Prolyl oligopeptidase (EC 3.4.21.26, prolyl endopeptidase) cDNA from bovine brain was cloned by PCR, and the amplified fragment was used as a probe to screen the cDNA library from bovine brain. The obtained clone contained a 2.7 kb DNA fragment with an open reading frame of 2130 nucleotides, and encoded a protein of 710 amino acids with a deduced molecular weight of 80640 Da. The deduced amino acid sequence is 95, 94, 51 and 48% homologous to those of human T-cell, porcine brain, Aeromonas hydrophila, and Flavobacterium meningosepticum prolyl endopeptidases, respectively. The bovine brain prolyl endopeptidase-encoding cDNA was expressed using an expression vector bearing a tac promoter, with an approximate yield of 20 μg/ml of cell culture.

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The pharmacokinetic characteristics, peritoneal permeability and hydrophobicity of three xanthine derivatives, theophylline, enprofylline and 1-methyl-3-propylxanthine (MPX), were investigated in rats. Isotonic saline (30 ml) containing xanthine (2.5, 5 and 10 mg/kg) and blue dextran (0.2%) was administered intraperitoneally. The pharmacokinetic parameters of these xanthines were estimated using concentration-time data obtained from the peritoneal cavity and systemic circulation. Disappearance of these xanthines from the peritoneum declined in almost a monoexponential manner regardless of the dose administered. The volume of distribution (33.9 ml) in the peritoneal cavity was similar to the injection volume, indicating that dialysate was not diluted by the fluid in the peritoneal cavity and the effect of drug adsorption on the peritoneal membrane was minimal. The pharmacokinetics of MPX was dose-dependent, but that of theophylline and enprofylline was not. The fraction of the administered dose absorbed through the peritoneal cavity was 0.71, 0.85, 0.93 for theophylline, enprofylline and MPX, respectively. The peritoneal clearance was significantly different (p<0.05) among the three xanthines by two-way analysis of variance, and a strong correlation was noted between their peritoneal clearance and hydrophobicity (r=0.98, p<0.01). These findings suggest that hydrophobicity is an important determinant in the peritoneal permeation of these xanthines.

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We investigated the effects of T-2591, a new ureidophenol derivative, on low density lipoprotein (LDL) oxidation, acyl CoA : cholesterol acyltransferase (ACAT) and foam cell formation of macrophages in vitro. T-2591 inhibited both copper ion-and endothelial cell-induced LDL oxidation with higher potencies than probucol did. It inhibited ACAT from rabbit intestine, liver and aorta, the respective IC₅₀ values being 0.26, 4.6 and 4.1 μM. It also inhibited ACAT from the mouse macrophage cell line J774 A.1, and its IC₅₀ value (0.067 μM) was much lower than that of CI-976 (4.1 μM). This probably accounts for the inhibition of foam cell formation measured as cholesteryl ester formation in both mouse peritoneal macrophages and J774 A.1 cells at low concentrations (IC₅₀; 0.06 and 0.44 μM, respectively). These observations suggest that T-2591 should be evaluated as a potential tool to retard atherosclerosis in animal models.

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In vivo antitumor effects of the conjugates of doxorubicin (DXR) with carboxymethylpullulan (CMPul) through tetrapeptide spacers were compared with those of DXR against tumor-bearing rats. CMPul-DXR conjugates bound through Gly-Gly-Phe-Gly and Gly-Phe-Gly-Gly spacers were found to be more potent than DXR after a single intravenous injection in rats bearing Walker 256 carcinosarcoma. These conjugates were also more effective than DXR in rats bearing Yoshida sarcoma. However, CMPul-DXR conjugate bound through Gly-Gly-Gly-Gly was less effective against Walker 256-bearing rats than DXR. Body weight loss of CMPul-DXR conjugates in rats, on the other hand, was less than that of DXR at a DXR dose of 10 mg/kg. Lethal doses of CMPul-DXR conjugates in CDF₁ mice were about 3-times higher than that of DXR. These data suggest that the therapeutic index of CMPul-DXR conjugates bound through appropriate peptide spacers was increased more than that of DXR. However, CMPul-DXR conjugates tested were all less effective than DXR against Walker 256 cells in vitro. Also, ¹²⁵I-labeled CMPul-DXR conjugate accumulated much less in the cells than ¹⁴C-DXR.

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Experiments were performed to examine the roles of γ-aminobutyric acid_B (GABA_B) and γ-hydroxybutyric acid (GHB) receptors in long-term potentiation (LTP) of the hippocampal CA1 region in vivo and in the genesis of the spike and wave discharges (SWDs) associated with absence seizures. When tetanic stimulation was delivered to the CA3 region, stable LTP was observed in the CA1 region in saline-treated mice. In mice treated with 5 mg/kg baclofen, the population spike amplitude was significantly potentiated by tetanic stimulation and the degree of potentiation was the same as that induced in saline controls. However, this potentiation decayed to the baseline level about 90 min after stimulation. The decay was reversed by pretreatment with 200 mg/kg P-[3-aminopropyl]-P-diethoxymethylphosphinic acid (CGP 35348), a selective GABA_B receptor antagonist. In mice treated with 50 mg/kg γ-butyrolactone (GBL), a prodrug of GHB, stable LTP was observed 90 min after tetanic stimulation and was greater than that in saline controls. GBL-induced potentiation of LTP was antagonized by 50 mg/kg NCS 382, a putative GHB receptor antagonist. Administration of baclofen (20 mg/kg) or GBL (70 mg/kg) induced absence-like seizures associated with 3-6 Hz SWDs, and CGP 35348 suppressed both baclofen- and GBL-induced SWDs. NCS 382 also attenuated SWDs induced by GBL and baclofen. These results suggest that baclofen and GHB have different effects on LTP in the CA1 region of the hippocampus in vivo, although they have a common mode of action on the thalamocortical functions related to the pathogenesis of absence seizures.

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MX-68 is a novel unpolyglutamatable antifolate. We here reported the in vitro and in vivo immunosuppressive properties of MX-68 compared with a polyglutamatable antifolate, methotrexate (MTX). MX-68 showed potent suppressive effects on mitogen-induced mouse splenic lymphocyte proliferation as well as immunoglobulin production from LPS-stimulated mouse splenic B cells. In in vivo studies, MX-68 significantly suppressed antigen-specific antibody production of both T cell-dependent antigen and T cell-independent antigen. Moreover, MX-68 inhibited a sheep red blood cell (SRBC)-induced delayed-type hypersensitivity (DTH) reaction by administration starting from the day of antigen immunization, but did not suppress the effector phase of the DTH reaction. MTX showed suppressive activities similar to MX-68 in all experiments. Interestingly, although MX-68 demonstrated somewhat stronger suppressive effects than MTX in vivo, the results from in vivo studies were reversed. These results suggest that polyglutamation is not always required to suppress immune responses and that MX-68 is a slightly stronger immunosuppressive drug than MTX in mice.

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This report describes the anti-platelet aggregation activity of 48 pyrazines. Among alkyl- and arylpyrazines tested, 2, 3-diphenylpyrazines showed the strongest anti-platelet aggregation activity. Then, various substituents were introduced into the phenyl groups, and the 2, 3-bis(p-methoxyphenyl)pyrazine derivatives were consequently found to possess considerably strong inhibitory activity.

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Enhancement of in vivo antiviral activity of 5, 7, 4'-trihydroxy-8-methoxyflavone (F36) against H3N2 subtype of influenza A virus by drug delivery system (DDS) with hydroxypropyl cellulose (HPC) was studied.Although in the absence of HPC F36 (0.5 mg/kg) showed no antiviral activity against mouse-adapted influenza virus A/Guizhou/54/89 (H3N2) in mice, when F36 solution containing HPC was administered intranasally 5 min after the virus inoculation, proliferation of the virus in both nasal and broncho-alveolar cavities was inhibited significantly.The relationship between concentration (0.2-0.5%) and deposition ratio of HPC was studied. When 10 μl of fluorescein isothiocyanate (FITC)-conjugated HPC solution was administered intranasally to BALB/c mine, deposition ratio of HPC at 6 h after inoculation in nasal cavity was dependent on its concentration. The deposition ratio of HPC in broncho-alveolar cavity, however, was reversely dependent on its concentration.Anti-influenza virus activity of F36 in nasal and broncho-alveolar cavities was dependent both on the concentration and deposition ratio of HPC. HPC was most effective at 0.5% in nasal cavity and at 0.3% in broncho-alveolar cavity. These results indicate that DDS with HPC enhances the anti-influenza virus activity of F36 in vivo.

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The 70% ethanol extract (KS-ext) from Kochiae Fructus (dried fruits of Kochia scoparia L.) was screened for its activity on nociceptive and inflammatory responses in experimental animals. Although KS-ext at an oral administration of 500 mg/kg had an antinociceptive effect on writhing responses induced by acetic acid, it was ineffective on nociceptive response in the hot plate test. Oleanolic acid oligoglycoside, momordin Ic isolated from Kochiae Fructus significantly decreased the frequency of licking behavior within a unit of time at the late phase without affecting that of the early phase in the formalin test. Also, KS-ext inhibited the rise of vascular permeability induced by acetic acid, the increase of paw edema induced by carrageenin, histamine, serotonin or bradykinin and ear swelling induced by arachidonic acid. Momordin Ic also exhibited an inhibitory effect on carrageenin-induced edema. These results indicated that Kochiae Fructus has a peripheral antinociceptive effect mediated by antiinflammatory action, and that its active component can be partially attributed to momordin Ic.

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We investigated the effects of escins Ia, Ib, IIa, and IIb isolated from horse chestnut, the seeds of Aesculus hippocastanum L., and desacylescins I and II obtained by alkaline hydrolysis of escins on acute inflammation in animals (p.o.). Escins Ia, Ib, IIa, and IIb (50-200 mg/kg) inhibited the increase of vascular permeability induced by both acetic acid in mice and histamine in rats. Escins Ib, IIa, and IIb (50-200 mg/kg) also inhibited that induced by serotonin in rats, but escin Ia didn't. Escins Ia, Ib, IIa, and IIb (200 mg/kg) inhibited the hind paw edema induced by carrageenin at the first phase in rats. Escin Ia (200 mg/kg) and escins Ib, IIa, and IIb (50-200 mg/kg) inhibited the scratching behavior induced by compound 48/80 in mice, but escin Ia was weakest. Desacylescins I and II (200 mg/kg) showed no effect. With regard to the relationship between their chemical structures and activities, the acyl groups in escins were essential. Escins Ib, IIa, and IIb with either the 21-angeloyl group or the 2'-O-xylopyranosyl moiety showed more potent activities than escin Ia which had both the 21-tigloyl group and the 2'-O-glucopyranosyl moiety.

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Conjugates of mitomycin C (MMC) with estradiol benzoate and estradiol via glutaric acid, abbreviated to EB-glu-MMC and E-glu-MMC, respectively, were investigated in vitro to determine their stability and MMC regeneration properties in biological media and on their binding to estrogen receptor. EB-glu-MMC and E-glu-MMC were added into a mixture of 1/15 M phosphate buffer, pH 7.4, (ionic strength=0.3), propylene glycol (PG) and rat plasma (4 : 5 : 1, v/v/v), named 10% plasma, or into a mixture of the buffer, PG and rat liver homogenate (9 : 10 : 1, v/v/w), named 5% liver homogenate, and each was incubated at 37°C. The conversion characteristics of EB-glu-MMC and E-glu-MMC were compared with those in the buffer-PG (1 : 1, v/v) mixture previously reported. In 10% plasma, the change of EB-glu-MMC to E-glu-MMC was accelerated enzymatically to some extent, while the enzymatic degradation of E-glu-MMC was not accelerated at all. In 5% liver homogenate, EB-glu-MMC changed quickly to E-glu-MMC, whereas the degradation of E-glu-MMC was accelerated very little. E-glu-MMC was considered to be rather stable against enzyme in the biological media. Competitive binding studies using the rat uterine estrogen receptor showed that the specific binding affinity of E-glu-MMC was 0.81% to that of estradiol, while E-glu-MMC hardly exhibited specific binding. E-glu-MMC was regarded as a hormone-drug conjugate showing a small specific binding affinity to the estrogen receptor. E-glu-MMC is considered to be an effective antitumor agent which gradually generates MMC in the body, and its receptor-mediated action to target cells such as estrogen receptor-positive tumor cells might be possible.

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(1→3)-β-D-Glucans remained in the liver and spleen for long time, i.e. more than a month, without major structural changes/because there is no specific metabolic pathway for it in the body. However, biological activities, such as priming activity to LPS, triggered TNF-α synthesis, and antitumor activity was reduced more quickly. In this paper, we demonstrated the contribution of protein binding in inactivating β-glucans. A particle β-glucan preparation, zymosan, was treated with serum or plasma at 37°C and their various biological activities were compared with zymosan alone. Such biological activities as antitumor activity, TNF-production, IL-6 production, complement activation and vascular permeability were significantly decreased by serum or plasma treatment. These results strongly suggested that the binding of serum or plasma protein(s) to β-glucans would be a key step in inactivating a particle β-glucan in the body.

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As previously reported, we have discovered that a novel compound, NO-1886 (diethyl 4-[(4-bromo-2-cyanophenyl)carbamoyl] benzylphosphonate) has a powerful lipoprotein lipase (LPL) stimulating activity. Oral administration of NO-1886 increased LPL activity in postheparin plasma of experimental animals, resulting in the reduction of plasma triglyceride with concomitant elevation of high density lipoprotein cholesterol. However, the mechanism of NO-1886 on LPL activity is not clearly understood. To address this problem, we examined the effect of NO-1886 on LPL activity in primary rat cell culture isolated from adipose and skeletal muscle tissue. NO-1886 increased total LPL activity 18% and 23% in adipocytes at a dose of 3 and 10 μg/ml, respectively, and 43% at a dose of 10 μg/ml in skeletal muscle cells. These results indicate that NO-1886 may act directly on LPL-producing cells such as adipose and skeletal muscle.

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The influence of diet on the single-dose pharmacokinetics of isosorbide 5-mononitrate (IS-5-MN) and sustained-release isosorbide dinitrate (ISDN) was studied in 8 healthy male volunteers. The subjects received either a low-calorie/low-fat diet (LCFD) or a high-calorie/high-fat diet (HCFD) according to a cross-over schedule and were administered the drug in tablet form after breakfast. The study was conducted first in 8 subjects who received a 20 mg IS-5-MN tablet and then in 6 of these individuals who received a 20 mg sustained-release ISDN tablet. After oral doses of IS-5-MN, the plasma drug levels declined monoexponentially and could be described by a one-compartment open model. There were no significant differences in the pharmacokinetic parameters for IS-5-MN between the LCFD and the HCFD. After administration of sustained-release ISDN, the plasma ISDN levels showed marked variations both between and within individuals. The increase in the mean AUC_0-12 values for ISDN with the HCFD was found to exceed 60%, although there was no significant difference between the two diets.

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Rectal absorption of 32-mer phosphorothioate deoxynucleotides (S-Oligo) in the rat was attempted with the aid of a suppository containing oleic acid and a surfactant. Although, the suppository without adjuvant did not show detectable blood levels, that containing over 10% oleic acid enabled the absorption of S-Oligo with t_max at 2 h postdosing.

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The hybrid liposomes (90 mol% DMPC/10 mol% C₁₂(EO)₁₀ or C₁₂(EO)₁₂) have a highly inhibitory effect on the growth of tumor cells (HL-60). The induction of apoptosis by the hybrid liposomes in HL-60 cells was revealed on the basis of flow cytometry and DNA electrophoresis.

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