Properties of the mercury and selenium complex (Hg-Se complex) produced in plasma after the simultaneous injection of mercuric chloride and sodium selenite were investigated by means of pronase digestion and isoelectric focusing. When the gel chromatographic fraction containing the Hg-Se complex was digested with Pronase E and chromatographed on a Sephadex G-200 column, the resulting derived proteins contained no mercury and very low percentage of selenium. Most of mercury and selenium in the digest of the Hg-Se complex remained at the top of column, but the content of proteins (derived proteins) found at the same site of column was only trace amount. In contrast, the derived proteins obtained from proteins containing mercury or selenium in serum by the same digestion contained mercury and high percentagae of selenium respectively. These results indicated that the Hg-Se complex in plasma did not contain protein as a component. When the isoelectric focusing of the serum containing the Hg-Se complex was carried out, the peak containing both mercury and selenium was present in the position of pH 2 and pH 4. The former peak was independent of the serum proteins, the latter peak overlapped the disribution of that. Similar behavior of mercury and selenium was observed in the isoelectric focusing of the mercuric selenide colloid dispersed in the solution containing bovine serum albumin. The properties of the Hg-Se comoplex observed in these studies supported the hypothesis that it is a mercuric selenide colloid.

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It was clearly shown that colestipol hydrochloride inhibits lymphatic absorption of not only endogenous but also exogenous cholesterol and triglyceride in thoracic duct cannulated rats. Inhibition of cholesterol absorption by colestipol hydrochloride was effectively blocked by administration of cholic acid. It was also suggested that colestipol hydrochloride inactivates bile acids which are essential for cholesterol absorption in the intestinal tract. Administration of colestipol hydrochloride to fasted rats decreased bile flow and biliary secretion of cholesterol and bile acids and increased fecal excretion of bile acids bound to colestipol hydrochloride. These results show that colestipol hydrochloride binds bile acids in the intestinal tract and interferes with bile acids reabsorption.

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Kinetics of the hydrolysis of amidinophenyl esters catalyzed by urokinase, kallikrein, plasmin and trypsins from various origins were studied. p-Amidinophenyl esters in which the arrangement of the specific group for bovine trypsin (charged amidinium) is reversed to that of normal substrates were characterized as specific substrates for the enzyme and named"inverse substrates" (K. Tanizawa, Y. Kasaba and Y. Kanaoka, J. Am. Chem. Soc., 99, 4485-4488). Behavior of these enzymes toward amidinophenyl esters was demonstrated to be very similar to that of bovine trypsin. These observations account for the structural similarity of their active sites. p-Amidinophenyl esters were found to be hydrolyzed also by clostripain which is known as a thiol protease with trypsin-like specificity. This is the first example of the "inverse substrates" for a thiol enzyme.

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The possibility of using mice in place of rats on the anti-inflammatory screening tests was investigated. The diversities of the responses between mice and rats on screening tests, that is, carrageenin, formalin-induced edema in the hind paw, adjuvant arthritis and cotton peller granuloma were observed with p.o. administration of prednisolone, 0.5 mg/kg and 5.0 mg/kg, and indomethacin, 0.5 mg/kg and 5.0 mg/kg. On the effects of these drugs on screening tests, a similar type of phenomena between rats and mice was found. Then the dose-response relationships of aspirin, flufenamic acid, phenylbutazone, cyproheptadine, prednisolone and indomethacin were examined in four kinds of screening tests mentioned above. Prednisolone, indomethacin, phenylbutazone and aspirin inhibited the carrageenin, formalin-induced edema, cotton pellet granuloma and adjuvant arthritis apparently, but flufenamic acid showed no inhibition on the carrageenin and formalininduced edema. Cyproheptadine inhibited the formalin and serotonin-induced edema. We have found that mice, instead of rats, can be used for the anti-inflammatory screening methods.

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A rapid and sensitive gas-liquid chromatographic procedure was developed for the measurements of trimethadione (TMO) and its metabolite, 5, 5-dimethyl-2, 4-oxazolidinedione (DMO) in plasma. TMO and DMO were extracted from plasma by a micro-extraction method and chromatographed on a 10% Carbowax 6000 column using paramethadione as an internal standard. Recoveries of TMO and DMO ranged from 96 to 104% using a 0.1 ml of plasma. The method is capable of measuring at least 5μg of TMO and DMO per mililiter. After an oral administration of TMO at a dose of 100 mg/kg, mean plasm levels of TMO and DMO in 6 rats reached a peak of 98μg/ml at 0.6 h and 163μg/ml at 8 h and declined with a half-life of 2.2 h and 39.4 h, respectively. After the intravenous administration of TMO at a dose of 100 mg/kg, half-lives of TMO and DMO were 2.5 h and 39.1 h, respectively. Pretreatment of rats with carbon tetrachloride, d-galactosamine and α-naphthyl isothiocyanate, prolonged T_1/2 and T_max of TMO, reduced the K_m value and increased the AUC. These results suggest that plasma levels of TMO and DMO might be useful as an index of drug metabolizing capacity in animal and man.

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Pharmacokinetic analysis of plasma concentration of acetyl procainamide ethobromide (APAEB) after single injection of 1.5, 8.8 and 20μmol/300 g rat of APAEB showed that its elimination from plasma followed saturation kinetics. The existence of saturable step in transport of APAEB from blood to bile was also evident from the data of the biliary excretion at three dose levels. The data of the biliary excretion were well fitted to two compartment model with two saturable processes at the hepatic uptake and the biliary excretion. The Michaelis-Menten parameters were calculated according to that model. The model was also applicable to the data of biliary excretion obtained by constant infusion of APAEB. Simulation of liver and plasma content gave a good agreement to the experimental data. All these results indicate that the pharmacokinetics of APAEB is satisfactorily represented by the proposed model.

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Distribution of isoniazid and its metabolites was observed in the liver, kidney, lung and plasma after the subcutaneous administration of isoniazid to rats. The tissue levels of isoniazid, acetylisoniazid, acetylhydrazine, 1, 2-diacetylhydrazine and hydrazine were determined by mass fragmentography using a gas chromatograph-mass spectrometer equipped with a multiple ion detecter-peak matcher. Using the compounds labeled with a stable isotope as an internal standard, namely the isotope dilution method, made it possible to estimate trace amounts of these metabolites in the tissues. The amount of hydrazine was much less than the other hydrazines, but the metabolite which is well known as a mutagen, could be successfully detected in the tissues and plasma. The greater part of free hydrazine is formed through a direct hydrolysis of isoniazid. The isoniazid-hydrolyzing activity was found to be significantly higher in the liver homogenate. This suggested that hydrazine formation is mainly caused by hepatic hydrolysis.

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Bilaterally nephrectomized rats were used to investigate the disposition of creatinine when renal function is acutely decreased. The percentage of radioactivity recovered in the expired air in 11 hours following intravenous administration of [carbonyl-¹⁴C] creatinine immediately and 24 hours after nephrectomy was 0.54 and 3.24% respectively, and these values were significantly different (p<0.05). As it was considered that expiratory excretion is one of the major elimination routes in nephrectomized rats, mechanism of expiratory excretion was investigated by incubating [carbonyl-¹⁴C] creatinine with the intestinal contents of the rats chronically loaded with creatinine. [carbonyl-¹⁴C] Creatinine was completely metabolized in that intestinal contents, but ¹⁴CO₂ was not produced. When the mixture of the metabolites was administered orally or inravenously to chronically creatinine loaded rats, however, about 50% of the total radioactivity was excreted into the expired air in 5 hours. Furthermore, biliary excretion of the radioactivity following intravenous administration of [carbonyl-¹⁴C] creatinine in the nephrectomized rats was much greater than that in normal rats. These results indicate that expiratory excretion following intravenous administration of [carbonyl-¹⁴C] creatinine to nephrectomized rats would arise following intestinal absorption of the creatinine metabolites, which seem to be produced by intestinal microflora after biliary excretion of creatinine. The change of disposition for urea by bilaterally nephrectomy was also studied to compare with that for creatinine.

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Interactions of Δ⁸-tetrahydrocannabinol (Δ⁸-THC) and its metabolites, 11-hydroxy-Δ⁸-THC, 11-oxo-Δ⁸-THC and Δ⁸-THC-11-oic acid, with hepatic microsomal drug metabolizing enzyme systems were studied in vitro and in vivo using mice. All the cannabinoids used were shown to produce a type I spectral change of cytochrome P-450 in hepatic microsomes. The apparent binding affinities (K_s) for Δ⁸-THC, 11-hydroxy-Δ⁸-THC, 11-oxo-Δ⁸-THC and Δ⁸-THC-11-oic acid were 5.2, 169.6, 33.2 and 148.8μM, respectively. Δ⁸-THC, 11-oxo-Δ⁸-THC and Δ⁸-THC-11-oic acid (100μM) caused a significant stimulation of NADPH oxidation in vitro with microsomes. In addition, Δ⁸-THC (20, 40 and 80μM) markedly inhibited p-nitroanisole O-demethylase and aniline hydroxylase in microsomes. 11-Hydroxy-Δ⁸-THC and 11-oxo-Δ⁸-THC also showed the inhibitory effect, but to lesser extents. Furthermore, single pretreatments with Δ⁸-THC and 11-oxo-Δ⁸-THC (30 mg/kg, i.v.) significantly decreased activities of p-nitroanisole O-demethylase and aniline hydroxylase in hepatic microsomes, accompanying a decrease of cytochrome P-450 content in case of Δ⁸-THC. On the other hand, all these pretreatments except for that with Δ⁸-THC-11-oic acid showed a stimulatory effect on p-nitrophenol glucuronidation with hepatic microsomes, in contrast to an inhibitory effect in vitro.

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We made a study on choleretic property and mechanism of action of iridoid compounds as well as dehydrocholate (DHC), cholate (CA), and salicylate (SA), examining their effects on factors such as bile flow, bile acids, electrolytes (Na⁺, K⁺, Cl^-, and HCO₃^-), and their metabolites. Each sample showed a characteristic property, respectively. Genipin and patrinoside decreased biliary concentrations of bile acids, Na⁺, Cl^-, and HCO₃^-, corresponding to their rapid choleretic actions which were due to bile acidsindependent fraction. The choleretic action of DHC is approximately twice as potent as that of CA. Their actions were due to bile acids-dependent fraction. CA gave a marked increase in Na⁺ concentration but DHC did not. And both compounds gave a marked diminution in Cl^- concentration and weakly decreased HCO₃^- concentration. SA showed a weak and durable choleretic action and also gave a marked increase in HCO₃^- concentration. The main metabolite detected from the bile given genipin was genipin-1-O-glucuronic acid (GGA). The periodical pattern of GGA level in bile was in agreement with that of genipininduced choleretic action, and quantitatively cation-anion gap produced was nearly compensated by biliary concentration of GGA. From our various results, the choleretic mechanism of iridoid compounds is considered to be as follows : The hemiacetal moiety of them undergoes conjugation in the liver to give glucuronide. Glucuronide thus formed is secreted into the biliary tree being coupled mainly with Na⁺ and water is passively excreted.

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Magnetic guidance of magnetic albumin microsphere for site specific delivery was investigated in mice and rats. After intravenous injection in mice, magnetic microspheres with 1 and 3μm diameter size were localized and retained in the target-site (lung) by application of two permanent magnets to the lungs. Injection into the renal artery in rats also indicated that the 1-μm microspheres were concentrated in the kidney by a magnetic field. When the magnets were not applied, however, the microspheres following intravascular injection were concentrated mainly in the liver, regardless of the route of administration. Such preferential localization by magnetic means suggested that magnetic albumin microspheres could become effective drug carriers with site specificity for the delivery of chemotherapeutic agents in cancer therapy.

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To further examine the mechanism of antihypertensive action, effects of a single (50 mg/kg) and repeated oral administration (50 mg/kg per day, for 14 days) of oxprenolol on mean arterial pressure (MAP) and heart rate (HR) were studied in spontaneously hypertensive (SHR) rats at rest and during handling stress. MAP was measured through a indwelling aortic cannula and HR was determined via chronically implanted electrodes. A single oral dose of oxprenolol produced a gradual fall in resting MAP. Although repeated dose of oxprenolol did not alter the developmental course of hypertension in SHR rats, a prompt and significant fall in MAP at rest was observed after the dose on the 14th day of the experiment. A single and repeated dose of oxprenolol attenuated the increase in MAP during handling stress, but these effects were less apparent when compared to the fall in resting MAP. Significant reductions in stress-induced tachycardia were observed both after a single and repeated dose, whereas resting HR tended to increase. These results indicate that some of the postulated antihypertensive mechanisms such as central inhibition of sympathetic outflow, peripheral inhibition of sympathetic nerve functions and suppression of cardiac output are not directly related to a fall in MAP observed in SHR rats after oxprenolol treatment. Time courses of the hypotensive effect of a single and repeated doses suggest that the accumulation of oxprenolol in active sites which appear to be located in deep compartments is required to develop hypotensive effect.

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The effects of phenobarbital on the plasma concentration of phenytoin and mortality of the phenytoin-treated mice were mainly studied. The plasma concentration of phenytoin estimated in the live mice after electroshock was significantly higher than that in the dead mice, suggesting that biological activity of phenytoin is dependent on its plasma concentration where individual difference seems to be large. Though the pretreatment of mice with phenobarbital lowered the plasma concentration of phenytoin, all the mice were alive after electroshock with further treatment with phenobarbital and phenytoin. These results suggest that combination of phenobarbital with phenytoin for the treatment of epileptic patients may be feasible.

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The biological fates of ethyl paraben were investigated after the simultaneous administration with salicylic acid. Those fates were compared with those of ethyl paraben alone obtained in the previous experiments. The biological fates of ethyl paraben after the simultaneous administration with salicylic acid were different from those of ethyl paraben alone as reported in the previous reports : The excretion of unconjugated p-hydroxybenzoic acid, which is a hydrolyzed product of ethyl paraben, increased and those of p-hydroxyhippuric acid, glycine conjugate of p-hydroxybenzoic acid, and p-hydroxybenzoyl glucuronide, its ester type glucuronide, decreased. The blood concentration patterns were considerably different from those of ethyl paraben alone, especially the elimination of every metabolite was delayed. Pharmacokinetic analyses on the data of blood concentration were carried out and the results also show the interaction of salicylic acid on the biological fate of ethyl paraben.

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