[Murine IgE antibody to recognize human major allergen, Mal f4].

To elucidate the specificity of murine IgE antibody against Malassezia furfur, the immunoblot patterns of IgE in sera obtained from mice inoculated repeatedly in the nasal cavity with M. f. cells, or injected intraperitoneally with M. f. cells mixed with Al (OH)3 gel were compared with those of IgE antibody detected in sera from patients with AD. Most of the murine IgE anti-Malassezia antibodies shared some antigenic bands with IgE in sera from patients with AD. The IgE antibody of murine also recognized Mal f4, one of the major allergens detected in patients with AD.

Pharmacological properties of (2R)-N-[1-(6-aminopyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamide: a novel mucarinic antagonist with M(2)-sparing antagonistic activity.

We evaluated the pharmacological profiles of (2R)-N-[1-(6- aminopyridin-2-ylmethyl)piperidin-4-yl]-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamide(compound A), which is a novel muscarinic receptor antagonist with M(2)-sparing antagonistic activity. Compound A inhibited [(3)H]NMS binding to cloned human muscarinic m1, m2, m3, m4, and m5 receptors expressed in Chinese hamster ovary cells with K(i) values (nM) of 1.5, 540, 2.8, 15, and 7.7, respectively. In isolated rat tissues, compound A inhibited carbachol-induced responses with 540-fold selectivity for trachea (K(B) = 1.2 nM) over atria (K(B) = 650 nM). In in vivo rat assays, compound A inhibited acetylcholine-induced bronchoconstriction and bradycardia with intravenous ED(50) values of 0.022 mg/kg and >/=10 mg/kg, respectively. Furthermore, in dogs, compound A (0.1-1 mg/kg p.o.) dose dependently shifted the methacholine concentration-respiratory resistance curves. In mice, compound A (10 mg/kg i.v.) did not inhibit oxotremorine-induced tremor. The brain/plasma ratio (K(p)) of compound A (3 mg/kg i.v.) was 0.13 in rats; this K(p) was less than that of scopolamine (1.7) and darifenacin (0.24). The inhibition of compound A (3 mg/kg i.v.) on ex vivo binding in rat cerebral cortex was almost similar to that of NMS. These findings demonstrate that compound A has high selectivity for M(3) receptors over M(2) receptors, displays a potent, oral M(3) antagonistic activity without inhibition of central muscarinic receptors because of low brain penetration. It is well known that central muscarinic antagonists may have diverse CNS effects, and M(2) receptors regulate cardiac pacing and act as autoreceptors in the lung and bladder. Thus, compound A may have fewer cardiac or CNS side effects than nonselective compounds.

Discovery of a muscarinic M3 receptor antagonist with high selectivity for M3 over M2 receptors among 2-[(1S,3S)-3-sulfonylaminocyclopentyl]phenylacetamide derivatives.

In the course of developing a metabolically stable M3 receptor antagonist from the prototype antagonist, J-104129 (1), introduction of certain substituents into the cyclopentane ring of 1 was found to be effective not only in improving metabolic stability but also in greatly enhancing the subtype selectivity. Among the cyclopentane analogues, sulfonamide derivatives (10f) and (10g) displayed 160- and 310-fold selectivity for M3 over M2 receptors, and both were significantly more selective than the prototype antagonist (120-fold). Subsequent derivatization of the sulfonamide series led to the highly selective M3 receptor antagonists (10h, 10i and 10j) with >490-fold selectivity for M3 over M2 receptors. Among them, p-nitrophenylsulfonamide (J-107320, 10h) exhibited 1100-fold selectivity for M3 receptors (Ki = 2.5 nM) over M2 receptors (Ki = 2800 nM) in the human muscarinic receptor binding assay using [3H]-NMS as a radio ligand.

[Suppressive effects of lanoconazole on arthus phenomenon in vivo and on production and functions of TNF in vitro].

The anti-inflammatory effect of lanoconazole (LCZ) was investigated in vivo and in vitro. The effect of LCZ was evaluated on the inflammatory reactions elicited by intradermal injection of ovalbumin to ovalbumin-immunized rabbits, as an Arthus phenomenon. A one or two % cream preparation of LCZ was topically applied on the lesion daily after challenging injection until the inflamation had diminished. By macroscopic observation and measuring the diameter of edema, erythema, hemorrhage and necrosis, the effects of LCZ on the reactions were compared with the reactions of the sites administered withcream vehicle as reference agent. Two % LCZ showed an anti-hemorrhagic effect. The in vitro effect of LCZ on production and functions of an inflammatory cytokine, TNF was also examined. LCZ suppressed the production of TNF by murine peritoneal macrophages at 20 micro g/ml and the adhesion of neutrophils at 100 micro g/ml. Moreover, LCZ significantly suppressed the growth inhibitory activity of TNF against L929 fibroblasts at 0.5 micro g/ml. A very low concentration of LCZ might protect the fibroblasts from immunological cytotoxicity in vivo. These findings suggest that LCZ has a suppressive activity to inflammatory responses and this suppressive action may be due to its protective activity to cells like fibroblasts.

A potent, long-acting, orally active (2R)-2-[(1R)-3, 3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamide: novel muscarinic M(3) receptor antagonist with high selectivity for M(3) over M(2) receptors.

A novel series of (2R)-2-[(1R)-3, 3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamides was designed and synthesized based on the structure and biological profiles of an active metabolite 2 of our prototype muscarinic M(3) receptor selective antagonist 1, to develop a potent, long-acting, orally active M(3) antagonist for the treatment of urinary tract disorders, irritable bowel syndrome, and respiratory disorders. Investigation of (2R)-2-[(1R)-3, 3-difluorocyclopentyl]-2-hydroxy-2-phenylacetamides containing a phenyl or heterocyclic ring as the piperidinyl side chain in place of the 4-methyl-3-pentenyl moiety of 15a revealed that this acid moiety was a versatile template for improving the selectivity for M(3) over M(2) receptors in comparison with the corresponding cyclopentylphenylacetic acid group. However, since the in vitro metabolic stability of these analogues was insufficient compared with that of 2, further derivatization was performed by introducing an appropriate hydrophilic group into the phenyl or 2-pyridyl ring. Thus, the 1-(6-aminopyridin-2-ylmethyl)piperidine analogue 15y exhibiting 190-fold selectivity for M(3) receptors (K(i) = 2.8 nM) over M(2) receptors (K(i) = 530 nM) in a human binding assay and good in vitro metabolic stability in dog and human hepatic microsomes was identified. This compound has excellent oral activity at 4 h after oral dosing (1 mg/kg), inhibiting methacholine-induced bronchoconstriction in dogs, and may be useful in clinical situations in which M(3) over M(2) selectivity is desirable.

Stereoselective synthesis of a new muscarinic M3 receptor antagonist, J-104129.

A diastereoselective synthesis of J-104129 (1) was developed. A key step of this synthesis was Michael addition of enolate generated from cis-chiral dioxolane 2 to cyclopentenone, followed by hydrogenolysis of the resultant enol triflate 4. A mixture of cyclopentyldioxolane (5, 6) was hydrolyzed with sodium hydroxide to yield carboxylic acid 7 in 86% ee.

[Taxonomy of genus Malassezia: progress and problems].

The genus Malassezia is composed of lipophilic basidiomycetous yeasts which were recently shown to consist of seven species, one lipid-independent species, M. pachydermatis and six lipid-dependent species, M. furfur, M. sympodialis, M. globosa, M. obtusa, M. restricta and M. slooffiae. Based on this classification, we will be able to analyze pathogenicity or relationship between Malassezia-related diseases and each species.

J-104129, a novel muscarinic M3 receptor antagonist with high selectivity for M3 over M2 receptors.

A new class of 4-acetamidopiperidine derivatives has been synthesized and investigated for human muscarinic receptor subtype selectivity. Introduction of a hydrocarbon chain of appropriate length into the piperidine nitrogen of the racemic N-(piperidin-4-yl)-2-cyclobutyl-2-hydroxy-2-phenylacetamide platform conferred up to 70-fold selectivity for human muscarinic M3 receptors over M2 receptors. Subsequent synthetic derivatizations resulted in highly potent M3 receptor antagonists with selectivity greater than two orders of magnitude for M3 over M2 receptors, from which the analogue 4r was selected. Preparation of both enantiomers of 4r led to the identification of (2R)-N-[1-(4-methyl-3-pentenyl)piperidin-4-yl]-2-cyclopentyl-2-hyd roxy-2-phenylacetamide (J-104129, (R)-4r), which exhibited 120-fold selectivity for M3 receptors (Ki = 4.2 nM) over M2 receptors (Ki = 490 nM). In isolated rat trachea, (R)-4r potently and specifically antagonized acetylcholine (ACh)-induced responses with a K(B) value of 3.3 nM. The highly subtype-selective profile was also seen in isolated rat tissue assays (50-fold) and in anesthetized rats (> 250-fold). Oral administration of J-104129 ((R)-4r) antagonized ACh-induced bronchoconstriction with an ED50 value of 0.58 mg/kg in rats. Thus, J-104129 ((R)-4r) may effectively facilitate bronchodilation in the treatment of obstructive airway disease.

Evaluation of plasma (1-->3)-beta-D-glucan measurement by the kinetic turbidimetric Limulus test, for the clinical diagnosis of mycotic infections.

The present multicentre clinical study was conducted to assess the clinical utility of a new diagnostic method for deep mycosis in which (1-->3)-beta-D-glucan, a fungal cell wall component existing in plasma, was quantitatively measured by the kinetic turbidimetric Limulus test (WB003). Plasma (1-->3)-beta-D-glucan concentrations were 0.57 +/- 0.10 microgram/l in 92 healthy subjects and 0.62 +/- 0.32 microgram/l in 26 patients with non-mycotic diseases (disease control group). In comparison with these healthy subjects and patients with non-mycotic diseases, patients with mycosis had significantly higher plasma (1-->3)-beta-D-glucan concentrations: 19.63 +/- 73.28 micrograms/l in 12 patients with candidaemia, 11.28 +/- 21.42 micrograms/l in 7 patients with urinary Candida infection, 4.84 +/- 12.71 micrograms/l in 5 patients with pulmonary candidiasis, and 12.21 +/- 31.31 micrograms/l in 4 patients with invasive pulmonary aspergillosis. On the statistical analysis of these data, a cut-off value was set at 1.0 microgram/l. Using this cut-off value, 3 patients with pulmonary cryptococcosis and 4 patients (4/6) with pulmonary aspergilloma were all negative with low plasma (1-->3-beta-D-glucan levels. The test WB003 provided equivalent or higher efficiency of diagnosis of candidiasis and aspergillosis, in comparison with commercially available antigen detection kits, demonstrating its utility as a diagnostic reagent. It may also be useful in assessing therapeutic effectiveness when used periodically after treatment.

[The antigen (CANDTEC antigen) detected by CAND TEC test for diagnosis of candidiasis].

Most guinea pigs inoculated with 5.4 x 10(9) of C. albicans intraperitoneally, produce CANDTEC antigen (GPCANDTECAG) in sera. The antigen is heat-labile (at 56 degrees C for 30 min) as is that in humans. According to gel filtration, the molecule size of the antigen from guinea pigs was 4000KDa or more. ELISA revealed the antigen-positive gel fractions to contain a small amount of mannan from the yeasts and C3. ELISA using rabbit anti-GPCANDTECAG serum indicated that the two CANDTEC antigens from guinea pigs and humans shared determinants. Gel filtration indicated that the CANDTEC antigen from patients was from 4000KDa to 3000KDa. In the antigen-positive gel fractions, IgM was detected by ELISA, but mannan and C3 were not detected. However, immunoblotting analysis on the antigen-positive fraction revealed a unique band of 200KDa, stained with concanavalin A-ALP. These findings indicate that CANDTEC antigens in guinea pigs and humans are immune complexes formed after infection of Candida, although the antigens have different components.

[Myocardial revascularization with bovine internal thoracic artery graft (BIOFLOW): a case report].

Bovine internal thoracic artery graft (BIOFLOW) was used for coronary artery bypass grafting (CABG). A 64-year-old woman who had undergone triple CABG with saphenous vein five years ago was referred to our hospital again with anginal attack. Coronary angiogram showed the obstruction of the graft for LAD and new stenosis at right coronary artery (RCA). Gastroepiploic artery was not available because the patient had a history of gastric ulcer several times, and saphenous vein had harvested initial operation. We planned re-CABG for LAD with left internal thoracic artery and RCA with BIOFLOW. But at the operation, LAD was not graftable because of severe calcification. So that we performed single CABG to RCA with BIOFLOW. The BIOFLOW was patent at the 28 th postoperative day. From our experience, BIOFLOW can be expected as a graft for CABG when saphenous vein, internal thoracic artery and gastroepiploic artery are not available.

Mouse IgE-mediated paw anaphylaxis in mice and rats.

Anaphylactic swelling is induced in the hind paws of mice and rats by intravenous challenge of antigen 72 h after subplantar injection of mouse IgE-rich antiserum into the paws, and can be quantified rapidly and accurately by measuring paw thickness. The edema model described may serve as an alternative to passive cutaneous anaphylaxis for studying immediate allergic reactions with mouse homocytotropic antibodies and screening potential antiallergic drugs.

Mouse paw anaphylaxis.

Anaphylactic swelling passively transferred to the hind paws of mice with homologous antibodies was investigated. The ascites fluid containing homocytotropic antibodies of the IgG1 class in a high concentration was obtained by repeated injections of bovine serum albumin in complete Freund's adjuvant into the peritoneal cavity of female ICR mice. The standard procedure for mouse paw anaphylaxis defined was as follows: female ICR mice received subcutaneously 25 microliters of a 1:20 diluted ascites in the hind footpad and 2 h later were given intravenously 1.0 mg of the antigen in saline, and subsequently at 5 min intervals paw thickness was assessed by means of a simple thickness gauge. Maximal paw edema occurred invariably 15 min after antigen challenge. Sex difference in response was not observed. Heating at 56 degrees C for 4 h resulted in a reduction of the activity of mouse ascites by approximately 40%. Histological examinations revealed that paw anaphylaxis was accompanied by mast cell degranulation and release of biologically active constituents from mast cell granules. Promethazine, pyrilamine, diphenhydramine, methysergide, cyproheptadine, ketotifen and phenoxybenzamine provided significant protection from the anaphylactic reaction in mice, while bromocriptine, dexamethasone and indomethacin were inactive. Relative drug effectiveness in paw anaphylaxis, passive cutaneous anaphylaxis and active and passive systemic anaphylaxis in mice was compared and discussed. Consequently, it was shown that mouse paw anaphylaxis was not only convenient for quantitative assessment of homocytotropic antibodies, but also useful in the routine work for screening of potential anti-allergic drugs.

Intraperitoneal systemic anaphylaxis in the mouse. I. Age-dependence of fatal anaphylactic shock.

Following the i.p. challenge of a shocking dose of BSA from 9 days up to 133 days after the s.c. injection of BSA in CFA, fatal anaphylaxis was induced regularly in female ICR mice that had been given the immunizing antigen when 8 weeks old. These immunized mice provided an antiserum to BSA that had the capacity to transfer fatal shock to normal recipient mice at a minimum Ab-N dose of 8 microgram when the i.p. route for challenge was employed. The optimal dose-range of antigen and antibody in order to elicit fatal shock following the i.p. challenge was much broader than that obtained by i.v. injection. Age is critical in producing fatal shock in mice; a 100% fatal anaphylaxis never occurred in groups of 6- and 7-week-old recipient mice although those at 8 weeks and older were sufficiently sensitized by the amounts of antibody given.