The fate of salicyluric acid (SU) after oral administration was examined in rabbits. Salicylic acid (SA) and unchanged SU were detected in the blood after oral administration of SU. However, SU only was found after intravenous administration of SU, suggesting that presystemic deconjugation of glycine was involved. After treatment of rabbits with kanamycin sulfate, complete inhibition of the formation of SA after oral administration of SU was demonstrated, indicating that the intestinal microflora was responsible for the bio-transformation. Furthermore, in vitro incubation of SU with contents of gut showed that the major source of the hydrolysis was the hind gut. Based on the findings described above, the time course data for blood concentration of SU and SA after oral administration of SU have been subjected to curve-fitting by a nonlinear least squares program. The SU and SA values obtained with the simplified model containing only first-order kinetic process which is proposed by the present authors were found to agree with those obtained from experiments.

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The mechanism of the enhancing effect of methyl viologen (MV) and flavin-adenine dinucleotide (FAD) on sulfoxide reduction which is mediated by a combination of aldehyde oxidase (AO) from guinea pig liver and one-electron reducing flavoenzymes, such as milk xanthine oxidase (XO), was examined. The activity of anaerobic reduction of diphenyl sulfoxide (DPSO) to diphenyl sulfide (DPS) was less than 1 nmol/min/mg protein of AO preparation in a system consisting of hypoxanthine, XO and AO. However, the sulfoxide reduction by this system was enhanced about 6-and 100-fold by the additions of FAD and MV, respectively. In the system containing MV or FAD, other one-electron reducing flavoenzymes such as nicotinamide adenine dinucleotide (reduced form) (NADH) dehydrogenase, lipoamide dehydrogenase and glutathione reductase with an appropriate electron donor, could replace XO. The ability of supplemented flavoenzymes to facilitate DPSO reduction correlated with their abilities to reduce MV and FAD. When AO was omitted from the combined system, no sulfoxide reduction was observed. Stoichiometric study revealed that MV semiquinone and FADH₂ were oxidized at ratios of 2 and 1 mol, respectively, permol of DPS formed. These results indicate that either MV or FAD serves as an electron carrier from the supplemented flavoenzymes to AO, a terminal reductase of sulfoxide.

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The effects of idebenone (CV-2619) and its metabolites on respiratory activity and lipid peroxidation in isolated brain mitochondria from rats and dogs were studied. CV-2619 was easily reduced by canine brain mitochondria in the presence of respiratory substrates. Reduced CV-2619 (2H-CV-2619) was rapidly oxidized through the cytochrome b chain, indicating that the compound functioned simply as an electron carrier of mitochondrial respiratory system. Both nicotinamide adenine dinucleotide (NADH)- and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent lipid peroxidations were examined in canine brain mitochondria in the presence of adenosine diphosphate (ADP) and Fe³⁺. NADH-cytochrome c reductase activity was sensitive to NADPH-dependent lipid peroxidation. CV-2619 (10^-5M) strongly inhibited both types of the lipid peroxidation reactions and protected the resultant inactivation of the NADH-cytochrome c reductase activity. Activities of succinate oxidase in rat and canine brain mitochondria were virtually unaffected by CV-2619 and its metabolites (10^-5-10^-6M). On the other hand, CV-2619 markedly suppressed the state 3 respiration in glutamate oxidation in a dose dependent manner without any effect on the state 4 respiration and the ADP/O ratio in intact rat brain mitochondria. The inhibitory effect of CV-2619 was also observed in NADH-cytochrome c reductase, but not in NADH-2, 6-dichlorophenolindophenol (DCIP) and NADH-ubiquinone reductases in canine brain mitochondria. These facts and results of inhibitor analysis suggest that the action site of CV-2619 is NADH-linked complex I in the mitochondrial respiratory chain and is different from that of inhibitors of oxidative phosphorylation such as rotenone, oligomycin and 2, 4-dinitrophenol. Finally, the above findings suggest that CV-2619 acts as an electron carrier in respiratory chains and functions as an antioxidant against membrane damage caused by lipid peroxidation in brain mitochondria. It appears likely that the inhibition of oxygen consumption caused by CV-2619 is related to the effect on non-respiratory systems such as lipid peroxidation which also consumes oxygen.

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The effect of 4-(4'-chlorobenzyloxy)benzoic acid (MII), a metabolite of a new hypolipidemic agent 4-(4'-chlorobenzyloxy)benzyl nicotinate (KCD-232), on lipid synthesis and cell growth was studied in human skin fibroblasts (HSF), mouse skin fibroblasts (MSF) and other rodent-derived cell. MII inhibited sterol synthesis in both HSF and MSF in a dose dependent manner. The drug also suppressed fatty acid synthesis, weakly in HSF and markedly in MSF. The growth of MSF was inhibited by MII in a dose dependent manner, whereas that of HSF was not inhibited at all by MII up to 1 mM. The MII-mediated inhibition of MSF growth was not prevented by the addition to the medium of intermediates of the cholesterol synthetic pathway, i.e., mevalonate, squalene or lanosterol. Furthermore, the growth of several cell lines derived from rodents (L, CHL and AH109A cells) was suppressed by MII. These results suggested a possibility that the different effects of MII on the growth of HSF and MSF might be due partly to differences in the effect of MII on fatty acid synthesis between human and rodent cells.

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A sensitive and specific radioimmunoassay for antiarrhythmic peptide (AAP) has been developed, utilizing 3-(4-hydroxy, 5-[¹²⁵I]iodophenyl)propionyl AAP and guinea pig anti-AAP serum. Synthetic AAP was used as a standard and the polyethylene glycol method was employed to separate free AAP from antibody-bound AAP. The minimum detectable dose of AAP was 0.2 pmol. In the assay system, immunoreactivity was shown not to be attributable to fragments derived from AAP, gelatin or collagen peptides, which sequences differ only by one amino acid from those of AAP. Immunoreactive (IR) AAP extracted from rat tissues and serum gave parallel dose-response curves to those of AAP. Thus, the AAP equivalents per g or ml of tissues measured in adult male rats were 203 pmol in heart, 166 pmol in kidney, 4 pmol in serum, and 2 to 6 pmol in the other tissues. IR-AAP was separated into two fractions by Sephadex G-25 chromatography. Fr. I was considered to have a larger molecular mass than AAP, while Fr. II appeared to have a molecular mass equal to AAP. Ratios of Fr. II in total IR-AAP were 50% in heart and 10% in kidney, while serum IR-AAP gave only Fr. II. The IR-AAP level in heart showed a positive correlation increase with age in rats.

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The application and usefulness of resealed erythrocyte as a cell carrier of dexamethasone were studied in rabbits. The erythrocytes were loaded with water soluble dexamethasone sodium phosphate by hypotonic dilution and dialysis methods. During in vitro incubation at 37°C, the level of dexamethasone held within the resealed erythrocytes declined according to first-order function. About one half of the dexamethasone-loaded cells, when returned to circulation, disappeared within the first 24 h. However, the survival of the remaining cells in circulation followed zero order function and declined for 7-8 d after administration. The apparent half-life of the remaining erythrocytes in circulation was 8.27 d by the hypotonic dilution method and 12.8 d by the dialysis method. This indicated that the encapsulated drug had a much longer half-life than when free drug (t_{1/2, β}=3.61±0.69 h) was administered intravenously. Dexamethasone phosphate entrapped within cells was slowly released from the loaded cells into plasma in in vivo circulation. When the encapsulated cells were administered to rabbits, the rabbits were protected from inflammation (the capillary permeability response) caused by histamine for approximately 2 to 5 d after administration. However by day 10 the protection was slight. This anti-inflammatory effect agreed reasonably well with the in vivo survival of loaded erythrocytes. Therefore, the results indicated that resealed erythrocytes act as a slow release system in vivo.

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Monodesmosides, saponin A, B and C, isolated from pericarps of Sapindus mukurossi(Enmei-hi) were shown to promote the rectal absorption of β-lactam antibiotics in rat, using an in situ loop method and an in vivo method. In the in situ loop method, monodesmosides were solubilized with a mixture of bisdesmosides, mukurozi-saponin X, Y₁ and Y₂, each of which was isolated from pericarps of Sapindus mukurossi. The promoting functions of the three monodesmosides for the rectal absorption of antibiotics were comparable and also suppressed by Ca²⁺ ion coexisting in the administered solution. Unlike the case of N-acylcollagenpeptides, which were also previously reported as absorption promoters, no influence of osmolarity of the administered solution on the absorption promoting action was observed. Absorption of β-lactam antibiotics from suppositories was enhanced with the aid of either of monodesmosides without solubilizing agent.

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Sulfotransferases (STs)active on androsterone (AD), cortisol (CS) and 4-nitrophenol (NP) were separated by diethylaminoethyl-cellulose chromatography from cytosolic fractions of female rat liver and were divided into five ST fractions (peaks I-V) with different activities toward three substrates. The precipitates obtained in the 68% of saturation of ammonium sulfate were passed through a Sephadex G-100 column and purified by agarose-hexane adenosine 3', 5'-bisphosphate affinity chromatography. AD-ST isoenzyme (peak I) was purified 85-fold, had low CS-ST activity, was devoid of NP-ST activity and appeared to correspond to hydroxysteroid ST 1.Peaks II and V appeared to consist mainly of hydroxysteroid ST and arvl ST, respectively.

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Forty kinds of bufotoxins and related compounds were tested for inhibition of Na⁺, K⁺-adenosine triphosphatase from guinea pig heart, and the structure-activity relationship has been discussed. The inhibitory activities of bufotoxins were dependent upon the dicarboxylic acid and amino acid components. The compounds having both the arginine and suberic acid moieties showed the higher inhibitory activities. The sulfates and glucuronides of cardiac steroids exhibited much less potency than the parent genins. The mode of inhibition was determined by means of the Dixon and Lineweaver-Burk plots.

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Effects of tolmetin, diclofenac Na, fenbufen, alclofenac, aminopyrine, mepirizole, thiaramide and aspirin as a positive control, which are widely used in this country as anti-inflammatory drugs, and on body and liver weights, triglyceride and cholesterol level and hepatic peroxisomal enzymes of normolipemic rats were examined. All of these drugs except diclofenac Na affected the enzyme composition of hepatic peroxisomes. Tolmetin (100 mg/kg) and fenbufen (50 mg/kg) increased carnitine acetyltransferase (CAT) and fatty acyl-coenzyme A oxidizing system (FAOS) activities, which participate in hepatic lipid metabolism. The latter also increased the activity of D-amino acid oxidase slightly. Alclofenac (300 mg/kg) increased the activites of FAOS, CAT and carnitine palmitoyltransferase which has been known as the rate-limiting enzyme of fatty acid oxidation in mitochondria, and decreased those of catalase and urate oxidase. Aminopyrine (300 mg/kg) increased the activities of catalase and FAOS. However, none of the above drugs influenced liver weight, serum or liver lipid levels. Mepirizole (300 mg/kg) increased the activities of FAOS and CAT about 2-fold, whereas the activites of catalase and urate oxidase and serum triglyceride level were decreased. Furthermore, these drugs showed no enhancement of the biosynthesis of peroxisome proliferation-associated polypeptide having a molecular weight of 80000. From these results, it is concluded that although these drugs have an influence on the enzyme composition of hepatic peroxisomes, they may not induce the peroxisome population in hepatic cells. Thus, the possibility of hepatocarinogenicity and lipid lowering effect through the peroxisome-proliferation would be excluded.

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An attempt was made to investigate the preventive effects of 20S-prosapogenin (20S-PG), a partial hydrolyzate of diol saponins isolated from Panax ginseng, on changes of peripheral tissue blood flow in diseased model animals with vagotonic-type autonomic imbalance [SART (specific alternation of rhythm in temperature)-stressed (repeated coldstressed) mice developed by us], and sympathicotonic-type stressed mice [restraint and water immersion-stressed (RWIS) mice], using the hydrogen clearance method. Decreases in gastric blood flow in both body and pyloric regions of the stomach and increases in dermal blood flow in both the shoulder and lumbar region in SART-stressed mice were prevented to a considerable extent by daily treatments with 20S-PG, 2.5-10mg/kg/d. Similarly, the marked decrease in gastric blood flow following RWIS loading was significantly blocked by 10 mg/kg/d of 20S-PG. 20S-PG had a minor preventive effect on decreases in hepatic and dermal blood flow in RWIS mice. The preventive effect of 20S-PG on changes in tissue blood flow, recognized in model animals with autonomic imbalance not only in the vagotonic state but also in the sympathicotonic state, is therefore regarded with considerable interest and may provide a clue to explain the pharmacological action of ginseng.

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