The distribution and relative proportions of the four isomers of α-, β-, γ-and δ-biliverdin-IX were investigated using urinary samples from seven healthy adults. The biliverdins from samples were adsorbed on talc, and the adsorbed biliverdins were then soaked in a solution of methanol and concentrated sulfuric acid for derivation to the dimethyl esters. The esters were passed to a reversed-phase cartridge for clean-up, then quantitated by HPLC on a reversed-phase column. The analyses revealed that the biliverdins in 24-h urine were comprised of 6.4±2.5 nmol α- and 9.3±5.4 nmol β-isomers. The HPLC analyses suggested the presence of a δ-isomer in a much smaller amount, but no γ-isomer was detected. The identifications of α- and β-isomers were performed with co-chromatography and absorption spectra.

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Aged mice (6 months of age) fed a conventional diet were shifted to diets containing 10% lard, high-linoleate safflower seed oil, high α-linolenate perilla seed oil or high-docosahexaenoate fish oil. A significant increase in whole body cholesterol (/g wt) was seen within 30 d after the shift (rapid response), followed by a gradual decrease in 60 to 120 d (slow response) ; similar changes occurred in all the dietary groups. Shortly after the shift, the serum cholesterol concentrations increased to higher levels in the lard and safflower oil groups than in the other groups, and the levels at 120 d were in the group order of safflower oil>lard>perilla oil>fish oil. Rapid and slow responses to dietary shifts were also seen in hepatic cholesterol levels (/g wt), which were higher in the lard group than in the other groups at 120 d. The arterial cholesterol contents of the fish oil group tended to be less than in the other groups at 120 d. Thus in aged mice after a relatively long-term feeding (>one tenth of the life-span), safflower oil was not hypocholesterolemic as compared with lard and other ω3-rich oils. Long-term feeding of fish oil maintained serum cholesterol concentrations at lower levels than feeding with safflower oil or lard and without accumulating cholesterol in the aorta, liver or whole body ; perilla oil was also hypocholesterolemic but to a lesser degree than fish oil. Saturated, monounsaturated and linoleic acids in diets accumulated relatively efficiently in the whole body but ω3 fatty acids were catabolized preferentially in aged mice. Our results do not support the concept that an increased intake of high-linoleate vegetable oils is useful for the prevention of hypercholesterolemia-associated diseases ; however, dietary oils rich in ω3 fatty acids may be useful.

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Reactions between ebselen and subcellular particles of rat liver were investigated by monitoring the activity of mitochondrial glutamate dehydrogenase and microsomal glucose 6-phosphate dehydrogenase. Rat small intestine lactate dehydrogenase was purified and was also used in the reaction between cytosolic protein and ebselen. Glutamate dehydrogenase in intact rat liver mitochondria was completely resistant to ebselen, but the enzyme was significantly inactivated in broken mitochondria mediated by Triton X-100, reflecting the fact that ebselen was not transported through the mitochondrial membrane into the matrix. Glucose 6-phosphate dehydrogenase in rat liver microsomes was inactivated by ebselen, accompanied by a slight decrease in the thiol groups of microsomal membrane protein. Purified cytosolic lactate dehydrogenase was inactivated concentration-and time-dependently by ebselen. The activity of rat small intestine lactate dehydrogenase abolished by ebselen was significantly restored by incubation with purified rat small intestine thioltransferase in the presence of reduced glutathione (GSH). The level of thiol groups in rat liver microsome membrane protein decreased by ebselen was partially restored by an incubation with purified rat liver thioltransferase in the presence of GSH. The results suggested that thioltransferase can cleave the Se-S conjugates between ebselen and cytosolic proteins or microsomal membrane proteins in the presence of GSH.

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We investigated whether endogenous nitric oxide (NO) has a role as an inhibitory modulator of norepinephrine (NE)- and angiotensin II (Ang II)-induced renal effects in anesthetized dogs. Intrarenal arterial infusion of NE (100 ng/kg/min) or Ang II (10 ng/kg/min) decreased renal blood flow (RBF), glomerular filtration rate (GFR) and urine formation. The NE- or Ang II-induced renal effects were augmented by the intrarenal administration of a NO synthase inhibitor, N^G-nitro-L-arginine (NOARG), at doses (10 and 40 μg/kg/min) which did not affect the mean arterial blood pressure and heart rate. The stimulating activity of NOARG on NE- or Ang II-induced renal effects were abolished by the simultaneous administration of L-arginine, a NO precursor. These findings suggest that endogenous NO, which is probably generated within the kidney, functions as an inhibitory modulator in NE- or Ang II-induced renal vasoconstriction and antidiuresis.

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In order to elucidate the role of neuropeptide Y (NPY) in cerebral circulation, we undertook the present study to examine the action of NPY itself, and the combined effects of NPY with other vasoconstrictor stimuli. NPY itself produced contractions of isolated canine basilar artery in a concentration-dependent manner, which was independent of the presence of absence of endothelium. C-terminal peptides of NPY (NPY_12-36) and (NPY_22-36) were weak agonists, while those without C-terminals were ineffective. The vasoconstriction produced by NPY was, however, strongly potentiated by increasing the K⁺ concentration in the medium up to 20 mM, or by pretreatment with tetraethylammonium, a K⁺ channel blocker and hemolysate containing oxyhemoglobin. NPY also augmented the contractile responses to prostaglandin F_2α, norepinephrine, and histamine, but not to serotonin. The contraction in response to NPY per se or in 20 mM K⁺ was effectively attenuated by Ca²⁺ antagonists such as d-cis-diltiazem, and in a Ca²⁺-free medium. These results suggest that in canine basilar artery, the activation of the Y₁ receptor modulates the availability of the L-type Ca²⁺ channel, leading to enhance Ca²⁺ influx from the extracelullar space and potentiate contractile effects of other cerebral vasoconstrictors. This might be involved in the cerebral vasospasm which occurs after subarachnoid hemorrhage.

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The effects of benidipine on contractions induced by contractile agents were studied in isolated dog basilar, middle cerebral and mesenteric arteries and compared with those of nicardipine. KCl (20-80 mM), U-46619 (3×10^-10-3×10^-6M) and 5-hydroxytryptamine (5-HT) (10^-9-3×10^-6M) produced concentration-dependent contractions in the three arteries. Benidipine (10^-11-8×10^-9M) inhibited non-competitively the KCl-induced contraction in the basilar, middle cerebral and mesenteric arteries with a pD'₂ of 9.1, 9.7 and 8.5, respectively. There was a significant difference between the pD'₂ values in the cerbral and mesenteric arteries. Benidipine (10^-6-3×10^-5M) attenuated significantly the contraction produced by both U-46619 and 5-HT in the three arteries, the pD'₂ being 4.4-4.9. Nicardipine inhibited the contraction induced by the three contractile agents in the same manner as benidipine. The contraction caused by 70 mM KCl in the cerebral and mesenteric arteries was reduced by 33-38 and 18%, respectively following treatment with 0.25 mM Ca²⁺ solution. Furthermore, pretreatment with a Ca²⁺-free solution containing 1 mM EGTA inhibited KCl-induced contractions in the basilar, middle cerebral and mesenteric arteries by 98.7, 95.6 and 92.1%, respectively. It also attenuated the contractions produced by both 10^-6M U-46619 and 3×10^-6M 5-HT in the cerebral and mesenteric arteries by 56-61 and 34-39%, respectively. These results suggest that the more effective inhibition of KCl-induced contractions by benidipine, as well as nicardipine, in the cerebral arteries may be due to a greater dependence of the KCl-induced contraction in the cerebral arteries on extracellular Ca²⁺, compared with the mesenteric arteries.

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Exposure of mitochondria to adriamycin (ADM)-Fe³⁺ induced formation of thiobarbituric acid reactive substances and fluorescent substances. Butylated hydroxytoluene (BHT) and the water soluble vitamin E analogue, trolox, not only strongly inhibited fluorescence formation but also mitochondrial lipid peroxidation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the formation of high molecular weight proteins when mitochondria were exposed to ADM-Fe³⁺. A mitochondrial protein with a molecular weight of approximately 30 kDa was very sensitive to ADM-Fe³⁺. BHT and trolox strongly inhibited mitochondrial protein cross-linking, indicating that the protein modification was due to ADM-Fe³⁺-induced lipid peroxidation. In addition, the susceptibility of ADM-Fe³⁺-exposed mitochondrial protein to proteases was unchanged. Bovine serum albumin (BSA) inhibited ADM-Fe³⁺-induced mitochondrial lipid peroxidation. Fluorescence emmited from BSA was detected during ADM-Fe³⁺-induced mitochondrial lipid peroxidation, and BHT strongly inhibited the oxidative modification of BSA. These results suggest that the oxidative modification of mitochondrial proteins and BSA is due to ADM-Fe³⁺-induced lipid peroxidation.

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DX-9386 is a traditional Chinese medicinal prescription consisting of ginseng (Panax Ginseng C. A. MEYER), polygala (Polygala Tenuifolia WILLDENEW), acorus (Acorus Gramineus SOLAND) and hoelen (Poria Cocos WOLF). We recently found that oral administration of the prescription at a dose of 500 mg/kg intensified the formation of long-term potentiation (LTP) in the dentate gyrus of anesthetized rats. To evaluate the individual contribution of separate ingredients in DX-9386 towards the observed biological activity, we investigated their direct influence upon LTP formation in vivo. A single oral administration of hoelen and ginseng (250 and 500 mg/kg) significantly increased the spike amplitude evoked by a subthreshold tetanic stimulation at time intervals up to 30 min after tetanus. Only minor effects of polygala (500 mg/kg) and no influence of acorus up to 500 mg/kg were observed. No drugs affected the basal spike amplitude induced by a test stimulus. In addition, we ascertained that DX-9386 was also active at a dose of 250 mg/kg. Taken together, these results indicate that hoelen and ginseng are the active components of DX-9386 with regard to the enhancement of hippocampal LTP.

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The study revealed that high molecular polyvinyl sulfate (PVP) or sulfonate (PVS), and low molecular alkyldisulfonates (NaO₃S (CH₂)_nSO₃Na, n=2-5 : EDS, TDS, BDS and PDS) can alleviate acute toxicity of the herbicide, paraquat dichloride (PQ) in mice. Their activity as antidotes and the mode of the action varied depending on molecular size. The survival rate for mice receiving PQ at 200 mg/kg alone was increasingly improved when the dose of antidotes was increased from 8 to 10 times the dose of PQ ; all the test compounds, except EDS (70% survival), achieved a survival rate of 100%. When test compounds were orally dosed to mice in a mixture with PQ, they improved LD₅₀ of PQ alone. With the low molecular compounds EDS, TDS, BDS and PDS, the value increased to about 2 to 3 times (300-458 mg/kg) over that of PQ alone (140 mg/kg). With high molecular PVS and PVP, the combination reached about a 7 fold (900-1000 mg/kg) increase in LD₅₀ value. The formation of lipid peroxide in lungs of rats due to PQ tended to be suppressed by concomitant administration of carbohydrate sulfate (DS and GS). PVP, BDS and TDS were more effective in depressing synthesis of lipid peroxide than DS or GS in the lungs, although BDS and TDS were less effective in suppressing PQ absorption from the rat small intestine than DS, GS or PVP. The results of these experiments indicate that the main mechanism for the detoxification of a high molecular alkylsulfate or sulfonate, and a low molecular alkyldisulfonate must be suppression of PQ absorption from the intestine, similar to that of carbohydrate sulfate. In addition, a low molecular alkyldisulfonate (BDS and TDS) was also proved to be significantly effective in suppressing the formation of lipid peroxide in the lungs.

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Carbon monoxide (CO) was generated in the process of hepatic microsomal oxidative metabolism of cannabidiol (CBD). After the generated CO was reduced to methane (CH₄) with a methanizer, CH₄ formed was determined by gas chromatography (GC) with a flame ionization detector. After oxidation with hopcalite, CO was also identified as CO₂ by gas chromatography/mass spectrometry (GC/MS). The reaction was NADPH-dependent and required molecular oxygen. It was inhibited by addition of some inhibitors of cytochrome P450-dependent monooxygenase. When CBD (191 μM) was incubated with hepatic microsomes of mice in the presence of an NADPH-generating system and oxygen, concentration of CO determined by GC was 4.7±0.5 ppm/nmol P450 in the incubation atmosphere. Pretreatment with phenobarbital (100 mg/kg, i.p. for 3 d) but not 3-methylcholanthrene (80 mg/kg, i.p.) increased the CO formation 78%, while pretreatment with cobaltous chloride (40 mg/kg, i.p. for 3 d) decreased the formation 56%. When CBD was incubated under oxygen-18 gas, molecular oxygen was not incorporated into the CO molecule. 8, 9-Dihydro- and 1, 2, 8, 9-tetrahydro-CBDs also produced CO to some extent, whereas CBD monomethyl- and dimethylethers reduced the ability to produce CO. In addition, cannabidivarin and olivetol produced CO, although none of Δ⁹-tetrahydrocannabinol, cannabinol and d-limonene did. Thus, the resorcinol moiety of CBD is important for CO formation.

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The involvement of the pituitary gland in the expression of sex-related differences in the activities of epoxide hydrolase (EH) in liver was examined in hypophysectomized and neonatally monosodium L-glutamate (MSG)-treated male and female mice. In hypophysectomized mice, the hepatic microsomal EH activity of males and females increased considerably above those of the respective sham-operated controls, and the female activity exceeded that of the sham-operated males. The hypophysectomy caused a decrease in soluble EH activity in males, while the operation led to an elevation of the activity in females. Neonatal treatment with MSG resulted in marked increases of microsomal EH activity in males and females at adulthood and the activity was about 2 times that of the male control mice. Treatment of hypophysectomized males and females with growth hormone (GH) resulted in a significant decrease in microsomal EH activity below those of the corresponding hypophysectomized controls, while the hormone treatment had no effect on the soluble enzyme activities. These results suggest that murine hepatic microsomal EH activity is principally under the suppressive control of the pituitary and GH is a causal hormone involved in the expression of sexual dimorphism in the enzyme activity.

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The properties of hemoglobin denaturation caused by two anionic surfactants (sodium dodecylsulfate and sodium lauroylmethyltaurate) were examined using spectrophotometry (multi-plate reader), circular dichroism (CD) and high-performance liquid chromatography (HPLC), and their denaturating action on hemoglobin and sorption to it were examined by various methods. Correlation and factorial analyses were applied to the experimental data and the following results were obtained : (1) High correlations were found among sorption, denaturation and α-helix content and the random structure of hemoglobin. (2) The α-helix of hemoglobin is randomized as the surfactants denature hemoglobin. (3) Factorial analysis indicates that there are two factors involved in hemoglobin denaturation, one related to the destruction of the α-helix and the other related to a change in the β-structure. These two factors are related to change in the environment around the heme group. (4) Destruction of the α-helix seems to be one of the causes of the eye irritation produced by anionic surfactants.

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The effect of an oral treatment with the Kampo formulation Juzen-taiho-to on the toxicity caused by the intraperitoneal administration of 15 mg/kg carboplatin (CBDCA) 9 times (on days 3, 4, 5, 6, 7, 8, 10, 11 and 12) was examined in ddY mice, which were subcutaneously inoculated with sarcoma 180 (S-180) cells on day 1. White blood cell counts, platelet counts, bone marrow cell counts, relative spleen and thymus weight, food intake and body weight decreased significantly, to about 29%, 13%, 14%, 59%, 36%, 42% and 72% of the control levels, respectively, and serum glutamic-oxaloacetatic transaminase, serum glutamic-pyruvic transaminase and relative stomach weight increased significantly, to about 4, 6 and 3 times the control levels, respectively, by the treatment with CBDCA. However, the blood urea nitrogen and serum creatinine were only slightly increased compared to the control value. Co-treatment with 1.7 g/kg of a lyophilized water extract of Juzen-taiho-to once a day 12 times (on days 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14 and 15) prevented both the decreases and increases caused by CBDCA to near the control levels without reducing the antitumor activity of CBDCA against S-180. The inhibitory effect of Juzen-taiho-to against CBDCA-induced myelosuppression was similar to that against 3.0 mg/kg cisplatin (CDDP) 9 times, while CBDCA-induced myelosuppression was more serious in comparison with CDDP. Therefore, these findings indicate that Juzen-taiho-to could be an effective drug for protecting against the side effects induced by CBDCA in the clinic as well as by CDDP.

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In this study, protein kinase C was demonstrated to operate as a down-regulator of glucose-6-phosphatase in the kidney, at least. Renal glucose-6-phosphatase activity reached a maximum level in 3 h after the administration of fluoride to rats. The incremental increase of renal glucose-6-phosphatase activity caused by fluoride administration was markedly amplified by the administration of staurosporine (66 μg/kg, i.p.), which has inhibitory activity against protein kinase C, or by the administration of H-7 (5 μmol/kg, i.p.), a specific and strong inhibitor of protein kinase C. Interestingly, the finding indicated that protein kinase C operates as a down regulator of renal glucose-6-phosphatase activity. The finding was reconfirmed by the result that fluoride-stimulated glucose-6-phosphatase activity was further enhanced by treatment with calphostin C (200 nmol/kg, i.p.), a specific and strong inhibitor of protein kinase C, but the change was small. Moreover, calmodulin was indicated as being possibly concerned with the down regulation of renal glucose-6-phosphatase activity by using W-7 (5μmol/kg, i.p.), a calmodulin specific inhibitor.

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19-Nor (2) and 5α-reduced (3) derivatives of androst-4-ene-3, 6, 17-trione (1) as well as 5α-androstan-17-ones 4-6 were tested for their abilities to inhibit aromatase in human placental microsomes. All the steroids except 5α-6-one 4 were fair to good competitive inhibitors of the enzyme, with apparent K_i's ranging from 50 to 820 nM in which 5α-3-one 5 was the most potent among them. The inhibitory activities of the 19-nor and 5α-reduced derivatives (2 and 3) were less potent than that of the parent compound 1. Inhibitor 2 caused a time-dependent, pseudo-first-order inactivation of aromatase activity with a rate constant for inactivation of 0.148 min^-1 in the presence of NADPH in air. The substrate androstenedione prevented the inactivation and L-cysteine did not protect aromatase from the inactivation.

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This study was carried out to examine the antiinflammatory effect of aqueous extracts of Kakkon-to (K), Kakkon-oren-ogon-to (KO), Kikyo-to (KK), Haino-to (H), Haino-san (HS), Mao-to (M) and Senkinkeimei-san (SK), which have been used for the treatment of stomatitis, tonsillitis, cold and chronic inflammatory diseases, and to elucidate the mode of their effects. Oral administration of K, KO, KK, H, HS, M and SK inhibited dose-dependently the increase of dye leakage induced by acetic acid in mice. Further antiinflammatory study was carried out on KK, H and HS which showed potent inhibition. All three extracts significantly inhibited the carrageenin-induced edema and the cotton pellet-induced granuloma formation. From these results, it is suggested that KK, H and HS may inhibit both the early exudative stage and the late proliferative stage in inflammatory processes. These extracts are comprised of Platycodon root as do other crude drugs, and the root may be partly responsible for the antiinflammatory effects induced.

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The effect of Saiko-ka-ryukotsu-borei-to (SRBT) on the stress-induced enhancement of serum corticosterone in various stress models was investigated in mice. The serum corticosterone was elevated significantly by immobilized stress, forced-swim stress, electric-shock stress, psychological stress and conditioned-fear stress, respectively. The concentration in the last two models was decreased in a dose-dependent manner by pre-administration of SRBT (10, 60, 100, 600 and 1000 mg/kg, p.o.). Therefore, this action seems to be effective in stress involving emotional factors but ineffective in physical stress models. These results indicate that the anti-stress effect of SRBT is dependent strongly on the degree of psychological change compared with physical change in mice.

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The promotional effect on rectal absorption of sodium ampicillin (ABPC) by the glycyrrhetinic acid derivative disodium glycyrrhetinic acid 3β-O-monohemiphthalate (GA MHPh) was studied in rats and compared with those of sodium caprate (CAP) and sodium glycocholate (GLY). Duration of the promotive effect of GA MHPh was also studied. Rectal absorption of ABPC was significantly enhanced by addition of GA MHPh at an optimum concentration of about 1.5%. The plasma maximum concentration of ABPC was 78.71 μg/ml 10 min after its rectal administration at 100 mg/kg with 1.5% GA MHPh. The bioavailability of ABPC with and without 1.5% GA MHPh was 82.12% and 3.92%, respectively. Thus, absorption of ABPC in the presence of 1.5% GA MHPh was about 21 times that of ABPC alone. GA MHPh was more effective as an absorption promoter than either CAP or GLY. Its promoting action on the mucosal membrane was apparent immediately, reached a maximum at 5 min and remained for at least 20 min after rectal administration of the solution. It is therefore suggested that GA MHPh is a very useful promoter absorption of the hydrophilic drug ABPC when administered rectally.

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The in vitro percutaneous permeation of diclofenac from various vehicles was examined using rat abdominal skin as a model membrane. The oleaginous vehicles used in this study consisted of three components : i.e. fatty acid, fatty acid ester and nonpolar oil. The solubilities of sodium diclofenac in formulated vehicles were above 0.2 M. The effect of each oleaginous component in the vehicle on the permeation of diclofenac across the skin was in the following order : oleic aicd>isostearic acid, diisopropyl adipate=diethyl sebacate>Panasate 875 and squalane>liquid paraffin. To clarify the reason for the differences in permeation among the fatty acid esters, the release of diclofenac through either porous or lipoidal membranes from these vehicles in vitro and the solubility of sodium diclofenac in the vehicles were studied. However, no relationship was observed between the release rate or solubility and skin permeability. The skin permeation of diclofenac increased following pretreatment with diisopropyl adipate or diethyl sebacate, but not with middle chain triglyceride (Panasate 875). These results suggested that the main reason may be the enhancement effect of fatty acid esters. Emulsions and creams containing 3% sodium diclofenac were prepared using the above oleaginous vehicles. A large flux and short lag time were observed in these preparations compared with an aqueous suspension of sodium diclofenac. The incorporation of urea significantly enhanced the permeation of diclofenac from these preparations. These results suggest that the emulsion and cream prepared in this study are useful for development for practical purposes.

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To ascertain the multiplicity of the cytochrome P-450 (P-450) species participating in the individual metabolic conversion of theophylline by 8-hydroxylation, 3-demethylation and 1-demethylation in mice, kinetics were studied under various conditions using untreated and inducer-treated mouse hepatic microsomes. Eadie-Hofstee plots of 1-demethylation in untreated microsomes exhibited a straight line, whereas those of 8-hydroxylation and 3-demethylation were curved lines. The biphasic kinetics indicated the contribution of two P-450 populations to the respective metabolic pathways ; one characterized by high affinity and low capacity, the other by low affinity and high capacity. The high affinity population was efficiently induced by β-naphthoflavone (β-NF), and was highly susceptible to inhibition by a specific CYP1A inhibitor. The low affinity population was sensitive to induction by phenobarbital (PB), and was markedly inhibited by preferential inhibitors for PB-inducible P-450 species. The present results indicated that two P-450 populations contributed to the theophylline metabolism in mouse hepatic microsomes, and that the high and low affinity populations corresponded, respectively, to CYP1A, and a PB-inducible P-450 species having a much higher capacity than CYP1A.

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Complement activation is important for removing foreign substances by the mononuclear phagocyte system in vivo. The interaction between liposomes and complement components is considered to affect the clearance of liposomes from the circulation. It has been previously demonstrated in our laboratory that multilamellar vesicles (MLV) with surfaces modified by cetylmannoside (Man) were eliminated from the circulation rapidly and showed an approximately 2-fold higher hepatic accumulation compared with control MLV (PC-MLV) (Yamashita et al., Int. J. Pharmaceut., 70, 225, 1991). In this study, we investigated the effect of Man-modification on complement system activation. As far as elimination from the blood is concerned, the initial values of blood liposome concentration were decreased and liposomes were removed from the circulation rapidly in accordance with the extent of the Man content into their membranes. The Man-modification also affected the organ distribution of injected liposomes and their stability in rat serum. Except for MLV containing 50 mol% Man, it was observed that the hepatic uptake of liposomes was enhanced according to the increasing Man content, whereas splenic uptake was decreased and the splenic clearance was comparable. The stability of liposomes in rat serum decreased with increasing Man content. Liposomal instability in rat serum was significantly reduced by preheating the serum at 56°C for 30 min, the treatment with anti-C3 antiserum and with EDTA but not abolished in serum treated with EGTA/MgCl₂. Thus, it is considered that the activation of the complement system through the alternative pathway is facilitated as a result of increasing the Man content in the liposomes. Furthermore, the contribution of the alternative complement pathway activation affected only the affinity of Man-modified MLV for the liver.

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The serum protein binding curve of chlorpromazine (CPZ) on the Scatchard plot in vitro was a two-phase downward curve. However, after i.v. administration of CPZ the curve was altered to an upward curve. To clarify the reasons for these in vivo changes, the influence of chlorpromazine S-oxide (CPZSO), chlorpromazine N-oxide (CPZNO), desmethylchlorpromazine (nor1-CPZ), chlorpromazine sulfone (sul-CPZ) and 7-hydroxychlorpromazine (7-OH-CPZ) on CPZ protein binding were studied in vitro. The results indicated that the characteristics of the CPZ protein binding are altered by the combination of CPZSO or CPZNO or by either of them. Since it was very difficult to explain the relationship between serum total and free concentrations of CPZ in vivo using mass-balance equations like Hill's equation or a competitive inhibition equation on the multiple binding sites for drug, the correlation between the ratio of total concentration of CPZ metabolites and CPZ (CPZSO/CPZ or CPZNO/CPZ) and the free fraction of CPZ was examined using the in vitro and in vivo data. The correlation between the ratio of CPZSO/CPZ and the free fraction of CPZ was good in both the in vivo and the in vitro studies. There was no statistically significant difference between the population regression coefficient of the two studies. The values of the slope and the intercept became almost the same as those obtained using the in vivo studies when combined with CPZNO. The results indicated that the influence of CPZNO on the correlation between the ratio of CPZSO/CPZ and the free fraction of CPZ is very small, and this correlation might be able to be used for the analysis of pharmacokinetic studies of CPZ in vivo.

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The time courses of the total concentration of chlorpromazine (CPZ) and its S-oxide (CPZSO) in serum after i.v. administration of CPZ are described by a two compartment model and a simple metabolism model, respectively. The time course of the free CPZ concentration in serum is predicted by the correlation between the ratio of CPZSO/CPZ and the free fraction of CPZ in serum which was established in the previous study. The time course of CPZ concentration in cerebrospinal fluid (CSF) is described by a basic physiological model in which the CSF compartment is connected with the serum compartment (free drug) by the apparent diffusion clearance. Equilibrium dialysis of the striatum homogenate was carried out to clarify the CPZ disposition in the striatum. Since the binding curves of CPZ in the striatum on the Langmuir plot and the Scatchard plot were a sigmoidal and an upward curve, the time course of the drug's concentration was analyzed on a simple kinetic model designed in conformity with a blood-flow limited model. The time course was described by a simple kinetic model for up to 8 h after CPZ administration. These pharmacokinetic models for CSF and the striatum will be used to analyze the relationship between the pharmacokinetics and pharmacodynamics of CPZ.

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The effects of bacteriohopane-32-ol (Monol) on the stability of various kinds of liposomal membranes composed of phosphatidylcholine (PC) were examined by measuring the release of calcein entrapped in the liposomes ; these effects were then compared with those of cholesterol (Chol). Incorporation of Chol enhanced the stability of both small unilamellar vesicles (SUV) and reverse-phase evaporation vesicles (REV) by increasing their content. However, the incorporation of Monol into PC membranes produced different effects on liposome stability due to the difference in vesicle size, fatty acyl chains of phospholipids and Monol contents. At a low Monol content of dipalmitoylphosphatidylcholine (DPPC) in SUV, incorporation of Monol exerted relatively major stabilizing effect at temperatures above T_m compared with those below T_m. Furthermore, the incorporation of Monol into REV of DPPC at about 12 mol% lowered the stability of REV below T_m of DPPC while at other concentrations this was increased. This peculiar effect was not observed in SUV membranes of DPPC and membranes composed of dimyristoylphosphatidylcholine (DMPC). These results are discussed with reference to the interaction between Monol and phospholipid molecules.

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The in vitro effect of asarone, the nematocidal principle of the rhizome of Acorus calamus, on second-stage larvae of Toxocara canis is composed of two independent actions : one is a fast acting inhibition of the larval mobility and the other is a slow acting larvicidal action. Mobility of the larvae was rapidly inhibited when they were incubated with asarone. Dye exclusion assay revealed that larvae were alive at this stage, and their mobility was restored after the first inhibition, suggesting that this effect was temporary and reversible. However, when the mobility decreased again during prolonged incubation, the cellular viability of larvae disappeared, showing that they were killed by the compound. The above two-stage effect of asarone was almost identical in two geometrical isomers ((E)- and (Z)- asarone). Di- and tri-methoxypropenyl or propylbenzenes carrying two methoxy groups at a vicinal position on a benzene ring showed, more or less, a two-stage effect of this type. These two actions were suggested to be separable by an appropriate modification of the structure.

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To clarify the mechanism (s) responsible for nausea and vomiting induced by L-dopa administration, dopamine levels in the plasma and lymph of rats were investigated in the 60-min period following an intravenous bolus of L-dopa (2.5 mg/kg body weight). The dopamine level in plasma from the femoral artery was the highest at 5 min immediately after the L-dopa injection, and was eliminated thereafter. Showing the same tendency as the plasma, the lymph from the thoracic duct showed a maximal increase of dopamine at 0 to 10 min, and a rapid decrease later. In contrast, the dopamine level in the lymph from the cervical lymph trunk increased, peaked at 10 to 20 min, and fell gradually thereafter. The dopamine level in the cervical lymph was higher than that in the thoracic lymph. When these data were kinetically analyzed, the cervical lymph had a larger area under the dopamine concentration-time curve than the thoracic lymph. Both the cervical lymph and the thoracic lymph had longer values of dopamine mean residence time than the plasma. Our findings revealed that when L-dopa was administered with an intravenous bolus, dopamine was higher and remained longer in the cervical lymph than in the rest of the body.

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Polymyxin acylase from Pseudomonas sp. M-6-3 can deacylate not only polymyxin group antibiotics, but also the long-chain fatty acyl group of proteins and peptides. We found the in vitro antitumor activity of polymyxin acylase against murine and human tumor cells, especially KB cells. The mechanism of the antitumor activity remains equivocal, but we speculate that it may result from the affinity of polymyxin acylase for long-chain fatty acyl proteins in human carcinoma cells.

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Phenylbutazone showed significant inhibition against the metabolic reduction of acetohexamide catalyzed by carbonyl reductase purified from rabbit kidney. Thus, the inhibitory effect of phenylbutazone was kinetically examined. Phenylbutazone was a competitive inhibitor for the enzyme with respect to NADPH, whereas it noncompetitively inhibited the enzyme activity with respect to acetohexamide. A fluorescence study revealed that phenylbutazone decreases the binding of NADPH to the free enzyme (apoenzyme). These results suggest that phenylbutazone causes the inhibition of carbonyl reductase by competing with NADPH in its coenzyme-binding domain.

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The present study investigated whether or not Sho-saiko-to (crude powder extract, TJ-9) can suppress nitric oxide (NO) generation by endotoxin-activated J774A.1 cells in order to study the preventive mechanism of Sho-saiko-to against endotoxemia. In this experiment, we estimated the NO^-₂ in the murine macrophage cell line J774A.1 using the Griess method. Our results clearly demonstrated that J774A.1 cells stimulated with endotoxin (0.01-10 μg/ml) can effectively produce NO, and the production was dependent on the dose of endotoxin. On the other hand, we investigated the suppressive effect of TJ-9 (10-100 μg/ml) on NO generation by endotoxin (0.1μg/ml)-activated J774A.1 cells. The NO level when the cells were incubated with endotoxin and TJ-9 (10-20 μg/ml) was slightly lower than that in cells treated with endotoxin alone. In contrast, treatment with TJ-9 (50-100 μg/ml) significantly inhibited endotoxin-activated NO generation in J774A.1 cells, whereas the treatment with TJ-9 (10-100 μg/ml) alone was ineffective in inducing NO formation and in inhibiting cell viability in the J774A.1 cells. These findings suggest that a Kampo presciption of Sho-saiko-to shows a suppressive effect on NO generation in macrophages stimulated with endotoxin, and that it may be useful in improving endotoxin-shock symptoms.

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The intraperitoneal administration of the methanol extract of Polygonati Rhizoma (OM) to Wistar fatty rats caused a significant decrease in the blood glucose level 4 h after its administration at 800 mg/kg (p<0.01). The hepatic content of facilitative glucose transporter isoform 2, liver type glucose transporter (GLUT2) mRNA content from rat liver significantly decreased in the intraperitoneally OM-treated rats compared to that in the controls (p<0.01). These results suggest that the hypoglycemic effect of OM is presumably due, at least in part, to the reduction of GLUT2 mRNA expression. Because of this unique therapeutic mechanism, OM could be a new category of therapeutic agent for non-insulin-dependent diabetes mellitus.

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In this study, disturbance of immune response as a pathogenic mechanism for human volume hypertension was investigated and compared to nephritis in its correlation with the metals such as zinc, iron and aluminum as environmental factors. Urinary γ-GTP excretions in patients with nephritis or hypertension were higher than in healthy people, whereas the plasma renin activity in these patients were lower on the average than in healthy individuals. Hypertensive patients participating in this study were diagnosed as the volume hypertension type from our clinical and other results. The serum IgM and IgA levels in renal patients showed a tendency to be lower than in the healthy people used as control. Urinary IgA excretion in hypertensive patients was increased in association with increasing excretions of aluminum and/or iron into urine. The values of regression coefficients in the urine samples for aluminum and iron vs. IgA, respectively, were very high at r=0.900 (n=9, p<0.05) and 0.736 (n=9, p<0.05). These correlations were shown to be very useful indicators in diagnosing volume hypertension. Moreover, hypertensive patients in this study were demonstrated to have a high regression coefficient (r=-0.702, n=7 ; p<0.05) for calcium vs. renin in the serum. In the hypertension, augmentation of serum calcium significantly decreased plasma renin activity.

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The entire amino acid sequence of Ac₁-Proteinase from Deinagkistrodon acutus venom was determined by using lysyl endopeptidase, metalloendopeptidase, trypsin and V8 protease. The hemorrhagic toxin had a typical zinc-chelating sequence His-Glu-X-X-His found in thermolysin.

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In a screening series of bioactive components in edible and medicinal plants, we found that Calumbae Radix (Colombo root, 1% powdered feed, for 5 d) and its component, columbin (20-40 mg/kg/d, for 5 d, orally), shortened the sleeping time induced by a urethane and α-chloralose mixture and prolonged the sleeping time induced by hexobarbital in mice.

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Water-insoluble typical intercalators, acridine and pyrene, were converted to water-soluble species, Dex-Pyr and Dex-Acr, respectively, by coupling with dextran polymer. By the use of these dextran-coupled intercalators, interactions of acridine- and pyrene-skeletones with various nucleotides in aqueous solutions were analyzed.

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A series of peptaibols, trichosporin (TS) -Bs, induced voltage-activated conductance in lipid bilayer membranes and catecholamine secretion from bovine adrenal chromaffin cells. The order of activities is the same as that of the lipophilicity rank of TS-Bs derived from the reversed-phase HPLC capacity factors.

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