A mouse inflammation model of chronic proliferative type was newly produced in this study. Among a great many methods examined in mice, only an air-pouch method with Freund's complete adjuvant with 0.1% croton oil obviously induced the granuloma pouch with well-developed vascularization, which a little differed from that seen in rats. Both granuloma formation and fluid exudation in this adjuvant pouch increased until one or two weeks after treatment, respectively, followed by reducing gradually to become nearly normal at 12 weeks after. The pathohistological study exhibited liquefaction necrosis in the earlier stage, followed by coagulation necrosis accompanied with inflammatory cells infiltration, The nascently sprouted capillaries were well-developed and than the fibrous tissue formation proceeded along with the vascularization. The granuloma finally changed to scars and callous tissues. Intrapouch injection of hydrocortisone and indomethacin decreased the pouch fluid and the pouch wall weight. In conclusion, the adjuvant pouch in mice may be a feasible experimental model for the pharmacological study on the vascularizaiton in the acute and chronic inflammatory processes.

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Metabolites of -[(4-amino-2-methyl-5-pyrimidinyl) methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU) in rat urine were investigated. After intravenous administration of ¹⁴C-ACNU into rats, four major radioactive metabolites and two minor ones were detected in the urine by two-dimensional thin-layer chromatograhic analysis. The main metablite was identified to be an imidazolidinone compound, 1-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-5-hydroxy-2-imidazolidinone (M-D). One of the other major metabolites was identified to be a nitrosated compound of the main metabolite i.e., 1-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-5-hydroxy-3-nitroso-2-imidazolidinone (M-C). These were new types of metabolites which have not been reported in the metabolic study of other chloroethylnitrosourea derivatieves. Compared with authentic compounds, two metabolites were identified to be a denitrosated derivative of ACNU i.e., 1-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-3-(2-chloroethyl) urea (M-B), and a cyclized pyrimidopyrimidine compound which lacks the ethylene moiety of ACNU, i.e., 3, 4-dihydro-7-methylpyrimido [4, 5-d] pyrimidin-2-(1 H)-one (M-A). The two minor metabolites were supposed to be compounds derived from M-A. Discussions were made on mechanism of formation of these metabolites in vivo.

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In vitro decomposition of ACNU, 3-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride, in various conditions was studied with the use of the ¹⁴C-labeled compound. Metabolite A, 3, 4-dihydro-7-methylpyrimido [4, 5-d] pyrimidin-2(1 H)-one (an intramolecular cyclized product), was formed spontaneously in the phosphate buffer (pH 7.4) with simultaneous liberation of the alkylating moiety. With rat liver enzyme preparations, formation of three metabolites was observed. Those were metabolite B, 1-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-3-(2-chloroethyl) urea (a denitrosated product), metabolite C, 1-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-5-hydroxy-3-nitroso-2-imidazolidinone (a product via oxidative dechlorination), and metabolite D, 1-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-5-hydroxy-2-imidazolidinone (a denitrosated product of metabolite C). Formation of metabolite B was catalyzed with both cytosolic and microsomal enzymes, not inhibited with SKF-525A, and partly dependent on nicotinamide adenine dinucleotide phosphate (NADPH). These results suggest that at least two enzymatic steps would be involved in the formation of this product. Metabolites C and D were produced by the microsomal preparation, being dependent on O₂ and NADPH, inhibited by CO and SKF-525A, and enhanced by phenobarbital pretreatment. When metabolite C was incubated with cytosolic and microsomal preparations, more efficient formation of metabolite D with the former than the latter was observed. Form these results, it was assumed that oxidative dechlorination of ACNU to metabolite C would be catalyzed with the microsomal mixed function oxidase, and metabolite D would be produced via denitrosation process of metabolite C.

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The action of a polysaccharide fraction obtained from hot water extracts of Angelica acutiloba KITAGAWA, termed as Angelica immunostimulating polysaccharide (AIP) fraction, on murine lymphocytes participating in antibody responses was investigated. When AIP fraction was injected concomitantly into mice immunized with antigens, immunoglobulin M (IgM) and IgG antibody responses against sheep erythrocytes (SRBC) increased significantly, but IgM response against T-independent antigens such as trinitrophenylated lipopolysaccharide (TNP-LPS) and TNP-Ficoll did not augment. Murine B lymphocytes were polyclonally activated in vitro and in vivo by AIP fraction to differentiate into antibody-forming cells as functionally matured cells. The differentiation of B lymphocytes to an intermediate stage capable of responding to helper T lymphocytes was also stimulated by the administration of AIP fraction into CDF₁ and C3H/HeJ mice. A concomitant injection of AIP fraction with SRBC for carrier priming resulted in the increment of anti-TNP IgM antibody response in cultured reconstituted with unprimed B and SRBC-primed T lymphocytes, indicating that AIP fraction can stimulate T lymphocytes.

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Effects of 3 oximes, diacetylmonoxime (DAM), pyridine-2-aldoxime (PAM) and pyridine-2-aldoxime methochloride (2-PAM), on the normal electrical and mechanical activities and on the slow response action potentials were examined in the guinea pig ventricular muscles. DAM and long-term exposure to high concentrations of PAM produced decreases in contractile force, action potential duration and slow response action potentials, whereas 2-PAM and low concentrations of PAM tended to increase these parameters. Thus, these 3 oximes did not act uniformly on cardiac muscle. It was speculated that DAM and high concentrations of PAM may act as slow channel inhibitors, whereas 2-PAM and low concentrations of PAM may act as slow channel activators.

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Effects of buthiobate (S-butyl S'-p-tert-butylbenzyl N-3-pyridyldithiocarbonimidate), a fungicide which inhibits lanosterol 14α-demethylation catalyzed by a cytochrome P-450 (P-450_14DM) of yeast, on cytochrome P-450 of rat liver microsomes were studied. Buthiobate bound to limited forms of cytochrome P-450 in the microsomes with three different K_d values, 0.38, 1.56 and 13.6 μM. Buthiobate inhibited lanosterol 14α-demethylase activity of the microsomes with a 50%-inhibition concentration of 0.4 μM. This concentration was comparable to the lowest K_d of buthiobate for the microsomal cytochrome P-450 and also to the 50%-inhibition concentration of the inhibitor for lanosterol 14α-demethylation by yeast P-450_14DM. Buthiobate partially inhibited 7-ethoxycoumalin O-deethylase activity of the microsomes but inhibited neither benzphetamine N-demethylation nor p-nitroanisole O-demethylation. These observations suggest that cytochrome P-450s catalyzing drug oxiations are rather insensitive to buthiobate. These observations indicate that buthiobate is an unique inhibitor for hepatic microsomal cytochrome P-450 system, which inhibits cholesterol biosynthesis effectively but causes a little effect on drug oxidations.

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The interactions between chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA) and tolbutamide including its displacement from plasma protein binding sites were investigated pharmacokinetically. An increasing concentration of unbound tolbutamide was observed in the in vitro experiment, conducted by equilibrium dialysis method at 30°C after the addition of CDCA and UDCA to human serum albumin (HSA), bovine serum albumin (BSA) and rabbit plasma containing tolbutamide. Small changes in total plasama concentration of tolbutamide were noted after high dose (0.167 mg/kg/min) intravenous infusion of CDCA to rabbits receiving a constant intravenous infusion of tolbutamide, but, such an observation was not obtained with low dose (0.083 mg/kg/min) of CDCA or with either high or low dose of UDCA. These results seem to indicate the displacement of high doses of CDCA. The coadiministration of sulfadimethoxine which not only displaces tolbutamide from binding sites but also inhibits its metabolism was investigated. A different plasma pattern was obtained under the same intravenous infusion conditions, as compared with the plasma pattern resulting from tolbutamide-CDCA or UDCA combination.

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Metabolic studies of idebenone (CV-2619), a new cerebral metabolism improving agent, in the rat and dog by thin-layer chromatography, gas-liquid chromatography-mass spectrometry and fast atom bombardment-mass spectrometry led to characterization of the following metabolites : the parent compound, 6-(9-carboxynonyl)-2, 3-dimethoxy-5-methyl-1, 4-benzoquinone (QS-10), 6-(7-carboxyheptyl)-2, 3-dimethoxy-5-methyl-1, 4-benzoquinone (QS-8), 6-(5-carboxypentyl)-2, 3-dimethoxy-5-methyl-1, 4-benzoquinone (QS-6), 6-(3-carboxypropyl)-2, 3-dimethoxy-5-methyl-1, 4-benzoquinone (QS-4), 1- or 4-phenyl sulfate of the hydroquinone derivatives fo CV-2619 and QS-4, and 1- and 4-phenyl glucuronides of the hydroquinone derivative of QS-10. These results indicated that CV-2619 was metabolized by oxidation of the side chain followed by β-oxidation of form successively QS-10, QS-8, QS-6 and QS-4, and reduction of the quinone ring and subsequent conjugation yielding the sulfates and glucuronides of the hydroquinone derivatives of CV-2619, QS-10, QS-8, QS-6 and QS-4.

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After oral administration of ¹⁴C-labeled idebenone (¹⁴C-CV-2619) to rats, the plasma ¹⁴C level reached a plateau at 15 min, which persisted till 8 h and then decreased with a half-life of 4.5 h. In dogs, after oral dosing, the plasma ¹⁴C peaked at 15 min, followed by biphasical decline with half-lives of 2.2 and 15.4 h. The plasma of both animals contained mostly metabolites, with a small amount of unchanged CV-2619, which was > 90% protein-bound. In rats given ¹⁴C-CV-2619 orally or intravenously, ¹⁴C was distributed widely in tissues, with relatively high concns. in the gut, liver and kidney. CV-2619 readily entered the rat brain to undergo subcellular distribution with a significant amount localized in mitochondria. The concn. of ¹⁴C in rat fetus was low, as was that in the milk. Oral ¹⁴C-CV-2619 was eliminated by rats and dogs mostly as metabolites within 48 h. In rats, more was excreted in urine than in feces, whereas in dogs excretion by these two routes was almost equal. Enterohepatic cycling of biliary ¹⁴C occurred in rats. Repeated oral ingestions of ¹⁴C-CV-2619 to rats resulted in no accumulation of ¹⁴C. The metabolites found in rats and dogs were QS-10, QS-8, QS-6 and QS-4 formed by oxidative shortening of the side chain of CV-2619, and desmethylated CV-2619 and QS-4. Glucuronides and sulfates of the dihydro (quinol) derivatives of the above metabolites were also detected. Dihydro QS-4 sulfate was the major metabolite in plasma and urine of both animals, while dihydro QS-10 glucuronide was predominant in rat bile.

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To investigate the disposition of fractionated ³H-heparin, commercial ³H-heparin was fractionated by affinity chromatography on protamine-Sepharose and subsequently by Sephadex G-100 chromatography. Effect of dose on the disposition of fractionated ³H-heparin was studied. At an early stage following the intravenous administration of fractionated ³H-heparin which had either affinity or non-affinity for protamine-Sepharose, elimination of the radioactivity from plasma was delayed with increasing dose. Even at 5 h after the administration when most radioactivity in plasma disappeared, the cumulative urinary recovery was only 4.8 to 8.4% of dose (1 to 15 μCi/kg), and 61 to 43% of dose was found in liver. Therefore, it was suggested that tissue (liver) distribution of radioactivity might be a predominant factor for the dose-dependent disappearance of the radioactivity from plasma at an early stage following the administration of fractionated ³H-heparin. Plasma radioactivity levels tended to be psudo-steady state or to increase slightly after about 1 to 2 h following the administration of fractionated ³H-heparin. Participation of entero-hepatic circulation in this tendency was rejected.

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The analgesic, anti-pyretic and anti-inflammatory effects of FI-302, N-(3-piperidinopropyl)-4-methyl-6-trifluoromethyl-furo [3, 2-b] indole-2-carboxamide, a newly synthesized tricyclic compound, were investigated in comparison with those of nonsteroidal anti-inflammatory drugs (NSAIDs). FI-302 showed potent analgesic and anti-pyretic effects in the various models used. FI-302 showed an inhibitory effect on acute inflammatory response such as carrageenin-induced edema, but did not show any effect on chronic inflammatory response such as cotton pellet granuloma. These effects of FI-302 were about 2 to 7 times more potent than those of non-acidic NSAIDs such as mepirizole and tiaramide. Moreover, FI-302 had little or no ulcerogenic activity. From these results, it is conceivable that the spectrum of FI-302's activities is similar to those of nn-acidic NSAIDs such as mepirizole and tiaramide. These results suggest that FI-302 is a new non-ulcerogenic anti-inflammatory compound, and should be suitably applicable for clinical purposes.

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