Effect of AD-5423 on animal models of schizophrenia: phencyclidine-induced behavioral changes in mice.

The antipsychotic efficacy of AD-5423, which has the properties of both a serotonin 5-HT(2) and a dopamine D(2) receptor antagonist, was evaluated using animal models of schizophrenia. Sensitization to phencyclidine (PCP)-induced hyperlocomotion is considered a model of the positive symptoms of schizophrenia, and was significantly antagonized by AD-5423 and haloperidol. The PCP-induced enhancement of immobility induced by the forced swimming test, a model of the negative symptoms of schizophrenia, was attenuated by AD-5423 but not by haloperidol. Since this attenuated effect of AD-5423 was antagonized by DOI, a serotonin 5-HT(2) receptor agonist, it is postulated to be mediated by serotonin 5-HT(2) receptors. These findings suggest that AD-5423 would be clinically effective against both the positive and negative symptoms of schizophrenia.

Maternal origin of a unique extra chromosome, der(9)(pter-->q13::q13-->q12:) in a girl with typical trisomy 9p syndrome.

We report on a girl with the typical trisomy 9p syndrome who had an additional E-sized metacentric chromosome. On the basis of GTG- and CBG-banding, her karyotype was considered to be 47,XX,+der(9)(pter-->q13::q13-->q12:) de novo. Results of a fluorescence in situ hybridization study using a chromosome 9-specific painting probe were compatible with this cytogenetic interpretation. Molecular analyses of six highly polymorphic dinucleotide repeat loci on the short arm and the proximal long arm of chromosome 9 demonstrated that the girl inherited one allele from her father and two identical or different alleles from the mother. We speculated that the extra chromosome may have resulted from either nondisjunction of chromosome 9 followed by a U-type exchange and a crossing-over between different sister chromatids during maternal meiosis I and subsequent breakage and malsegregation during meiosis II, or nondisjunction during meiosis II followed by isochromosome formation in one of the two maternal chromosomes 9 and subsequent breakage.

Isoforms of caveolin-1 and caveolar structure.

The relationship between caveolin-1 isoforms alpha and beta and caveolar ultrastructure was studied. By immunofluorescence microscopy of human fibroblasts, caveolae were observed as dots positive for caveolin-1, but many dots labeled by an antibody recognizing both isoforms (anti-alphabeta) were not labeled by another antibody specific for the alpha isoform (anti-alpha). Immunogold electron microscopy of freeze-fracture replicas revealed caveolae of different depths, and indicated that anti-alpha labeled deep caveolae preferentially over shallow ones, whereas anti-alphabeta labeled both forms with an equivalent frequency and intensity. The presence of the beta isoform in deep caveolae was confirmed by labeling epitope-tagged beta-caveolin. When made to be expressed in HepG2 cells lacking endogenous caveolins, the alpha isoform formed caveolar depressions efficiently, but the beta isoform hardly did so. Caveolae were also formed in cells expressing the two isoforms, but their frequency was variable among cells of the same clone. Coexpression of caveolin-1 and caveolin-2 caused more efficient formation of deep caveolae than caveolin-1 alone. The result indicates that the two isoforms of caveolin-1 have a different potential for forming caveolae structure, and more importantly, that deep and shallow caveolae may be diversified in their molecular composition.

Pharmacologic properties of a novel Ca2+ entry blocker, AJ-2615, in vitro.

We studied the in vitro vascular relaxant properties of AJ-2615, (+/-)-N-[6,11-dihydrodibenzo[b,e]-thiepin-11-yl]-4-[4- fluorophenyl]-1-piperazinebutanamide monomaleate, a novel compound with long-lasting antihypertensive activity. AJ-2615 inhibited the high K(+)-induced contractile response in rat aorta with an IC50 of 2.08 x 10(-8) M. It was 13 times less potent than nifedipine and 3, 10, and 15 times more potent than verapamil, diltiazem, and fluanarizine, respectively. AJ-2615 also inhibited the high K(+)-induced 45Ca influx in rat aorta at almost the same concentration as that for inhibition of the contractile response. The inhibition of 45Ca influx was reversed by Bay k 8644, a Ca2+ channel agonist. The effects of AJ-2615 on the contractile response and Ca2+ influx persisted for at least 120 min after AJ-2615 was removed from the medium. These results indicate that AJ-2615 acts directly on the potential-dependent Ca2+ channel in a long-lasting manner. AJ-2615 inhibited [3H]prazosin binding to dog aortic membranes (IC50 = 1.25 x 10(-8) M) and phenylephrine-induced contractile response in superior mesenteric artery (SMA) of rabbits (IC50 = 3.87 x 10(-8) M), indicating that AJ-2615 has potent alpha 1-adrenoceptor blocking activity. AJ-2615 at 10(-6) M did not inhibit the caffeine-induced contractile response in rabbit SMA in Ca(2+)-free medium, nor did it inhibit calmodulin (CAM) activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological profile of AD-5423, a novel antipsychotic with both potent dopamine-D2 and serotonin-S2 antagonist properties.

The pharmacological properties of AD-5423 [2-(4-ethyl-1-piper-azinyl)-4- (4-fluorophenyl)-5,6,7,8,9,10-hexahydrocycloocta[b]pyridine] were studied in biochemical and behavioral tests. In vitro, AD-5423 bound preferentially to dopamine (DA)-D2 (Ki, 14.8 nM; cf. haloperidol, 8.79 nM; and clozapine, 149 nM) and serotonin (5-HT)-S2 (Ki, 3.98 nM; cf. haloperidol, 26.8 nM; and clozapine, 8.66 nM) receptors. It displayed low affinity for adrenaline (Ad)-alpha-1 (Ki, 56.3 nM) receptors and was virtually devoid of binding to DA-D1 (Ki, 2870 nM), 5-HT-S3, Ad-alpha-2, Ad-beta, muscarine, tau-aminobutyric acid and benzodiazepine receptors. In addition, AD-5423 was only a weak inhibitor of DA, 5-HT and noradrenaline uptake systems. When administered p.o., AD-5423 (0.3-10 mg/kg) increased brain contents of the DA metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid in mice and rats and the 5-HT metabolite 5-hydroxyindoleacetic acid in mice. Behaviorally, AD-5423 (0.2-2 mg/kg p.o.) decreased exploratory activity in mice, suppressed conditioned avoidance responding and methamphetamine-induced hyperactivity in mice and rats, antagonized apomorphine-induced gnawing in rats and vomiting in dogs and reduced hostile responses in monkeys. In these effects, AD-5423 was more or less equi-potent to haloperidol. However, AD-5423 (10 mg/kg p.o.), unlike haloperidol, did not antagonize SKF38393-induced vacuous oral movements in rats. Head twitches induced by 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane in mice and by para-chloroamphetamine in rats were antagonized by AD-5423 at much lower doses (0.5-2 mg/kg p.o.) than those of haloperidol and clozapine.(ABSTRACT TRUNCATED AT 250 WORDS)

Improvement of 5-HT3 receptor binding assay: enhancement of specific [3H]quipazine binding with Triton X-100-treated membranes from rat cortex.

The 5-hydroxytryptamine (5-HT)3 receptor binding assay using [3H]quipazine was examined. It was impossible to obtain specific [3H]quipazine binding with the membrane fractions from rat cortex prepared by the usual procedure. When the membranes were pretreated with detergent Triton X-100, the ratio of specific [3H]quipazine binding markedly increased, depending upon the concentration of Triton X-100 in the range of 0.01-0.1% (w/v). At a concentration of more than 0.05%, the specific binding reached a maximum of 55 to 60% of the total binding. The specific [3H]quipazine binding to the Triton X-100-treated membranes was reversible and was potently inhibited by several 5-HT3 antagonists, while 5-HT1, 5-HT2 receptor antagonists and other receptor-specific ligands had no effect on the binding. Scatchard analysis indicated a single class of binding sites with a Kd of 0.62 nM and Bmax of 97 fmol/mg protein. Thus, the Triton X-100-treated membranes retained the characteristics of 5-HT3 binding sites, making it possible to use [3H]quipazine for a 5-HT3 receptor binding assay with a high ratio of specific binding.

Significance of the methyl group on the oxazine ring of ofloxacin derivatives in the inhibition of bacterial and mammalian type II topoisomerases.

A study was made of the correlation between the in vitro inhibitory effects of several quinolones, including four ofloxacin derivatives, on bacterial DNA gyrase from Escherichia coli KL-16 and on topoisomerase II from fetal calf thymus. No correlation was observed between the inhibitions of DNA gyrase activity and topoisomerase II activity. On the other hand, the inhibitory effects of these quinolones against topoisomerase II were closely correlated with their inhibition of cell growth. Furthermore, among the oxazine derivatives tested, the derivative with a methyl group at position 3 in an S configuration showed the highest activity against DNA gyrase and derivatives without a methyl group on the oxazine ring were more potent against topoisomerase II than those with a methyl group. Among these derivatives, DR-3355, the S isomer of ofloxacin, showed the highest activity against DNA gyrase and low activity against topoisomerase II. These results indicate that the methyl group on the oxazine ring plays an important role in the inhibitory activities of ofloxacin derivatives for these enzymes.

Inhibitory effects of quinolones on murine hematopoietic progenitor cells and eukaryotic topoisomerase II.

The in vitro inhibitory effects of 12 quinolones, including clinically available compounds such as nalidixic acid, ofloxacin and enoxacin, on the murine hematopoietic progenitor cells and topoisomerase II of fetal calf thymus were compared. The quinolones tested inhibited the proliferation of the murine cells in a dose-dependent manner, and the cytotoxicity of quinolones correlated well (r = 0.910) with their inhibitory potency against the topoisomerase II.

In-vitro and in-vivo activity of DR-3355, an optically active isomer of ofloxacin.

DR-3355 [S-(-)-ofloxacin], an optically active isomer of ofloxacin, showed a broad spectrum of antibacterial activity against both Gram-positive and Gram-negative bacteria and was generally twice as potent as ofloxacin and considerably more active than DR-3354 [R-(+)-ofloxacin]. The activity of DR-3355 was largely unaffected by the type of culture medium, inoculum size and addition of human serum, but decreased under acidic condition at pH 6.0. The protective effect of orally administered DR-3355 in experimental infections in mice with various bacterial pathogens was superior to those of ofloxacin and ciprofloxacin.

Stimulation of macrophages by muroctasin to produce colony-stimulating factors.

Murocatasin (N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine, MDP-Lys(L18], a muramyl dipeptide derivative, has been reported to increase the number of peripheral granulocytes and monocytes after subcutaneous administration to animals and humans. When macrophage cell lines such as P388D1 and J774.1 cells were incubated with muroctasin in vitro, the production of colony-stimulating factor (CSF) from these cells was increased significantly. By Northern blot analysis, expression of the M-CSF gene, but not the G-CSF gene, in these macrophage cell lines was found to be enhanced by treatment with muroctasin. However, expression of the G-CSF gene in NFSA cells, a fibrosarcoma cell line established as a G-CSF producer, was actually enhanced by incubation with the conditioned medium from P388D1 cells stimulated with muroctasin. Thus, the hematopoietic activity of muroctasin was suggested to be attributable primarily to the enhanced production of M-CSF from macrophages. The enhanced G-CSF production from NFSA cells may be due at least to interleukin-1 released from muroctasin-stimulated macrophages.

Influences of cephem antibiotics on the immune response in mice.

The effects of six cephem antibiotics, including ceftezole, cefmetazole, cefoxitin, cefotiam, cefoperazone, and cefotaxime, on murine humoral immunity were examined. In female BDF1 mice each cephem antibiotic was administered at a dose of 800 mg/kg/day i.v. for 7 consecutive days. Among the antibiotics tested, only ceftezole and cefoperazone induced a significant increase in serum total IgM, but not in serum total IgG. Especially in case of ceftezole, the mice developed splenomegaly due to the proliferation of IgM-producing cells in the germinal centers. The proliferation of splenic IgM-producing cells was also observed in female thymus-deficient Balb/c-nu/nu mice receiving intravenous ceftezole. Thus, the drug was indicated to enhance the polyclonal IgM production in mice by acting as a B cell mitogen. This is consistent with the in vitro finding that ceftezole exhibited a mitogenic effect on whole spleen cells from BDF1 mice, but not on B cell depleted spleen cells.

Protective role of complement in the development of experimental pneumococcal pneumonia in mice.

To evaluate the role of complement in the lung defense against Streptococcus pneumoniae, mice decomplemented with multiple injections of cobra venom factor were challenged with type 3 pneumococci by inhalation. Without injection of cobra venom factor, the organisms were eliminated rapidly from the lungs in the majority of mice, accompanied by a significant but transient decrease in the serum C3 level. Focal pneumonia developed occasionally in some mice retaining the organisms in the lungs. By decomplementation with cobra venom factor, on the other hand, pneumococci were not eliminated completely from the lungs during the early stage of infection and afterward proliferated extensively. Consequently, the mice developed typical pneumococcal pneumonia with attendant bacteremia, while the serum C3 level has recovered compensatory during the course of infection. Thus, the complement was indicated to play an important role in the lung defense against pneumococci in mice, especially during the early stage of infection.

Purification and some properties of a hemolysin from the poisonous mushroom Rhodophyllus rhodopolius.

A hemolytic protein which causes diarrhea and death to mice was purified from the fruit bodies of a poisonous mushroom species Rhodophyllus rhodopolius (Fries) by DEAE-cellulose column chromatography, ammonium sulfate precipitation, gel filtration on Sephadex G-200, and DEAE-Sephadex A-25 column chromatography. The mol. wt of the purified hemolysin was estimated to be 40,000 by SDS-polyacrylamide slab gel electrophoresis. The hemolytic activity of the purified hemolysin was destroyed by heating at 60 degrees C for 10 min, and partially reduced by pepsin, papain and 2-mercaptoethanol. Cholesterol and phosphatidylcholine did not inhibit the activity. The hemolysin was unstable below pH 7.0 but stable at pH 8.0. The optimal pH for hemolysis was 6.0. Hemolysis did not occur below 4 degrees C even though the hemolysin bound to the erythrocyte. Mouse, chicken, rat, horse and human erythrocytes were sensitive in this order, but sheep and cow erythrocytes were not lysed by the toxin.

In-vitro activity of DR-3355, an optically active isomer of ofloxacin, against bacterial pathogens associated with travellers' diarrhoea.

The in-vitro activity of DR-3355, the S-(-)-isomer of ofloxacin, was determined against bacterial pathogens associated with travellers' diarrhoea. DR-3355 was highly active against isolates of enteropathogenic Escherichia coli (MIC90 0.05 mg/l), Salmonella spp. (MIC90 0.10 mg/l), Shigella spp. (MIC90 0.10 mg/l), Campylobacter jejuni (MIC90 0.39 mg/l) and Vibrio parahaemolyticus (MIC90 0.39 mg/l). The activity of DR-3355 against these bacteria was generally two- to eight-fold greater than that of ofloxacin and equal to, or only two-fold less than, that of ciprofloxacin with the exception of isolates of C. jejuni, which were two-fold less susceptible to ciprofloxacin than to DR-3355.

Structure-epileptogenicity relationship of quinolones with special reference to their interaction with gamma-aminobutyric acid receptor sites.

The relationship between the chemical structure and epileptogenic activity of quinolones was investigated. When the quinolones were administered intravenously to mice concomitantly with oral biphenylacetic acid, a major metabolite of the nonsteroidal antiinflammatory drug fenbufen, enoxacin, norfloxacin, ciprofloxacin, and pipemidic acid, which have an unsubstituted piperazine moiety at the 7 position of their parent nuclei, provoked clonic convulsions and subsequent death at doses of 6.25 mg/kg or more in a dose-dependent manner. AM-1091 and T-3262, which have an unsubstituted aminopyrrolidine moiety at their 7 positions, were less epileptogenic than the compounds listed above were. In contrast, ofloxacin, AT-4140, and nalidixic acid, which have piperazine substituted with methyl group(s) or no piperazine moiety at their 7 positions, never induced convulsions, even at doses of 100 mg/kg. Lomefloxacin, which has a 3-methyl piperazine, however, provoked convulsions at doses of 6.25 mg/kg or more. In the presence of biphenylacetic acid, all the test quinolones except nalidixic acid competitively inhibited [3H]muscimol binding to receptor sites for gamma-aminobutyric acid (GABA) in vitro. Nalidixic acid did not inhibit the binding at all, even at the highest concentration tested, i.e., 10(-4) M. The 50% inhibition doses for [3H]muscimol binding varied within 4 orders of magnitude or more, between 10(-8) to more than 10(-4) M for various compounds, and there was a close correlation between the epileptogenic activities of quinolones and their inhibitory potencies for [3H]muscimol binding to GABA receptor sites. These results indicate that the epileptogenic activity of quinolones possibly relates to the GABA-like structures of substituents at their 7 positions, which act as antagonists of GABA receptors.

Inhibitory effects of quinolones on DNA gyrase of Escherichia coli and topoisomerase II of fetal calf thymus.

The in vitro inhibitory effects of quinolones on the bacterial DNA gyrase of Escherichia coli KL-16 and topoisomerase II of fetal calf thymus were compared. All the quinolones tested required higher concentrations to inhibit the topoisomerase II than to inhibit the DNA gyrase, and no correlation existed among their inhibitory activities against both enzymes. However, there was a large difference among the quinolones in their selectivities between the bacterial enzyme and its eucaryotic counterpart. The selectivity of ofloxacin was highest, and the selectivities of CI-934 and nalidixic acid were lowest.

Sub-inhibitory and post-antibiotic effects of an optically active isomer of ofloxacin.

The sub-inhibitory and post-antibiotic suppressive effects of DR-3355, an optically active isomer (S-(-) form) of (+/-)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methylpiperazin-1-yl)-7- oxo-7H- pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid (ofloxacin) were compared with those of ofloxacin, ciprofloxacin and ceftazidime by observing changes in bacterial multiplication and morphology in vitro. DR-3355 affected the proliferation of Staphylococcus aureus E46, Escherichia coli E77156 and Pseudomonas aeruginosa PI-III at 1/4 to 1/2 times the minimal inhibitory concentration (MIC) for each strain. The compound also affected the morphology of these strains at 1/32 times the MICs. Upon MIC basis, these sub-inhibitory effects of DR-3355 were almost identical to those of the reference compounds. On the other hand, exposure of E. coli to 1 and 4 times the MIC of DR-3355 for 3 h produced post-antibiotic effects of 0.7 and 1.9 h, respectively, and the effects of the compound were almost equal to those of ofloxacin and ciprofloxacin and much greater than that of ceftazidime.