This paper demonstrates that the stable isotope tracer technique using NMR spectroscopy and the selective ¹³C labeling of protonated carbons can provide a relatively sensitive method to investigate pharmacokinetic problems in man. The urinary excreted [1, 3, 5-¹³C₃] hippuric acid ([¹³C] HA) formed from orally administered [1, 3, 5-¹³C₃] benzoic acid ([¹³C] BA) as a model substrate was successfully quantitated without any separation procedures by proton-decoupled ¹³C-NMR spectroscopy of 10-fold concentrated urine in a 10 min accumulation time. In spite of the low dosage (10mg BA), the C3, 5 resonances of [¹³C] HA were detected with favorable signal-to-noise ratios to quantitate [¹³C] HA concentration. The administered [¹³C] BA was found to be quantitatively biotransformed to HA and excreted in urine within 4h. The lower limit of detection was estimated to be 50 nmol in an NMR tube, which was improved about one order of magnitude over that of BA labeled in the quaternary carbon (C7). The potential of an inverse detection experiment using heteronuclear multiple quantum coherence was also investigated in order to detect [¹³C] HA in urine with a higher sensitivity. The inverse experiment improved the sensitivity by a factor of 2-3 over ¹³C{¹H}-NMR, although the specificity of detection was relatively poor.

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A hydrolytic enzyme which catalyzes hydrolysis of the ester-linkage of a series of 17-O-acyl derivatives of 7-ethylcamptothecin-21-(2-dimethylamino) ethylamide [acyl derivatives of 22E] was purified from rat liver and its properties were characterized. It hydrolyzed the ester-linkage of all 22E derivatives tested as well as p-nitrophenyl acetate at pH 8-9 but had no effect on 7-ethyl-10-[4-(piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11 : irinotecan), unlike CPT-11 converting carboxylesterase, which was previously purified from rat serum [Tsuji T. et al., J. Pharmacobio-Dyn., 14, 341 (1991)]. The enzyme had no effect on either acetyl choline or butyrylcholine. It was inhibited by several organophosphorous compounds such as diisopropyl fluorophosphate (DFP), bis-(pnitrophenyl) phosphate and paraoxon, but was insensitive to inhibitors specific for choline esterases. These results indicate that this liver esterase is clearly distinct from choline esterase and serum CPT-11 converting enzyme and is able to convert pro-drugs, O-acyl derivatives of 22E, to an antitumor agent.

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Dehydrochlorination of D-glucosamine (2-amino-2-deoxy-D-glucose) hydrochloride with an anion exchange resin made its DNA breaking activity in plasmid pBR322 much higher, especially in the presence of Cu²⁺. The sample of anion exchanger-treated D-glucosamine hydrochloride, i.e., HCl-free D-glucosamine sample, showed an absorption maximum at 274 nm on the UV absorption spectrum in water as seen in the case of fructosazine [2, 5-bis (D-arabino-tetrahydroxybutyl) pyrazine], one of the dimers of D-glucosamine. On a positive-ion fast atom bombardment (FAB) mass spectrum, the sample showed an ion peak at m/z 323 as a base peak, corresponding to dihydrofructosazine [2, 5-bis (D-arabino-tetrahydroxybutyl) dihydropyrazine], which was a precursor of fructosazine, as well as those of D-glucosamine itself (m/z 180) and fructosazine (m/z 321). The DNA strand breaking activity of HCl-free D-glucosamine sample was directly proportional to the peak intensity of m/z 323 ion, while the DNA breaking activity of fructosazine was much weaker than that of HCl-free D-glucosamine sample. 2, 5-Dihydro-3, 6-dimethylpyrazine and 2, 3-dihydro-5, 6-dimethylpyrazine having a dihydropyrazine skeleton the same as dihydrofructosazine showed the same extent of DNA strand breaking activity as did the HCl-free D-glucosamine sample. These results indicated that dihydrofructosazine produced during the dehydrochlorination is closely involved in the DNA breaking activity of HCl-free D-glucosamine sample.

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Young and old human red blood cells (RBC) were separated from freshly collected human blood by Percoll density gradient centrifugation. The amounts of lipid peroxidation products in the lipid fractions of these RBC were determined by high performance liquid chromatography-chemiluminescence, thiobarbituric acid and LPO-586 methods. The levels of phosphatidylcholine and phosphatidylethanolamine hydroperoxides in old RBC were higher than in young RBC. The products reflecting the advanced stage of lipid peroxidation, malonaldehyde (free and bound forms), 4-hydroxyalkenals (free and bound forms) and other aldehydes, were also higher in old RBC than in young RBC. The levels of these lipid peroxidation products increased when whole RBC were mildly oxidized with ferric ion. These results indicate that oxidative damage of RBC in the circulation takes place during the aging process, and lipid hydroperoxides and other lipid peroxidation products accumulate in old RBC.

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Semi-purified diets supplemented with either a high α-linolenate (n-3) (perilla) oil or a high linoleate (n-6) (safflower) oil were fed to rats through two generations. Rats fed safflower oil showed a decrease in docosahexaenoic acid (n-3) and a compensatory increase in docosapentaenoic acid (n-6) in all the brain regions and organelles examined, when compared with rats fed perilla oil. As reported previously, the safflower oil-fed rats exhibited inferior learning ability compared with the perilla oil-fed rats (N. Yamamoto et al., J. Lipid Res., 28, 144 (1987)). Using brains of rats in these dietary groups, the activities of several enzymes, Na⁺, K⁺-ATPase, Ca²⁺-ATPase, 5'-nucleotidase, 2', 3'-cyclic nucleotide phosphodiesterase, acetylcholinesterase, and choline acetyltransferase in membranes, were compared. The 5'-nucleotidase activity in cortex and hippocampus, and the Na⁺, K⁺-ATPase activity in myelin decreased slightly but significantly in the safflower oil group. None of the other membrane-associated enzyme activities in all the brain regions and organelles examined was affected significantly by the dietary fatty acids under optimal assay conditions in vitro. However, in the safflower oil group, the Na⁺, K⁺-ATPase activity of synaptosomes at a suboptimal concentration of ATP was 78% that in the perilla oil group. These results suggest that relatively large changes in the proportions of n-3 and n-6 polyunsaturated fatty acids in brain membranes caused by dietary manipulation do not provoke significant alterations in most membrane-bound enzyme activities. However, a small but significant change in Na⁺, K⁺-ATPase activity at a suboptimal concentration of ATP may be implicated in the altered learning behavior reported earlier.

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Changes in carbonyl reductase (CR) activity towards seven substrates in adrenal glands of female rats were investigated during estrous cycle and the circadian patterns of adrenal CR activity towards seven substrates in male rats were examined. The localization of the enzyme protein in the adrenal gland was carried out by immunohistochemistry. In female rats, adrenal CR activity was lowest between 1200 and 2000 h on the day of proestrus, the phase in which a large, significant increase in ovarian steroids, gonadotropins and prolactin is observed. Immunoreactivity in the zona reticularis of the adrenal cortex to anti-adrenal CR, CR-K2, antibody on the day of proestrus was faint or negative compared with that on other days of the estrous cycle. In male rats, no major changes in adrenal CR activity were observed compared with females, although these were slight changes in all the enzyme activities. These results suggest that adrenal CR may be negatively regulated by the pituitary-adrenal axis in female rats and, in particular, on the day of proestrus.

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The bioavailability of calcium from various calcium sources in humans and animals has been the subject of investigation for many years and there is considerable controversy as to the relative bioavailability of different calcium salts. Most of the studies have used a calcium balance technique which has numerous problems in terms of performance and interpretation. Using a method for evaluating the efficacy of calcium from calcium salts used for plasma calcium metabolism and bone mineralization, we examined the bioavailability of calcium from four commercially available calcium salts, namely calcium carbonate, DL-calcium lactate, L-calcium lactate and powdered oyster shell-calcium in vitamin D-deficient or -replete rats. Among the calcium salts, the differences in bioavailability were small and not statistically significant as tested by analysis of variance in both groups of rats. Thus, we conclude that calcium is utilized to the same extent from calcium carbonate, DL-calcium lactate, L-calcium lactate and powdered oyster shell-calcium in both vitamin D-deficient and -replete rats.

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The antiallergic constituents of oolong tea stem were examined. The stem extracts inhibited the 48 h homologous passive cutaneous anaphylaxis (PCA) reactions of rats in a dose-dependent manner and showed the same extent of inhibitory activity as ketotifen. All antiallergic constituents from the stem were concentrated into chloroform and ethyl acetate fractions, when extracted by various solvents. These fractions were treated with polyvinylpolypyrrolidone (PVPP), which resulted in the elimination of antiallergic activity in the ethyl acetate fraction, suggesting that one of the antiallergic constituents may be tea catechins. Then, six kinds of catechins, (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), (-)-epicatechin (EC), (+)-catechin (C) and (-)-gallocatechin gallate (GCG), were isolated from the ethyl acetate fraction, and the inhibitory activity of these catechins on histamine release from rat peritoneal mast cells passively sensitized with anti-egg albumin (EA) IgE antibody was investigated. Among these catechins, significant inhibitory activity was observed in all the catechins except for EC. In addition, the inhibitory activity of GCG was greater than that of EGCG, which is well known to be an antiallergic constituent in tea. These results suggest that GCG may be a novel antiallergic constituent among tea catechins, and also the most potent.

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Induction of acute nephritis in the rat by injecting anti-glomerular basement membrane (GBM) antiserum is accompanied by a transient increase in angiotensin II generation in blood circulation within the first 24 h and a subsequent elevation of transforming growth factor-β1 (TGF-β1) mRNA levels in kidney cortex with a peak at days 7-8. Studies were carried out to determine whether the increased generation of angiotensin II plays a role in the elevation of TGF-β1 mRNA. Elevation of TGF-β1 mRNA levels 7d after injection of antiserum was significantly inhibited by a successive daily administration of TCV-116, angiotensin II type 1 receptor antagonist, at 1 mg/kg/d from days 0 to 2 or from days 0 to 6, while it was not influenced by a single administration of this dose on day 0. In addition, angiotensin II infusion for 24 h at a rate of 50 ng/min did not alter the level of TGF-β1 mRNA which was measured 6 d after the infusion. These results suggest that the anti-GBM antiserum-induced increase in TGF-β1 expression in the kidney is not responsible for angiotensin II generated in the blood circulation during the early phase of acute nephritis, but is probably mediated by angiotensin II generated locally in the kidney.

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The mode of the neuromuscular blocking action of coryneine (a quaternary ammonium derivative of dopamine) derived from aconite root was investigated in isolated phrenic nerve-diaphragm muscles and denervated diaphragm muscles of mice. Coryneine (20-150μM) blocked the nerve-evoked twitch response without affecting the contraction evoked by electrical stimulation of the muscle. The blocking effect was reversed by neostigmine, a cholinesterase inhibitor. The electrical charge-response curve on depolarization produced by iontophoretically applied acetylcholine (ACh) at the endplate regions in normal muscles was shifted to the right on decreasing the maximal response by 40μM coryneine. The double-reciprocal plot revealed that coryneine reduced the apparent affinity of ACh for its receptor on decreasing the maximal response. Coryneine (20μM-2mM) itself depolarized the endplate membrane and this effect was reversibly suppressed by 1 and 5μM pancuronium. Coryneine (30μM-10mM) produced contractions of denervated muscles in a concentration-dependent manner and the effects were reduced by 70nM pancuronium. These results indicate that coryneine is a depolarizing agent and a mixed-type competitive and noncompetitive neuromuscular blocker.

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3, 4, 5-Trimethoxyphenylacetaldehyde, an intermediate metabolite of mescaline, was oxidized to 3, 4, 5-trimethoxyphenylacetic acid by mouse hepatic microsomes. The reaction was NADPH-dependent, and inhibited by SKF 525-A, metyrapone and disulfiram. A P450 isozyme in mouse hepatic microsomes, P450 MUT-2 (CYP2C29), catalyzed the reaction (0.96 nmol/min/nmol P450) in which NADPH and NADPH-cytochrome c reductase were essential for the catalytic activity. The reaction was confirmed to be an oxygenation since molecular oxygen was incorporated into the carboxylic acid metabolite formed under oxygen-18 gas by GC-MS analysis. By addition of antibody against CYP2C29 to the microsomes (3.2 mg/mg microsomal protein) the MALDO activity was inhibited by 35% of the control value with preimmune serum, suggesting that CYP2C29 or an immunologically-related isozyme (s) plays a major role in the NADPH-dependent oxidation of 3, 4, 5-trimethoxyphenylacetaldehyde to 3, 4, 5-trimethoxyphenylacetic acid by mouse hepatic microsomes. Pharmacological experiments on mescaline and its deaminated metabolites using mice indicated that the metobolites were much less active or were inactive in cataleptogenic effect and pentobarbital-induced sleep prolongation as compared with the parent compound.

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To investigate the contribution of a cardiac renin-angiotensin system to cardiac hypertrophy due to volume overload, the effects of losartan, a non-peptide angiotensin (Ang) II type 1 (AT₁) receptor antagonist, on left ventricular hypertrophy (LVH) was studied. LVH was produced in male Wistar rats by volume overload secondary to aortic insufficiency (AI). Losartan (10 mg/kg/d) was orally administered for 2 weeks after surgery to both AI and sham-operated (control) rats. Two weeks after surgery, aortic pulse pressure and left ventricular (LV) weight were markedly increased in the AI rats as compared with the control group, whereas cardiac angiotensin converting enzyme (ACE) activity remained unchanged. The effects of the chronic administration of losartan on AT₁ receptors were verified by the blockade of Ang II pressor response. Losartan treatment produced a significant reduction in LVH in AI rats without affecting the systolic blood pressure. In separate groups of rats, to elucidate the mechanisms of the attenuation of LVH by treatment with losartan, we determined plasma and LV immunoreactive Ang II content and plasma renin activity (PRA). LV Ang II content increased in AI rats, while plasma Ang II content, PRA and serum ACE activity did not. Losartan significantly reduced the LV Ang II content, whereas PRA and plasma Ang II concentration were increased by the treatment. There was a significant positive correlation between LV weight and LV Ang II content. These results suggest that cardiac Ang II, rather than circulating Ang II, plays an important role in the LVH due to volume overload via the AT₁ receptor.

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Effect of cytochrome P450 (P450) inducers on liver microsomal metabolism of 3, 4, 3', 4'-, 3, 5, 3', 5'-and 2, 5, 2', 5'-tetrachlorobiphenyl (TCB) was studied using male Wistar rats, male Hartley guinea pigs and male Golden syrian hamsters. In metabolism of 3, 4, 3', 4'-and 3, 5, 3', 5'-TCB, liver microsomes from 3-methylcholanthrene (MC)-or 3, 4, 5, 3', 4'-pentachlorobiphenyl (PenCB)-treated hamsters showed hydroxylase activities for both TCB isomers, although the activities were much less than those of rats. In contrast, liver microsomes from untreated and phenobarbital (PB)-, MC- or PenCB-treated guinea pigs showed no hydroxylase activity. In 2, 5, 2', 5'-TCB metabolism, 3-hydroxylase activity was observed in untreated guinea pigs and hamsters, but not in untreated rats. The activity was induced by PB treatment in all three species, at rates of 324, 19 and 20 pmol/min/mg protein in rats, guinea pigs and hamsters, respectively. This activity was not enhanced by treatment with either MC or PenCB. Only in hamsters was 4-hydroxylated metabolite formed in all microsomes used in addition to the 3-hydroxylated one, and the formation was accelerated 2.0-, 2.7- and 4.8-fold by treatment with PB, MC and PenCB, respectively. These results suggest that different P450 isoforms in hamster liver microsomes are involved in 3- and 4-hydroxylation of 2, 5, 2', 5'-TCB. Thus, there are species differences in the basal ability to hydroxylate TCB isomers, and in the extent of effect of P450 inducers on the metabolism of these isomers among the three species.

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The direct effect of 12(S)-hydroxyeicosatetraenoic acid [12 (S)-HETE] on rat crystalline lens was investigated in this study. 12 (S)-HETE lowered the glutatione (GSH) level and GSH reductase activity in the lens, while accelerating aggregation and insolubilization of lens proteins and production of thiobarbituric acid-reactive substrances. The study also indicated that 12 (S)-HETE insolubilized α-crystalline and induced opacification of the lens when the lens was incubated with 12 (S)-HETE. From these results, we presumed that 12 (S)-HETE may be oxidized or peroxidized easily and automatically in the air. The substances derived from 12 (S)-HETE by oxidation or peroxidation may give the action disordering lens normalcy and induced cataract formation. Thus, the direct effect of 12 (S)-HETE may of no benefit to the crystalline lens.

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When rats with streptozotocin (STZ)-induced diabetes were given a daily intraperitoneal (i. p.) injection of VOSO₄ (+4 oxidation state of vanadium), their serum glucose dropped from hyperglycemic level to normal level within 2 d and serum free fatty acid (FFA) level also dropped to normal level. Vanadium was incorporated in most organs as well as in the adipose tissues, as detected by neutron activation analysis (NAA). The mechanism for the insulin-like action of vanadium in terms of FFA release from isolated rat adipocytes was investigated : (1) Vanadyl (IV) and vanadic (III) ions normalize the FFA release in the adipocytes treated with epinephrine ; (2) vanadate (V) ion treated with ascorbic acid, cysteine or glucose is effective in normalizing the FFA release but vanadate ion alone has no effect on FFA release ; (3) vanadyl ion is incorporated into the adipocytes, while vanadate ion is not, as indicated by ESR spectroscopy ; and (4) vanadyl ion can act on the glucose transporter, as indicated by experiments using cytochalasin B which is an inhibitor of this transporter. From these results, the normalization of both serum glucose and FFA levels by vanadyl ion was concluded to be due to the incorporation of vanadyl ion into the adipocytes, in which the metal ion acts on the glucose transporter and induces both the promotion of glucose uptake and the decrease of FFA release from peripheral adipocytes. The vanadyl state was suggested to be a possible pharmacologically active form of vanadium allowing the insulin-like action. We further propose that the monitoring of serum FFA level is another sensitive index in addition to serum glucose level by which to know the degree of the diabetes, and the FFA release from adipocytes is a good in vitro evaluation system to find a compound which shows an insulin-like action.

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Bryonolic acid was isolated in high yield from transformed hairy root cultures of Tricosanthes kirilowii var. japonica. Bryonolic acid exhibited cytotoxic activity to various tumor cells in vitro, independent of cell type. Normal cells such as rat hepatocytes are less sensitive to bryonolic acid. The appearance of a DNA ladder was detected in the bryonolic acid-treated HL-60RG cells, indicating that cell death triggered by bryonolic acid is due to apoptosis.

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We report the inhibitory effect of topical application of extracts of a traditional Chinese herbal prescription on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in mice. Methanol and water extracts obtained from 22 traditional Chinese herbal prescriptions were assayed and their inhibition ratios calculated. In general, the methanol extracts produced more effective inhibition than the water extracts. Of the various traditional Chinese herbal prescriptions, the methanol extract of Rikkunshi-to was more effective than other prescriptions as far as inhibition of TPA-induced inflammation was concerned. Hoelen, Glycyrrhizae Radix, Atractylodis Rhizoma, components of Rikkunshi-to markedly inhibited the inflammatory activity induced by TPA in mice. Furthermore, topical application of the methanol extract of Rikkunshi-to markedly inhibited TPA-induced tumor promotion in two-stage carcinogenesis in mouse skin.

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The effects of dose, pH and osmolarity on the intranasal absorption of a recombinant human erythropoietin (rEPO) solution were studied in male Wistar rats. The intranasal administration of rEPO was evaluated by measuring percentage circulating reticulocytes of red blood cells on a stained blood smear (smear method), and also by measuring residual circulating reticulocyte counts using a microcell counter (sysmex method). Both results suggest that rEPO solution was absorbed through the nasal mucosa of rats without enhancers after a single intranasal administration. The pharmacological availabilities of rEPO after intranasal administration compared with intravenous administration were about 7% and 4%, when estimated by smear method and sysmex method, respectively. The pharmacological activity was enhanced in low pH and hypotonic mannitol solution.

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We modified the surface of liposomes with galactosyl ligands. At first we determined whether or not the galactosyl moiety was exposed on the liposomes. We then investigated the effect of the ligands on the hepatic accumulation of liposomes in rats. We introduced an oligoethylene glycol moiety as a spacer. Among the various ligands tested, those with a tri-or tetraethylene glycol moiety as a spacer caused the greatest accumulation of liposomes in the liver. Liposomes bearing ligands with a tri-or tetraethylene glycol moiety as a spacer, were aggregated by Ricinus communis agglutinin. On the other hand, those modified with ligands with a mono- or diethylene glycol spacer did not clearly agglutinate. These results show the importance of a spacer between the homing device and the ligand anchor.

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We investigated the influence of anesthetic regimens on the intestinal absorption of 5-fluorouracil (5-FU), which is known to be absorbed by concurrent Na⁺-dependent, carrier-mediated transport and passive transport, in single-pass perfusion experiments in rats. Compared with the absorption in unanesthetized rats, the regular dose of urethane (1.13g/kg) reduced the maximum transport rate (J_max), the Michaelis constant (K_m) and the membrane permeability coefficient of passive transport (P_{m, d}); a low dose of urethane (0.7g/kg) reduced J_max and K_m, but did not affect P_{m, d}; pentobarbital sodium (50mg/kg) increased J<max> without affecting K_m, and reduced P_{m, d}. The reductions in J_max and K_m were comparable for the regular and low doses of urethane. Thus, urethane and pentobarbital, which have been most commonly used in laboratory animal experiments, exerted qualitatively different effects on the carrier-mediated transport of 5-FU, although they similarly inhibited the passive transport. For urethane, the effect on the passive transport was avoided by reducing the dose, but the effect on the carrier-mediated transport was not. This influence of anesthetic regimens on intestinal drug absorption may not be easily scaled for normalizing absorption data. When compiling them for such purposes as establishing in situ-in vivo quantitative correlation, the absorption data in perfusion (in situ) should be categorized on the basis of anesthetic regimens, to avoid ending up with poor outcomes. We also examined the effect of urethane on the exsorption of Na⁺ in the intestinal loop where Na⁺-free buffer was introduced, and found a minimal effect. Therefore, urethane may not significantly affect Na⁺ concentration at the intestinal surface, ruling out the possibility of its indirect effect on the carrier-mediated transport of 5-FU through a reduction in the Na⁺ concentration. Urethane may directly affect the carrier and/or membrane environment.

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The cerebral metabolic rate of glucose (CMRglc) and cerebral blood flow (CBF) in rats were estimated noninvasively with a high-resolution animal positron emission tomography (PET) system that we recently developed. Quantifications of CMRglc and CBF were attempted with radiotracers, ¹⁸F-2-fluoro-2-deoxyglucose and ¹⁵O-water, respectively. From the data obtained with PET, the CMRglc and CBF values under pretreatment conditions were respectively calculated as 51±14μmol/min/100g and 61±7ml/min/100g under pentobarbital-anesthesia. These values were in close agreement with published data obtained with the autoradiographic technique. Moreover, and increase of 69% (mean) in CBF was detected after i.v. acetazolamide administration. The results indicate that this high-resolution animal PET scanner holds great potential for the assessment of drug-related metabolic and circulatory effects in intact and experimental animals.

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Bupleuri Radix is a commonly used medicinal plant in Kampo medicine, and its hot water extracts show mitogenic activity to murine lymphocytes. In this paper the mitogenic substances in the hot water extracts of Bupleuri Radix (Bup-HWE) were fractionated and characterized physicochemically and immunologically. Most of these substances were recovered from mol. wt of more than 200 kDa fraction (fr. C-13). Separation of fr. C-13 by phenol-water fractionation method gave water soluble and phenol soluble mitogenic substances. These substances showed the activity even in C3H/HeJ mice, and polymyxin B or lysozyme treatment did not abrogate the activity, suggesting that the active substances are not related to bacterial lipopolysaccharide. Treatment of the mitogenic substances recovered from the phenol layer with NaClO₂, a polyphenol degrading chemical, significantly reduced the activity, but pronase and pectinase treatments were not effective. The mitogenic substances in the water layer were active even after NaClO₂ treatment. These findings suggested that the mitogenic substances of Bup-HWE are large molecular weight polyphenolic compounds and polysaccharide. The mitogenic substances are suggested to be B cell mitogens.

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Octastatin (RC-160, vapreotide INN) is a somatostatin analogue being developed for use in oncological, enterologic and neuroendocrine applications. The pharmaceutical form is a freeze-dried preparation for parenteral injection use. Three dosage forms containing 0.5, 1.5 and 15 mg of vapreotide base have been investigated. Various freeze-drying conditions, stabilizing agents, membranes for sterile filtration and heating procedures have been examined. The formulation with glutamic acid-sodium glutamate buffer as a stabilizing agent, the type of membrane for filtration and the freeze-drying procedure have been found appropriate for subsequent industrial production. No evident degradation was observed either after manufacturing, or after a three-week accelerated stability study at 50°C and 70% relative humidity.

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The action of carrageenan (CAR), a representative blocking reagent for phagocytes, on the antitumor effect and tissue distribution of highly branched (1→3)-β-D-glucan, SSG, was examined. CAR inhibited the antitumor effect of intraperitoneally administered SSG only when applied before inoculation of the tumor, and had little effect when applied after tumor inoculation. A similar result was observed when SSG was administered intralesionally. In contrast, CAR had considerable effect on tissue distribution of i.p. SSG. The differences with respect to the results in normal mice were : 1) the distribution of SSG from the peritoneal cavity to the rest of the body was inhibited, 2) large numbers of peritoneal exudate cells (PEC) were produced and a relatively high concentration of ³H-SSG was found in the PEC fraction 48 h after administration of ³H-SSG, 3) one week after administration, ³H-SSG was distributed to throughout the body but the amount of ³H-SSG distributed was lower than in normal mice, 4) a significant amount of ³H-SSG was recovered from ligaments (containing omental milky spots, peritoneum, mesentery and associated fat) in which negligible amounts were found in normal mice. These results suggest that the inhibition of the antitumor effect of SSG by CAR probably results from the prevention of the natural resistance of mice which is related to phagocytic function, and that the distribution of SSG to throughout the body is significantly modulated by CAR.

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The effect of the lighting schedule on the pharmacological action of theophylline was studied in chick embryos. Fertile eggs of White Leghorns were incubated and investigated, on two occasions, under constant light conditions or under constant dark conditions. A sigle injection of theophylline 2.14, 4.29, 8.57 and 17.14 mg/egg into the air sac of fertile eggs was carried out on the 16th day of incubation. Electrocardiograms (ECGs) were recorded 0 to 60 min after drug injection. After drug injection, heart rate increased under constant light conditions, but decreased under dark conditions. In addition, arrhythmia was produced by theophylline, 17.14mg/egg, under constant dark conditions. These results indicate that the manipulation of the lighting schedule may have a marked influence on the pharmacological effects of theophylline in chick embryos.

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6-(10-Hydroxydecyl)-2, 3-dimethoxy-5-methyl-1, 4-benzoquinone (idebenone) has been found to have a membrane-stabilizing activity in studies using lysosomes. Using dimyristoyl L-α-phosphatidylcholine (DMPC) as a substrate, the effects of idebenone and related benzoquinones on phospholipid digestion by phospholipase A₂ (PLA₂) was investigated. Free myristic acid, released from DMPC upon PLA₂ treatment, was anthrylmethylated with 9-anthryldiazomethane (ADAM) and determined by reversed-phase HPLC. Idebenone and a related benzoquinone, 2, 3-dimethoxy-5-methyl-6-(10-morpholinodecyl)-1, 4-benzoquinone hydrochloride (QS-10·Mor), inhibited the hydrolysis of the substrate with PLA₂ in a dose-dependent manner. It is suggested that the effect of idebenone on PLA₂, in addition to its antioxidant activity against lipid peroxidation, can be attributed to membrane-stabilizing activity.

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Conformational analysis of potent competitive inhibitors of aromatase, androst-4-enes 5, as well as 2, 4-diene steroids 3, 4, and 6 was carried out, using theoretical calculations, to determine the stereochemistry of their aromatase-catalyzed oxygenation. In the steroids examined, both the 19-alcohols and the 19-aldehydes favor the above-A ring conformation among the possible three in each. The results suggest that the 3-deoxy steroid 5a as well as the 2, 4-diene steroids 4a and 6a would be oxygenated at C-19 by aromatase through the same stereomechanism as that involved in the androstenedione aromatization.

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The hypoglycemic effect of the rhizomes of Ophiopogonis Tuber (Liliaceae) was investigated in normal and streptozotocin-induced diabetic mice. The n-butanol extract of rhizomes of Ophiopogonis Tuber (BM) (100 mg/kg) reduced the blood glucose of normal mice from 201±13 to 151±7mg/100ml 4h after intraperitoneal administration (p<0.05), and also significantly lowered the blood glucose of streptozotocin-induced diabetic mice from 590±28 to 470±37mg/100ml under similar conditions (p<0.05). BM also tended to suppress epinephrine-induced hyperglycemia in mice. We concluded that the hypoglycemic effect of BM does not alter the insulin concentration.

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Diadenosine 5', 5'''-p¹, p⁴-tetraphosphate (Ap4A) degradation in dog plasma was inhibited by the addition of EDTA. The Ap4A degrading activity seemed to be recovered by the addition of metal ions, Ca²⁺, Co²⁺, Cu²⁺, Mg²⁺, Mn²⁺ or Zn²⁺ to plasma in which the activity was lost by the addition of EDTA. Especially, the degradation was accelerated by the addition of Ca²⁺ to the plasma, and the degradation rate was dependent on the concentration of Ca²⁺. The results of this study suggested that the enzymatic degradation of Ap4A in dog plasma was modulated by the concentration of Ca²⁺.

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The permeation and accumulation of lauric acid (12 : 0) in rat skin as an enhancer for the percutaneous absorption of thiamine disulfide (TDS), using propylene glycol as a vehicle, were determined in vitro. 12 : 0 barely permeated through skin, and accumulated in skin. The accumulated amount increased with increasing concentrations in the vehicle (1-20%). The proportion of the accumulation to total dose was highest at a dose of 5%. A positive relationship was found between the accumulated amount of TDS and 12 : 0. It was suggested that the penetration of 12 : 0 into skin contributes to the enhanced partition of TDS to skin.

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We studied the absorption enhancement effects of three types of protease inhibitors, aprotinin, bacitracin and soybean trypsin inhibitor, in the rat intestine. Of these protease inhibitors, bacitracin enhanced the absorption of FD-4 and phenol red from the rat small and large intestine without mucosal toxicity. Thus, it was suggested that bacitracin has not only a protease-inhibitory but also an absorption-enhancing capability.

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