We found that NADPH-dependent ubiquinone reductase (NADPH-UQ reductase) in rat liver cytosol reduces ubiquinone (UQ) to ubiquinol (UQH₂) in lipid membranes and consequently inhibits lipid peroxidation [Takahashi T., et al., Biochem. J., 309, 883-890 (1995)]. Here we examined whether or not this UQH₂-regenerating system functions as a cellular antioxidant defense in animals. Rats were given UQ-10 for 2 weeks, and were then exposed to carbon tetrachloride (CCl₄). The UQ-10 supplement increased only in the NADPH-UQ reductase and the UQH₂-10 pool of rat liver without any appreciable change in the levels of other antioxidant factors. On the other hand, CCl₄ markedly increased plasma aspartate aminotransferase and alanine aminotransferase, liver weight and thiobarbituric acid reacting substances formation, which are indicators of CCl₄-hepat itis, and it decreased the liver levels of L-ascorbic acid, reduced form of glutathione (GSH), α-tocopherol, NADPH-UQ reductase and glutathione S-transferase. However, all the above indicators of CCl₄-induced hepatitis were significantly improved in rats given UQ-10. Furthermore, α-tocopherol, but neither L-ascorbic acid nor GSH, was significantly saved. UQ-10 supplement also was recovered glutathione S-transferase and NADPH-UQ reductase activities slightly. These results indicated that UQ-10 given to rats increased the cellular UQH₂-10 pool and cytosolic NADPH-UQ reductase activity in their livers, resulting in the inhibition of lipid peroxidation in the biomembranes, and consequently protected the rats from the CCl₄-hepatotoxicity.

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Basal cells were prepared from the newborn rat skin epidermis and cultured on a type I collagen-coated filter. This cultured basal cell layer showed an amiloride-sensitive polarity, and the apical surface of the cell layer was more acidic than the basal surface. When the basal cells were cultured with ascorbic acid, a polarity reverse of the cultured cell layer was observed. In this case, synthesized and accumulated type IV collagen was detected on the whole apical surface of the cultured cell layer. An inhibitor of collagen synthesis, cis-hydroxyproline (cis-OH-Pro), suppressed the ascorbic acid-induced polarity reversal.Furthermore, when basal cells were cultured on a type IV collagen-coated filter, the amiloride-sensitive polarity of the cultured cell layer resembled the cultivation on a type I collagen-coated filter. In this case, the polarity was reversed by the addition of ascorbic acid, and cis-OH-Pro suppressed this reversal.Based on these results, we concluded that cultured skin basal cell layer showed an amiloride-sensitive polarity. Moreover, newly synthesized and accumulated type IV collagen was deduced to affect the polarity reversal in the cultured basal cell layer.

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We investigated the effects of natural and synthetic estrogens on estrogen receptor-mediated signal transduction and transcription. To assess estrogen receptor-dependent gene expression, we constructed a reporter plasmid that can express the luciferase gene under the control of the estrogen-responsive element. A transcriptional assay was performed by transfection of the reporter plasmid into estrogen-responsive MCF-7 cells. Transcription was completely dependent on the natural estrogen, 17β-estradiol (E₂), in a dose-dependent manner. Retinoic acids and other steroid hormones had no effect on gene expression. The effect of synthetic estrogens on estrogen receptor-mediated signal transduction was also examined using the specific reporter assay system. A synthetic estrogen, diethylstilbestrol, exhibited higher activity than E₂ at lower concentrations. In addition, transcriptional activities of the (+)- and (-)-enantiomers of indenestrol A, a metabolite of diethylstilbestrol, could be distinguished using the reporter assay system.

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Using a new technique of vortex-stirring of cells with high molecular weight polyacrylic acid (A-119), non-permeant fluorescein isothiocyanate-labeled dextrans (FDs) of various molecular weights were tested to determine the degree of their internalization into cultured murine leukemia L1210 cells. Flow cytometric analyses of the treated cells revealed that FD4 with a molecular weight (MW) of 4400 was incorporated in an amount 13.2 times greater than that of the control and that FDs with MW of 19000 and 38260 were incorporated at levels several times greater than control levels, by vortex-stirring cells with A-119 for 10s followed by maintenance at 0°C for 10 min. Results of the present study indicate that this permeabilization procedure might be useful to introduce poorly permeable materials into cultured mammalian cells, which would be beneficial in various fields of biological research.

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A cDNA clone encoding the rabbit cysteine proteinase cathepsin K, which is predominantly expressed in osteoclasts and is closely related to cathepsins L (EC 3.4.22.15) and S (EC 3.4.22.27) [Tezuka K., Tezuka Y., Maejima A., Sato T., Nemoto K., Kamioka H., Hakeda Y., Kumegawa M., J. Biol. Chem., 269, 1106 (1994)], was expressed at high levels in Escherichia coli in a T7 expression system. The insoluble recombinant enzyme was solubilized in urea and refolded at an alkaline pH. Cathepsin K (37-kDa) was purified by gel filtration and its enzymatic characteristics were determined. The enzymatic activity of cathepsin K was strongly inhibited by cysteine proteinase inhibitors and its optimal pH was pH 5.5. Synthetic substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumaryl-amide, which is hydrolyzed by cathepsins L and S, was also cleaved by cathepsin K. On the other hand, benzyloxycarbonyl-Gly-Pro-Arg-7-(4-methyl)coumaryl-amide was the most suitable substrate for cathepsin K, but was hardly hydrolyzed by cathepsin L. The substrate specificity of cathepsin K, as determined using various chemogenic substrates, showed different characteristics from cathepsins L and S.

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Aconitine and higenamine are the major cardioactive compounds obtained from processed aconite. The chronotropic interaction between these two compounds was investigated in isolated right atria of mice. Both aconitine and higenamine potentiated the action of the other. Practolol (1 nM), a selective β₁-adrenergic antagonist, but not butoxamine (1 μM), a β₂-adrenergic antagonist, blocked the potentiation by higenamine (5 nM) of the aconitine-induced positive chronotropic effect and, at high concentrations (30 and 300 nM) also shifted the aconitine concentration-response curves to the right. The potentiating interaction between aconitine and higenamine was reversed by pretreating with cholera toxin (CTX) and forskolin. In CTX (100 nM, 1 h)- and forskolin (30 and 100 nM)-treated atria, higenamine significantly depressed the aconitine-induced response, which was abolished by pertussis toxin (PTX, 150 μg/kg, i.p., 3 d). Neither CTX (50 and 100 nM) nor forskolin (15-100 nM) significantly affected the aconitine-induced positive chronotropic effect, while PTX (150 μg/kg) depresesd it. These results suggest that the potentiating interaction between aconitine and higenamine involves "cross-talk" between the β₁-adrenergic signalling pathway and G_i-protein.

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Effects of the intracerebroventricular administration of 1-[[[5-(4-nitrophenyl)-2-furanyl]methylene]imino]-2-, 4-imidazolidinedione (dantrolene, an inhibitor of intracellular Ca²⁺ mobilization) on learning/memory were investigated in mice using step-through passive avoidance and radial-arm maze tests. In the passive avoidance test, the administration of 6 nmol of dantrolene shortened the response latency in the retention test. The number of times of acquisition training required to achieve the criterion latency (300 s) did not change in the acquisition test. Ten nmol of administration of dantrolene increased the number of times of acquisition training required to achieve the criterion latency in the acquisition test and shortened the response latency in the retention test. In the radial-arm maze tests, 20 nmol of administration of dantrolene disrupted maze-choice accuracy and increased error numbers. These results suggest that intraneuronal Ca²⁺ mobilization plays important roles in learning and memory.

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The effects of 70% methanolic extract (CC-ext) from Cinnamomi Cortex on acute and chronic inflammation were investigated.CC-ext inhibited the rise in vascular permeability induced by acetic acid and the increase of paw edema induced by carrageenin in mice. It was ineffective on edema derived by histamine or bradykinin, and exhibited only weak inhibitory effect on the edema derived by serotonin. CC-ext also showed inhibitory effects on the prekallikrein enzyme activity and ear edema induced by arachidonic acid. It also had an inhibitory effect on cotton pellet-induced granuloma but showed no atrophying action against the adrenal or thymus glands. Little effect was shown on secondary lesions in the development of adjuvant-induced arthritis (the arthritis reappeared from 11 to 27d after injection of the adjuvant). These results suggest that some active component having an inhibitory effect on acute inflammation is contained in Cinnamomi Cortex.

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This study was carried out to elucidate the antiinflammatory effect of 50% ethanol extract obtained from the fruit of Trichosanthes kirilowii MAXM. and its effective parts. The cut dried fruit was separated into the whole fruit, seeds and remaining parts, and each part was extracted with 50% ethanol. The antiinflammatory activity of these extracts was investigated on acetic acid-induced vascular permeability in mice, carrageenin-induced edema and cotton pellet-induced granuloma formation in rats, as well as writhing symptoms in mice. The whole fruit and seed extractions showed an antiinflammatory activity and analgesic activity, but the other part did not when given perorally. These results suggest that the antiinflammatory effect and analgesic effect induced by ethanol extract of the fruit of T. kirilowii may be due to the seeds contained in the fruit.

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This study was designed to evaluate the percutaneous absorption of insulin in an attempt to develop an efficient transdermal therapeutic system (system) for the treatment of diabetes. First, the dissociation of porcine insulin existing mainly as hexamers was examined. Next, enhancement of the percutaneous absorption of the hormone was studied by the combined use of two or more kinds of enhancers which exert their enhancement effects by different mechanisms, or by preparing the liposomal formulation of insulin. Porcine insulin dissociated in 0.1 M glycine-HCl buffer (pH 4.0), probably to a dimer, this being demonstrated by the notable attenuation of the maxima at 221 and 274 nm of the circular dichroism (CD) spectra. Thus, 0.1 M (or partly 1 M) glycine-HCl buffer (pH 4.0) was selected for the preparation of all gel formulations. The in viv o absorption of insulin through Wistar rat skin was estimated by blood glucose level. System 3 containing liposomal insulin, D-limonen and taurocholate gave the greatest hypoglycemic response, out of the formulations used, with its response persisting over a 10 h period and resulting in the highest pharmacological availability (20.7±4.6%). The combination of n-octyl-β-D-thioglucoside (OTG), cineol and deoxycholate (system 6) or D-limonen and OTG (system 5) also produced a high hypoglycemic effect. The in vitro penetration of insulin was investigated using system 5 and 6. The percutaneous penetration of insulin was demonstrated by an in vitro experiment, but was small in quantity. Our data present unambiguous evidence that this hydrophilic macromolecule was absorbed through the stratum corneum of rat skin under selected conditions.

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Dipalmitoylphosphatidylcholine (DPPC) liposomes with a soybean-derived sterol mixture (SS) or cholesterol (Ch) were examined as a carrier for the oral administration of insulin in rats. Four kinds of liposomes were prepared : liposomes containing SS or Ch (molar ratio of DPPC/X=7 : 2 or 7 : 4, X=SS or Ch), respectively. The pharmacological availability was greater and the lag time for the glucose reduction was later in the order of DPPC/SS (7 : 4)>DPPC/SS (7 : 2)>DPPC/Ch (7 : 4)»DPPC/Ch (7 : 2)-liposomes. This order appears to correspond well with that of the rigidity of the liposomal membrane. In particular, DPPC/SS (7 : 4)-liposomes reduced blood glucose levels for up to 21 h in rats after oral administration. The highest absolute pharmacological availability was 31.6% at a dose of 20.0 IU/kg of DPPC/SS (7 : 4)-liposomes among the liposomes in comparison to intravenous administration.

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The pharmacodynamic activities (leukopoietic effect) and pharmacokinetics of recombinant human granulocyte colony-stimulating factor (rhG-CSF) administered rectally in various doses as hollow-type suppositories were investigated in rabbits. Three kinds of rhG-CSF hollow-type suppositories were employed : suppository I containing rhG-CSF (100-1000 μg/kg) in 10 mM acetate buffer solution (ABS) at pH 4.0, suppository II containing rhG-CSF in ABS with α-cyclodextrin (α-CyD) as an absorption-enhancing agent, and suppository III containing lyophilized rhG-CSF powder. We found that the total count of leukocytes in peripheral blood (total blood leukocyte count) and serum granulocyte colony-stimulating factor (G-CSF) concentration showed a dose-dependent increase. Consequently, the area under the increased total blood leukocyte count-time curve (AUL), an index of pharmacodynamic activity, and the area under the serum G-CSF concentration-time curve (AUC), a pharmacokinetic parameter, increased with an increase in the rhG-CSF dose administered rectally. Good correlation was found between AUL and AUC;thus, it is concluded that an increase in AUC leads to an increase in the effect of rhG-CSF in inducing leukopoiesis in rabbits following rectal administration. We report for the first time the rectal absorption of rhG-CSF using a new rectal dosage vehicle (rhG-CSF hollow-type suppository) and its leukopoietic effect. The rhG-CSF hollow-type suppository is found to be a promising drug delivery system.

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The pharmacodynamic activity (leukopoietic effect) and pharmacokinetics of recombinant human granulocyte colony-stimulating factor (rhG-CSF) administered in various doses as hollow-type suppositories were investigated in rabbits with leukopenia induced by cyclophosphamide (CPA-treated rabbits). We found that serum granulocyte colony-stimulating factor (G-CSF) concentration was increased following the rectal administration of rhG-CSF. Therefore, rhG-CSF could be absorbed from the rectal mucosa in leukopenic rabbits who received CPA at a dose of 30 mg/kg/d. When rhG-CSF of at least 300 μg/kg was administered rectally once a day for 3 d in CPA-treated rabbits 1 d after the induction of leukopenia (the total count of leukocytes in peripheral blood [total blood leukocyte count] was below 5000/μl), decreased total blood leukocyte count returned to the normal physiological level and a maximum (7000-16000/μl) was obtained 3 d after the start of the rhG-CSF multiple-dosing regimen. We report for the first time that rhG-CSF administered rectally induces leukopoiesis in CPA-treated rabbits and reduces the period of leukopenia induced by CPA. Thus, the rhG-CSF hollow-type suppository offers a promising means of reducing the risk of leukopenia, an adverse side effect of CPA therapy.

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The absorption, distribution and excretion of intravenously and orally administered β-cyclodextrin (β-CD) and glucosyl (G)-β-CD in rats were studied using HPLC with pulsed amperometric detection. Within 10 h after intravenous administration, unchanged β-CD and G-β-CD recovered in urine were about 90% of each dose. The nephritic accumulation of G-β-CD after the intravenous administration of G-β-CD was slightly lower than that of β-CD using the same treatment. The maximum plasma concentrations of β-CD and G-β-CD after the oral administration of CDs (500 mg/kg) were observed within 40 min. After oral administration, 0.6% of β-CD and 0.3% of G-β-CD were excreted in urine. The pharmacokinetic behaviour of both CDs after the intravenous administration of CDs (50 mg/kg) was almost the same. Furthermore, the inclusion complexes of estriol and betamethasone with CDs were prepared, and their absorption was evaluated after oral administration in rats. The plasma concentrations of CDs after oral administration of drug-CD complexes were significantly decreased in comparison with those after the oral administration of CDs alone. On the other hand, the plasma concentrations of drugs after the oral administration of drug-CD complexes were higher than those after the administration of drugs alone.

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To evaluate whether lipofection using Lipofectin^TM is suitable for delivering foreign genes into skin fibroblasts as target cells, we performed experiments using human superoxide dismutase (hSOD) and neomycin-resistance (Neo^r) genes as models in rat skin fibroblasts (FR and primary cells) in vitro. The amounts of DNA used in the lipofection procedure significantly affected the transfection efficiencies, and the optimal amounts were determined for all cells used. However, the efficiencies in rat skin fibroblasts were about 20-fold higher than that in rat lung epithelial-like cells (L2 cells). The differences in plasmid vectors (pRc/RSV-SOD and pRc/CMV-SOD) hardly affected the transfection efficiencies. The amounts of Lipofectin^TM significantly affected the transfection efficiencies, and the optimal amounts were determined for both types of skin fibroblasts However, cytotoxic effects in both skin fibroblasts. were observed with high doses of Lipofectin^TM. On the other hand, with optimal amounts of DNA and Lipofectin^TM, the reporter gene (Neo^r) introduced into cells was mainly integrated into the host cell chromosome. Western blot analysis showed the continuous expression of hSOD protein for at least 45 d in skin fibroblasts transfected with the expression plasmid for hSOD by Lipofectin^TM under the optimal conditions, and the cellular SOD activity fluctuated in parallel with the expression of hSOD protein. Differences in the type of cells also affected the expression of hSOD. These results indicate that it is necessary to set up optimal conditions for transfection using Lipofectin^TM for each cell type, and that transfection with Lipofectin^TM under optimal conditions may be an efficient method for introduction of foreign genes into skin fibroblasts for use as a clinical delivery system of therapeutic protein.

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We have determined Hemoglobin A_1C (HbA_1C) in normal, neonatal streptozotocin-induced diabetic animals (NSZ), a model of lean-type non-insulin-dependent diabetes mellitus (NIDDM) with hypoinsulinaemia, and KK-Ay mice, a model of obese-type NIDDM with hyperinsulinaemia. The HbA_1C of NSZ mice was slightly increased from 5 to 20 weeks while the HbA_1C of KK-Ay mice was markedly increased from 5 to 10 weeks. Our findings showed a difference between experimentally-induced (NSZ) and genetically (KK-Ay) diabetic animals.

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We previously investigated the role of the Lys108 residue of ribonuclease (RNase) Rh from Rhizopus niveus, and suggested that Lys108 probably acts to stabilize the pentacovalent intermediate, and that an Arg residue could replace the role of Lys108. In RNase Le2 from Lentinus edodes, a homologous enzyme of RNase Rh, Lys108 is replaced by Thr. In this paper, the enzymatic properties of a K108T mutant and its analogous enzyme, K108S, were investigated to determine the effect of Thr and its analog, Ser at the 108th position on enzyme activity. The enzymatic properties of these mutant enzymes were compared with those of other mutant enzymes at this position (K108M, K108A, K108L). The results showed that Thr and Ser could replace Lys108 but resulted in only 2-20% of the activity of the native enzyme depending on the substrates used.

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Basic human urinary arginine amidase (or esterase, called BHUAE) which is only found in male urine, was measured from normal volunteers between the age of 4 and 70 years using D-valyl-L-leucyl-L-arginine-p-nitroanilide as a substrate. BHUAE increases during early adolescence, between 8 to 17 years of age. Then, BHUAE decreases in the twenties and takes a certain range of value in the mature age group, between the late thirties and fifties.In patients with prostate cancer, a significant increase in BHUAE was demonstrated in comparison with the healthy male group (control) over 55 years old. On the other hand, patients with benign prostatic hypertrophy showed no significant elevation of this enzyme activity. It would appear that the measurement of BHUAE in urine can be used as a marker of prostate cancer in an advanced age group.

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Datura metel L. var. muricata (BERNH.) DANERT was found to be double recessive with respect to the genes concerning the color and form of the corolla by breeding experiments involving four varieties, i.e. var. metel (white, simple corolla), var. rubra (purple, simple), var. fastuosa (purple, double or triple) and var. muricata (white, purple). The results support the proposal by Danert and others that these variants should be considered as varieties or forms of a single species, Datura metel. The analysis of tropane alkaloids in the seeds, flowers, and leaves of these four varieties showed that scopolamine was always dominant over hyoscyamine. The range of the scopolamine content (% of dry weight) of seeds, flowers, and leaves was 0.294 (var. rubra)-0.631 (var. fastuosa), 0.190 (var. metel)-0.698 (var. rubra), and 0.042 (var. rubra)-0.255 (var. metel), respectively. These findings proved that all the varieties, including var. muricata, which exhibited medium scopolamine content among the varieties, can be utilized as sources of scopolamine.

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Zonisamide (ZNS), a new anti-epileptic drug that exhibits a wide anti-epileptic spectrum, is commonly prescribed concomitantly with other anti-epileptic drugs. The interaction between ZNS and other anti-epileptic drugs was investigated in epileptic patients. The steady state plasma levels of all the anti-epileptic drugs were measured by high-performance liquid chromatography. The concomitant administration of phenobarbital (PB), phenytoin (PHT), or carbamazepine (CBZ) with ZNS significantly decreased the ratio of the steady state plasma concentration to the administered dose (C/D ratio) of ZNS, whereas clonazepam (CZP) and valproic acid (VPA) when administered concomitantly with ZNS did not change the C/D ratio. Significant negative correlations were observed between the C/D ratio of ZNS and the doses of PB, PHT, CBZ, and VPA. On the other hand, the ratio of the plasma concentration of carbamazepine-10, 11-epoxide (CBZ-E), the major active metabolite of CBZ, to the plasma concentration of CBZ was significantly decreased by concomitant administration with ZNS, indicating that ZNS inhibits the metabolism of CBZ. These findings show that when ZNS is administered concomitantly with these anti-epileptic drugs, it is necessary to monitor the plasma concentration of ZNS in order to adjust its dosage.

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Nitroglycerin (GTN) has been used as the drug of choice in the treatment of angina pectoris. It has been shown that some glutathione S-transferases (GSTs) catalyze the metabolic conversion from GTN to glyceryl dinitrates (GDNs). In this study, we examined the substrate specificity of GSTs for GTN. Alpha and mu GSTs were isolated from porcine liver and intestinal mucosa by means of CM-cellulose and glutathione-affinity column chromatography. Mu GSTs degraded GTN time-dependently and formed 1, 3-GDN in preference to 1, 2-GDN at a ratio (1, 2-GDN/1, 3-GDN) of 0.61, whereas alpha GSTs formed twice as much 1, 2-GDN as 1, 3-GDN. These results showed that two GST families participate in the metabolic conversion of GTN at different hydrolyzing portions of the nitrogroups.

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The relationship between the plasma drug concentration and the antihypertensive effect of felodipine was analyzed by an ion-channel binding model which takes into consideration the slow association/dissociation process of a drug at the calcium channel. The in vitro dissociation constant (K_d) of felodipine to the calcium channel in the heart of rats was determined, and was compared to the in vivo dissociation constant (D_{d, calc}) estimated by the pharmacodynamic analysis of the concentration-effect data in Japanese essential hypertensive patients obtained from literature. The relative relationship between K_d and K<d, calc> of felodipine was substantially identical with eight other calcium channel blocking agents reported previously. This result suggested the possibility that we can predict the pharmacodynamic behavior of newly developed calcium channel blocking agents from the in vitro K_d value and plasma concentration-time profile in human using the ion-channel binding model.

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We inserted human Cu, Zn-superoxide dismutase (hSOD) cDNA into the eukaryotic expression plasmid (pRc/CMV) under the control of the cytomegalovirus promoter. The hSOD expression plasmid (pRc/CMV-SOD) was transfected in L2 cells by mean of lipofection. The intracellular SOD activity in pRc/CMV-SOD transfected cells (CMV-SOD cells) was about twice that in host cells. However the level of extracellular SOD activity was similar in CMV-SOD and host cells. When exposed to xanthine (X)/xanthine oxidase (XO) to generate active oxygen species, significantly more CMV-SOD cells than host cells survived. The production of lipid peroxidation in host cells significantly increased in the presence of X/XO, but that in CMV-SOD cells did not change. Thus, transfection with SOD gene effectively prevented X/XO-induced cytotoxicity. The results indicated that increasing the level of intracellular SOD activity protected cells against extracellular superoxide anion stress.

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We examined the effect of CYP2C9/19 polymorphisms on the pharmacokinetics of phenytoin in 17 Japanese patients with epilepsy. The maximal elimination rate (V_max) of phenytoin was slightly decreased (up to 14%) in patients with CYP2C19 mutations for the defective allele. The V_max values in patients with a CYP2C9 mutation for the heterozygous Ile/Leu³⁵⁹ allele were 40% lower than those in patients with wild-type CYP2C9 for the homozygous Ile³⁵⁹ allele. These findings suggested that the genetic polymorphism of CYP2C isoenzymes plays an important role in the pharmacokinetic variability of phenytoin, and that the mutation in CYP2C9 proteins is a determinant of impaired metabolism of the drug.

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Crude extracts containing the enzymes obtained from mouse liver were incubated with 3-deoxyglucosone (3-DG), and then subjected to assay of the activities of enzymes responsible for glucose metabolism. Hexokinase and glucose-6-phosphate dehydrogenase activities were decreased by 3-DG and hexokinase activity was strongly inhibited time and concentration dependently, while glucokinase, glucose-6-phosphatase, and phosphofructokinase activities were scarcely affected. These results suggest that 3-DG inhibits the intake of glucose in the liver and a connection with development of diabetes.

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Mouse brain microsomes contain an amidase that catalyzes the hydrolysis of N-arachidonylethanolamide to arachidonic acid and ethanolamine. The enzymatic activity is dependent on the protein concentration of the microsomes and observed over a wide range of pH, 7.4 to 9.5. Kinetic analysis indicated that Km (μM) and Vmax (nmol/min/mg protein) were 17.7 ± 4.1 and 1.81 ± 0.32, respectively. Cannabidiol (CBD), cannabinol (CBN) and Δ⁹-tetrahydrocannabinol (Δ⁹-THC) significantly inhibited the hydrolysis of the amide by mouse brain microsomes. At a concentration of 160 μM, the inhibitory potency decreased in the following order, CBD(66%)>CBN(46%)>Δ⁹-THC(31%).

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A hand-held spectrophotometer was used for the quantitative evaluation of PCA caused by anti-TNP-IgE and TNP-BSA. A good relationship existed between the dilution of sera and the value of ΔE^*ab (a numerical expression of color) obtained by a hand-held spectrophotometer. In addition, the value of ΔE^*ab and the amount of Evans blue measured by the conventional colorimetric method correlated well. Because the method using a hand-held spectrophotometer provides a simple and objective analysis, it appears that the method is suitable as a substitute for the conventional method, which is time-consuming and requires killing animals cruelly.

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