8-THC from delta8-THC by hepatic microsomes of rats. These results indicate that there is a sex-related difference in the oxidation of 7-hydroxy-delta8-THC to 7-oxo-delta8-THC and the reaction is mainly catalyzed by P450 enzyme(s) belonging to the 3A subfamily as major enzyme(s) of microsomal alcohol oxygenase in rats.

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.

Characterization of gastric Na+/I- symporter (NIS) of the rat was carried out. Sequencing of the open reading frame of gastric NIS mRNA showed only three nucleotide changes when compared with FRTL-5 NIS cDNA, and two of these changes led to amino acid changes. The results of Northern blot analysis showed that abundant NIS mRNA was expressed in the stomach when compared with other organs. Western blot analysis using gastric mucosa and FRTL-5 lysates detected the difference in molecular weight between FRTL-5 and gastric mucosa lysates, suggesting abnormal posttranslational modification of gastric NIS protein. Immunohistochemically, gastric NIS protein was located in the cornification layer of the stratified squamous epithelium of the pars proventricularis and in parietal cells and on the apical border of surface epithelial cells of the pars glandularis. Gastric NIS protein was present in tubulovesicular structures and lysosomes in parietal cells by immunoelectron microscopy. Gastric NIS protein exists to trap I- from the gastric lumen, except in parietal cells. Results indicated that a very large amount of gastric NIS mRNA is expressed to be translated, whereas only a small amount of immature gastric NIS protein is detected. This may indicate that immature gastric NIS protein rapidly degrades to peptides.

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.

Twenty four thiouracil derivatives, including N3-allyl- (19) and N1-allyl-2-thiouracil (20) were synthesized and their pharmacological effects [sedative-hypnotic activity (loss of righting reflex and spontaneous activity), convulsant activity, effect on pentobarbital (PB)-induced sleep and mortality] were evaluated in mice at doses of 320 mg/kg, i.p. and 2 μmol/mouse by intracerebroventricular (i.c.v.) injections, respectively. N3-Allyl-6-propyl-2-thiouracil (3), N3-allyl-5, 6-dimethyl-2-thiouracil (10), N3-allyl-1, 2, 3, 4, 5, 6, 7, 8, 9-nonahydro-4-oxo-2-thiocyclohepta[d]pyrimidine (16) and N3-allyl-5-methyl-2-thiouracil (18) exhibited sedative-hypnotic activity, whereas N3-allyl-6-ethyl-5-methyl-2-thiouracil (11), N1-allyl-5-methyl-2-thiouracil (21), N1-allyl-1, 2, 3, 4, 5, 6, 7, 8, 9-nonahydro-4-oxo-2-thiocyclohepta[d]pyrimidine (23) and N1-allyl-5, 6-dimethyl-2-thiouracil (24) conversely displayed clonic- and/or tonic-convulsant seizures. N3-Allyl-6-propyl-2-thiouracil (3) and N3-allyl-5-methyl-2-thiouracil (18) decreased spontaneous activity. Other compounds examined were inactive, or only slightly active in the sedative-hypnotic assay even at high doses. Fifteen compounds (1-4, 7, 10, 11, 14-16, 18-21, and 23) significantly prolonged the PB-induced sleeping time. Interestingly, only N1-allyl-5, 6-dimethyl-2-thiouracil (24) shortened the PB-induced sleeping time. These results showed that these thiouracils possessed many different effects such as sedative-hypnotic, anticonvulsant and/or convulsant, and that N3-allyl-5-methly-2-thiouracil (18) and N1-allyl-5, 6-dimethyl-2-thiouracil (24) had the most potent hypnotic activity and antagonistic effect against PB, respectively.

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.

Tetrahydrocannabinol (THC), a psychoactive component of marijuana, is known to exert cytotoxicity in immune cells. In the present study, we examined the cytotoxicity of Δ⁸-THC in mouse macrophage J774-1 cells and a possible involvement of cannabinoid receptors and stress-responsive mitogen-activated protein kinases (MAPKs) in the cytotoxic process. J774-1 cells were treated with Δ⁸-THC (0-20 μM) for up to 6 h. As measured by the MTT and LDH assays, Δ⁸-THC induced cell death of J774-1 cells in a concentration- and/or exposure time-dependent manner. Δ⁸-THC-induced cell damage was associated with vacuole formation, cell swelling, chromatin condensation, and nuclear fragmentation. The cytotoxic effect of Δ⁸-THC was significantly prevented by a caspase-1 inhibitor Ac-YVAD-cmk but not a caspase-3 inhibitor z-DEVD-fmk. The pretreatment with SR144528, a CB₂ receptor-selective antagonist, effectively suppressed Δ⁸-THC-induced cytotoxicity in J774-1 cells, which exclusively expressed CB₂ receptors as indicated by real-time polymerase chain reaction analysis. In contrast, AM251, a CB₁ receptor-selective antagonist, did not affect the cytotoxicity. Pertussis toxin and α-tocopherol significantly attenuated Δ⁸-THC-induced cytotoxicity suggesting that G(i/o) protein coupling signal transduction and oxidative stress are responsible for the cytotoxicity. Δ⁸-THC stimulated the phosphorylation of p38 MAPK and c-Jun N-terminal kinase (JNK) in J774-1 cells, which were effectively antagonized by the pretreatment with SR144528. In addition, SB203580, a p38 MARK inhibitor, significantly attenuated the cytotoxic effect of Δ⁸-THC, whereas SP600125, a JNK inhibitor, significantly enhanced the cytotoxicity. These results suggest that the cytotoxicity of Δ⁸-THC to J774-1 cells is exerted mediated through the CB₂ receptor followed by the activation of p38 MAPK.

The stereoselectivity in 4-hydroxylation of bunitrolol (BTL), a beta-adrenoreceptor blocking agent, was examined in liver microsomes from monkeys (marmosets and Japanese monkeys) and compared with the results of human liver microsomes. The formation of (+)-4-OH-BTL from (+)-BTL from (+)-BTL was significantly higher than that of (-)-4-OH-BTL from (-)-BTL in the liver microsomal fractions from the two kinds of monkeys. The 4-OH-BTL-forming activity from racemic BTL was significantly lower than from enantiomeric BTL, indicating a possible metabolic interaction between BTL enantiomers. The in vitro profiles observed in the monkeys were very similar to those in humans, but the stereoselectivity in BTL metabolism [(+)-BTL > (-)-BTL] in the primates was found to be reverse to that in rats [S. Narimatsu et al., Anal. Biochem., 222, 256-261 (1994)]. The 4-OH-BTL-forming activity from BTL enantiomers was significantly suppressed by quinidine and quinine, while the former was more potent than the latter, and also by alpha-naphthoflavone. Furthermore, the activity was also suppressed by antisera against rat cytochromes P450-2D2 and -1A2 in concentration-dependent manners. However, kinetics showed that enantiomeric BTL 4-hydroxylation was monophasic in liver microsomes from marmosets of both genders and from male Japanese monkeys. These results suggest that cytochrome P450-2D and -1A enzymes with similar Km values are involved in BTL 4-hydroxylation in monkey liver microsomes.

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.

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.

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.

The effect of the viscosity of sodium hyaluronate solution on the rectal absorption of morphine was determined in rabbits. Hollow-type suppositories containing 10 mg morphine in viscous sodium hyaluronate solution of various mean molecular weights (MWs) were prepared. Rectal absorption of morphine in the sodium hyaluronate solution (MW 2.1 x 10(6) daltons) was dependent on sodium hyaluronate concentrations in the range of 0.1 to 3% (w/v). Bioavailability after rectal administration of morphine in 0.1% sodium hyaluronate solution is consistent with that of morphine solution in the absence of sodium hyaluronate and the sustained-release plasma profile was observed for morphine in 3% sodium hyaluronate solution. Administration of hollow-type suppositories containing 10 mg morphine in 1% sodium hyaluronate solution resulted in the highest bioavailability of approximately 2-fold that after administration of morphine in physiological saline solution. Five kinds of sodium hyaluronate solution with MWs of 2.4 x 10(5), 1.0 x 10(6), 1.8 x 10(6) and 2.1 x 10(6) daltons and respective viscosities of 70, 3.3 x 10(3), 3.2 x 10(6), 5.1 x 10(3) and 5.7 x 10(3) cP (20 degrees C) were examined. Optimal viscosity of the sodium hyaluronate solution was found to enhance rectal morphine absorption. These results indicate that the selection of relevant viscosity of the sodium hyaluronate solution may contribute to the improvement of bioavailability of morphine on rectal administration.

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.

Reaction of 9-[3,5-di-O-(tetrahydropyran-2-yl)-beta-D-arabinofuranosyl]uracil (2) with diethylaminosulfur trifluoride in the presence of pyridine afforded 2'-deoxy-2'-fluororiboside 3a, from which 2'-deoxy-2'-fluorocytidine (4b) has been synthesized in good yield. Compound 3a was deprotected and subsequently treated with various benzyl halides or 2-chloro-4-fluoroacetophenone to give corresponding N3-substituted 2'-deoxy-2'-fluorouridines 5a-c and 6. Compounds 5a-c, as well as 6, showed weak hypnotic activity in mice. Compound 4b showed moderate antiviral activity against human immunodeficiency virus-1 but 3b, 5a-c, and 6 were virtually inactive.

The bioavailability of morphine after rectal administration using three types of suppositories containing morphine hydrochloride (10 mg) in different added forms was evaluated in rabbits. Three types of suppositories were constructed with a base material (Witepsol H-15): a conventional suppository containing morphine hydrochloride mixed with a base material, a hollow-type suppository containing morphine in an aqueous solution in its cavity, and a hollow-type suppository containing morphine as a powder (hydrochloride salt) in its cavity. The plasma concentrations of morphine and its metabolites, morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G), were determined. The mean AUC0-6 of morphine after rectal administration of the hollow-type suppository containing powdered morphine was significantly higher than that after the administration of a conventional suppository, whereas the mean AUC0-6 of M-3-G was lower than that after administration of a conventional suppository. The mean AUC0-6 of M-6-G by a hollow-type suppository containing powdered morphine was higher than that by a conventional suppository. Although the mean AUC0-6 ratio of M-3-G to morphine after administration of a conventional suppository was three times larger than that of a hollow-type suppository containing powdered morphine, the mean AUC0-6 ratios of M-6-G to morphine after use of the three types of suppositories were all approximately 1.0. This study demonstrates that the hollow-type suppository containing powdered morphine is a more effective rectal dosage vehicle than the conventional suppository.

Methyl (2), ethyl (3), propyl (4), butyl (5), allyl (6), benzyl (7), o-, m-, p-xylyl (8-10), and alpha-phenylethyl (11) derivatives of arabinofuranosyluracil (1) were synthesized and their pharmacological effects in mice were examined by using hypnotic activity and synergism with pentobarbital as indices for the CNS depressant effects. At a dose of 2.0 micromol/mouse by intracerebroventricular injection, the values of mean sleeping time induced by 7-11 were 144, 154, 117, 33, and 34 min, respectively, whereas the alkyl (2-6) derivatives did not cause any hypnotic activity. N3-o-Xylylarabinofuranosyluracil (8) displayed the most potent hypnotic activity among the derivatives tested. Certain derivatives (6-11) significantly prolonged the pentobarbital-induced sleeping time compared to control. The present study indicated that substitution with benzyl and/or related groups on the N3-position of arabinofuranosyluracil produced CNS depressant effects.

Uridine analogue binding sites, the so-called uridine receptor, were observed in the experiments on specific [3H]N3-phenacyluridine binding to bovine synaptic membranes using two isomers, N3-(S)-(+)- and N3-(R)-(-)-alpha-hydroxy-beta-phenethyluridine, as ligands. The potent hypnotic, N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine, but not the (R)-isomer, strongly inhibited [3H]N3-phenacyluridine binding. The racemate had inhibitory activity intermediate between that of the two alpha-hydroxy-beta-phenethyluridines ((R)- or (S)-isomers). The inhibitory constants of these compounds were determined. The Ki values of N3-phenacyluridine, alpha-hydroxy-beta-phenethyluridine (racemate), N3-(R)-(-)-, and N3-(S)-(+)-alpha-hydroxy-beta-phenethyluridine were 0.65, 397.4, 1908, and 10.2 nM, respectively. The present results indicate the existence of uridine receptors in the central nervous system in relation to their hypnotic activities reported previously.

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.

Fifteen kinds of uracil derivatives, which were substituted with functional groups such as benzyl (Bn), methoxymethyl (MOM), n-propyl (Pr) or allyl (A) group at the N1 and/or N3 position of uracil, were synthesized. Sedative-hypnotic activity, pentobarbital (PB)-induced sleep prolongation and acute toxicity as indices of their pharmacological activities were evaluated using mice. Spontaneous activities of mice treated with N1-benzyluracil (N1-BnU, 7), N3-benzyluracil (N3-BnU, 8), N1,N3-dibenzyluracil (DBnU, 9), N1-benzyl-N3-allyluracil (BnAU, 14) and N1-allyl-N3-benzyluracil (ABnU, 15) were measured. Ten kinds of uracil derivatives showed hypnotic activity. ED50's values of ABnU (15) and N1-propyl-N3-benzyluracil (PrBnU, 13) were 155 and 172 mg/kg, i.p., respectively. Those effects were more potent than that of barbital (179 mg/kg, i.p.) Ten kinds of uracil derivatives tested significantly prolonged the PB-induced sleeping time. N3-BnU (8) increased the spontaneous activity of mice at a dose of 80 mg/kg, i.p., while ABnU (15) depressed the spontaneous activity at a dose of 160 mg/kg, i.p. The other compounds did not show any significant effect on the spontaneous activity of mice. LD50's values of ABnU (15), PrBnU (13) and N1-methoxy-methyl-N3-benzyluracil (MOMBnU, 11) were 199, 229 and 363 mg/kg, i.p., respectively. LD50's values of the other derivatives were more than 480 mg/kg, i.p. These results indicate that the benzyl group at the N3 position of uracil is important for exhibiting sedative-hypnotic activity of uracil derivatives, and that ABnU (15) has the most potent central depressant activity among the uracil derivatives tested.

Successful engraftment of human T-lymphotropic virus type 1 (HTLV-1)-infected cells and a marked increase of proviral DNA loads (PVLs) in non-obese diabetic/severe combined immunodeficient (NOD/SCID)/gammac(null) (NOG) mice have been reported. Whether the increased PVL in transplanted mice is due to the new infection of HTLV-1 was examined.Mononuclear cells from 3 NOG mice with primary engraftment from asymptomatic HTLV-1 carriers were transplanted into a second group of NOG mice. HTLV-1 PVL, proviral integration by fluorescence in situ hybridization assay, expression of viral antigen, and T-cell clonality were analyzed.The PVLs in the secondarily transplanted NOG mice were significantly higher than those of primarily transplanted NOG mice. Multiple signals of HTLV-1 proviruses in the nucleus of the infected cells were revealed by fluorescence in situ hybridization analysis. Expression of HTLV-1 tax/rex mRNA and antigen was observed. The variety of T-cell clones was limited in the transplanted NOG mice.Multiple proviral integrations were considered to be due to the new infection from HTLV-1-infected cells to the other cells. Only a certain fraction of T cells seemed to have selectively survived in NOG mice after engraftment.

Effects of pentobarbital pellet implantation on [3H]baclofen binding in the frontal cortex of cerebellum of rat brains were examined. In the frontal cortex, pentobarbital tolerance caused an increase in the number of binding sites (Bmax) without changing their affinity (KD). Twenty-four hours after withdrawal of the pentobarbital pellets, there was a significant increase in the KD and Bmax values. Cerebellar binding, in contrast, was not significantly changed in any of the treatment groups. Addition of 1 mM of pentobarbital directly to binding assays using cortical membrane produced as increase in KD without a change in Bmax. In vitro, pentobarbital affected neither the KD nor the Bmax in the cerebellar [3H]baclofen binding. These results suggest that like the GABAA receptor, [3H]baclofen binding to the GABAB receptor in rat frontal cortex was affected by pentobarbital tolerance and dependence, and that there are regional differences in the properties of the GABAB receptor.

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.

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.

N3-Substituted derivatives of 2',3'-O-isopropylideneuridine (1) were synthesized and their pharmacological effects on the central nervous system (CNS) examined using mice. Methyl (2), ethyl (3), propyl (4), butyl (5), allyl (6), benzyl (7), o-, m-, p-xylyls (8, 9, 10), and alpha-phenylethyl (11) derivatives of 1 were administered to mice by intracerebroventricular (i.c.v.) injection for evaluating hypnotic activity, pentobarbital-induced sleep prolongation, and spontaneous activity as indices. Only 3 possessed hypnotic activity by i.c.v. injection at the dose of 2.0 mumol/mouse. Compounds 3, 4, and 10 significantly showed synergism with a barbiturate, indicating that the derivatives have some CNS depressant effects. Moreover, 3 and 4 caused decrease in the spontaneous activity of mice, even at low doses. The present study indicated that substitution by ethyl, propyl, and p-xylyl groups at the N3-position of 2',3'-O-isopropylideneuridine imparted the CNS depressant effects.

We investigated whether cannabidiol (CBD) and cannabidiol hydroxy-quinone (CBDHQ) generate reactive oxygen species (ROS) during metabolism with mouse hepatic microsomes. CBD and CBDHQ (91.5 microM) significantly suppressed lipid peroxidation in the mouse hepatic microsomes. CBDHQ also significantly decreased NADH-cytochrome b5 reductase (fp1) activity by 25% of the control activity in the hepatic microsomes, and tended to increase NADPH-cytochrome c (P450) reductase (fp2) activity. CBDHQ also significantly inhibited superoxide dismutase and catalase activities in mouse hepatic 105,000 xg supernatant. Moreover, CBDHQ significantly increased glutathione reductase activity and significantly inhibited NAD(P)H-quinone reductase activity. CBD exhibited similar effects on these enzymes, except that cannabinoid significantly inhibited glutathione reductase activity in mouse hepatic 105,000 xg supernatant. These results suggest that CBDHQ is easily converted to the semiquinone form rather than the hydroquinone form. It was also suggested that CBDHQ and CBD were capable of generating ROS as superoxide anion radicals during their metabolism with mouse hepatic microsomes or with purified fp2 by electron spin resonance spin trapping methods with 5,5-dimethyl-1-pyrroline-N-oxide. The present results suggest that CBDHQ formed during hepatic microsomal metabolism of CBD is capable of generating ROS and inducing cell toxicity.

N3-(2',5 '-Dimethoxyphenacyl)arabinofuranosyluracil (N3-(2',5 '-DiMeOPhAc)AraU) is a pyrimidine nucleoside derivative which possesses antinociceptive effect by intracerebroventricular (i.c.v.) administration to mice. The compound (100 microM) significantly decreased the specific binding of [3H]D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin (DAMGO) and [3H]D-Pen2, D-Pen5-enkephalin (DPDPE) at the mu- and delta-opioid receptor, respectively, but has no effect on the binding of [3H]U-69,593 at the kappa-opioid receptor of rat brain slices. The autoradiographic studies also demonstrated that [3H]DAMGO and [3H]DPDPE bindings on the rat brain slice were displaced by N3-(2',5'-DiMeOPhAc)AraU. These results indicate that N3-(2',5'-DiMeOPhAc)AraU interacts with mu- and delta-opioid receptors.

Pyrolysates of cyanocobalamin, thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, and ascorbic acid were tested for mutagenicity in the histidine-requiring mutants Salmonella typhimurium TA98 and TA100. Each vitamin was sealed in a glass tube and heated at 100–600°C in a muffle furnace. Methanol-chloroform extracts of the pyrolysate of each vitamin tested did not show any mutagenicity in either TA98 or TA100 without rat liver 9000 × mg supernatant fraction (S9) added. In the presence of S9, the B-group vitamins (cyanocobalamin, thiamine hydrochloride, riboflavin, and pyridoxine hydrochloride) were all mutagenic in TA98 and TA100, with the highest activity among the vitamins tested found in the pyrolysate of cyanocobalamin. The pyrolysate of 0.25 μmole cyanocobalamin produced 3200 revertants, while the pyrolysates of 0.25 μmole thiamine hydrochloride and riboflavin produced only 910 revertants, and the pyrolysate of pyridoxine hydrochloride did not show any mutagenicity at that amount. The mutagenicity was generally more active to TA98 than to TA100, indicating that frameshift-type mutagens were contained in the pyrolysates. The pyrolysate of ascorbic acid did not show any mutagenic activity in either TA98 or TA100 under the present experimental conditions.

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.

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.

Seventy eight N(3)-substituted derivatives of uridine (1), thymidine (2), 2'-deoxyuridine (3), 6-azauridine (4), 2',3'-O-isopropylideneuridine (5), and arabinofuranosyluracil (6) were synthesized and their antinociceptive effects were evaluated. N(3)-(2',4'-Dimethoxyphenacyl)uridine (1l), N(3)-(2',4'-dimethoxyphenacyl)2'-deoxyuridine (3l), and N(3)-(2',5'-dimethoxyphenacyl)arabinofuranosyluracil (6m) possessed 93, 86, and 82% of the antinociceptive effects tested by hot plate, respectively. The antinociceptive effects of three derivatives were 5.8, 5.4, and 5.1-folds of the effect of N(3)-phenacyluridine (1h) (16%), respectively. The structure-activity relationship of N(3)-substituted pyrimidine nucleosides was also discussed.

The antinociceptive activity of N3-phenacyluridine, a novel hypnotic, was examined with tail pinch, hot plate and acetic acid-induced abdominal constriction methods by intracerebroventricular (i.c.v.) injection to mice. In the first method, N3-phenacyluridine exerted antinociceptive activity after the i.c.v. injection at a dose of 0.5 micromol/mouse, although this activity was comparable to the activity of morphine (0.01 micromol/mouse, i.c.v.). In the second method, the activity of N3 -phenacyluridine was continued until 120 min after the injection at a dose of 0.5 micromol/mouse. In last, N3-phenacyluridine (0.1 micromol/mouse, i.c.v.) significantly decreased in the numbers of acetic acid-induced abdominal constriction as compared to the control (1% Tween 80 saline). The ED50 value of the antinociceptive activity of N3-phenacyluridine was 0.02 imol/mouse, i.c.v. The present paper demonstrated for the first time that N3-phenacyluridine belonging to oxopyrimidine nucleoside analogue possesses not only the hypnotic and sedative activities previously reported, but also antinociceptive activity.