In vitro antifungal activity of KP-103, a novel triazole derivative, and its therapeutic efficacy against experimental plantar tinea pedis and cutaneous candidiasis in guinea pigs.

The in vitro activity of KP-103, a novel triazole derivative, against pathogenic fungi that cause dermatomycoses and its therapeutic efficacy against plantar tinea pedis and cutaneous candidiasis in guinea pigs were investigated. MICs were determined by a broth microdilution method with morpholinepropanesulfonic acid-buffered RPMI 1640 medium for Candida species and with Sabouraud dextrose broth for dermatophytes and by an agar dilution method with medium C for Malassezia furfur. KP-103 was the most active of all the drugs tested against Candida albicans (geometric mean [GM] MIC, 0.002 microg/ml), other Candida species including Candida parapsilosis and Candida glabrata (GM MICs, 0.0039 to 0.0442 microg/ml), and M. furfur (GM MIC, 0.025 microg/ml). KP-103 (1% solution) was highly effective as a treatment for guinea pigs with cutaneous candidiasis and achieved mycological eradication in 8 of the 10 infected animals, whereas none of the imidazoles tested (1% solutions) was effective in even reducing the levels of the infecting fungi. KP-103 was as active as clotrimazole and neticonazole but was less active than lanoconazole and butenafine against Trichophyton rubrum (MIC at which 80% of isolates are inhibited [MIC(80)], 0.125 microg/ml) and Trichophyton mentagrophytes (MIC(80), 0.25 microg/ml). However, KP-103 (1% solution) exerted therapeutic efficacy superior to that of neticonazole and comparable to those of lanoconazole and butenafine, yielding negative cultures for all samples from guinea pigs with plantar tinea pedis tested. This suggests that KP-103 has better pharmacokinetic properties in skin tissue than the reference drugs. Because the in vitro activity of KP-103, unlike those of the reference drugs, against T. mentagrophytes was not affected by hair as a keratinic substance, its excellent therapeutic efficacy seems to be attributable to good retention of its antifungal activity in skin tissue, in addition to its potency.

Synthesis and antifungal activities of (2R,3R)-2-aryl-1-azolyl-3-(substituted amino)-2-butanol derivatives as topical antifungal agents.

2-Aryl-1-azolyl-3-(substituted amino)-2-butanol derivatives I were prepared by ring-opening reaction of epoxides II with excess amine, and their antifungal activities were evaluated as topical agents. Azolyl-cyclic amine derivatives with a methylene group showed extremely strong activity with a broad spectrum in vitro. In general, anti-Trichophyton mentagrophytes activities of most of the topical antifungal agents are substantially reduced by addition of keratin (a major constituent of the keratinized tissue). However, the triazole derivative (2R,3R)-2-(2,4-difluorophenyl)-3-(4-methylenepiperidino)-1-(1H-1,2 ,4- triazol-1-yl)-2-butanol ((-)-40, KP-103) showed very little deactivation by addition of keratin. This biological characteristic of triazole derivative (-)-40 resulted in excellent therapeutic efficacy on dermatophytosis superior to that of the corresponding imidazole derivative ((-)-41).

Synthesis and antibacterial activity of novel 7-(3-substituted-3 or 4-trifluoromethyl-1-pyrrolidinyl)-8-methoxyfluoroquinolones.

The titled compounds were synthesized and evaluated for in vitro antibacterial activity. The (3R, 4S)-3-aminomethyl-4-trifluoromethyl derivative (S-34109) was confirmed to be optimal because of its superior activity against quinolone and methicillin-resistant Staphylococcus aureus and low side effect potential.

Acceleration of natural killer (NK) cell recovery by a glucan, sizofiran, in anti-asialoGM1 antibody-treated mice.

The proportion of asialoGM1 positive cells and NK activity of murine splenic cells was reduced to almost zero one day after intravenous injection of rabbit anti-asialoGM1 antibody. The cells and the activity started to increase at the latest 3 days after the injection, although the proportion was far below that of the normal control. The proportion of asialoGM1 positive cells and NK activity increased more remarkably when 1,3-beta glucan, sizofiran, was administered intramuscularly one day after the antibody injection. The increases were dose related (50-1000 micrograms/mouse). The fact that sizofiran hastened the recoveries of splenic NK activity and asialoGM1 positive cells suggests sizofiran may have the activity to accelerate the differentiation of asialoGM1 positive NK cells.

Inhibitory effects of sizofiran on anticancer agent- or X-ray-induced sister chromatid exchanges and mitotic block in murine bone marrow cells.

The inhibitory effects of a biological response modifier (BRM), sizofiran, on sister chromatid exchanges (SCEs) in the bone marrow cells of mice treated with various anticancer agents or irradiation were investigated. Sizofiran (10 mg/kg i.m.) inhibited SCEs induced by mitomycin C (2 mg/kg i.v.), adriamycin (20 mg/kg i.v.) and cyclophosphamide (20 mg/kg i.v.) by about 20%, respectively. Analysis of the SCEs in vivo after irradiation plus sizofiran indicated that SCE levels were significantly lower than those observed in mice exposed to irradiation without sizofiran. Moreover, the effects of sizofiran were dependent on the timing of administration. Our results indicated that sizofiran should be administered simultaneously or soon after irradiation in order to minimize damage. Sizofiran also markedly restored the bone marrow cell mitosis which had been suppressed by anticancer agents, and this action was closely correlated with the prevention of increase in SCEs. These results indicate that in addition to immunopotentiating activity, sizofiran may play a role in preventing chromosomal damage induced by cancer chemotherapy and radiotherapy.

Two mechanisms of butenafine action in Candida albicans.

The mechanism of action of a new benzylamine antimycotic, butenafine hydrochloride, was studied in Candida albicans by using the thiocarbamate antimycotic tolnaftate as a reference drug. Butenafine completely inhibited the growth of a test strain of C. albicans at 25 micrograms/ml and was cidal at 50 micrograms/ml. Tolnaftate did not show any growth-inhibitory activity up to 100 micrograms/ml. Both butenafine and tolnaftate inhibited squalene epoxidation in C. albicans, with 50% inhibitory concentrations being 0.57 and 0.17 microgram/ml, respectively. Butenafine, but not tolnaftate, induced the release of appreciable amounts of Pi from C. albicans cells at 12.5 micrograms/ml. This effect of butenafine was augmented when the cells were pretreated with tolnaftate. The results suggest that the direct membrane-damaging effect of butenafine may play a major role in its anticandidal activity and that the drug-induced alteration in the cellular sterol composition renders the cell membrane more susceptible to the membrane-damaging effect of this drug.

Anti-Trichophyton mentagrophytes activity and percutaneous permeation of butenafine in guinea pigs.

We examined anti-Trichophyton mentagrophytes activity, cutaneous penetration, and skin localization of butenafine, a novel benzylamine antifungal agent. The following results were obtained. (i) In the guinea pig dorsal skin trichophytosis model, butenafine produced complete eradication of fungi from infected sites. Clotrimazole was active when animals were infected with 10(4) or 10(5) cells but was almost inactive when the inoculum size was 10(6) cells. (ii) The MICs of butenafine and clotrimazole against arthrospores of T. mentagrophytes KD-04 were 0.025 and 0.39 microgram/ml, respectively. (iii) When 0.2 ml of a 1% 14C-butenafine solution was applied for 23 h/day for 7 days, high radioactivity corresponding to 250 to 500 micrograms of butenafine per g of skin in the epidermis, including the horny layer, was observed. (iv) Butenafine penetrates through transepidermal and transfollicular routes. The excellent therapeutic efficacy of butenafine on experimental dermatophytosis may be attributed to its low MIC and good penetration and distribution in the horny layer and hair follicles, where fungi reside.

Topical treatment with butenafine significantly lowers relapse rate in an interdigital tinea pedis model in guinea pigs.

Butenafine is a novel antifungal agent of the class of benzylamines. The incidence of relapse after topical treatment with butenafine or bifonazole was investigated in a guinea pig interdigital tinea pedis model. One percent butenafine or bifonazole cream was applied on the infected site of animals for 20 consecutive days starting on day 10 postinfection. On day 30 posttreatment, relapse of the infection occurred in 11 of the 12 feet treated with bifonazole but in only 3 of the 12 feet treated with butenafine. The lower relapse rate after butenafine treatment might be attributable to its potent fungicidal activity and long retention time in the skin.

Combination therapy of radiation and Sizofiran (SPG) on the tumor growth and metastasis on squamous-cell carcinoma NR-S1 in syngeneic C3H/He mice.

The efficacy of Sizofiran(SPG), a highly purified beta-1,3-D-glucan from the culture broth of basidiomycetes Schizophyllum commune Fries, in combination with local irradiation was investigated using squamous-cell carcinoma NR-S1 and syngeneic hosts of C3H/He mice. NR-S1 tumor was implanted sc in the thigh of C3H/He mice. When tumor grew to 4 mm in diameter, the local irradiation of 55 Gy was delivered. SPG was injected im at a dose of 5 mg/kg. When SPG was administered after irradiation, remarkable inhibition of tumor growth was observed in comparison with the radiation alone group. Furthermore, the combination effect of radiation and active immunotherapy using mitomycin C-treated NR-S1 cells as vaccine was examined. When radiotherapy and active immunotherapy were combined with SPG, suppression of tumor growth was observed from an early stage in comparison with the group which was not administered SPG. SPG also inhibited the pulmonary metastasis of NR-S1 tumor after radiotherapy.

[Synthesis and antifungal activity of butenafine hydrochloride (KP-363), a new benzylamine antifungal agent].

In screening of new antifungal agents, bis(naphthalenemethyl)amines were found to have more potent antifungal activity than clotrimazole. Studies on their structure-activity relationships indicated that benzylamines had potent antifungal activity. Among them, butenafine hydrochloride (N-p-tert-butylbenzyl-N-methyl-1-naphthalenemethylamine hydrochloride, KP-363) has proved to show the strongest activity. It exhibits a wide spectrum activity in vitro against particularly dermatophytes (87 strains; minimal inhibitory concentration (MIC) range, 0.0015 to 0.05 microgram/ml), and also against Aspergillus (15 strains; MIC range, 0.025 to 0.78 microgram/ml), Cryptococcus neoformans (4 strains; MICs 0.78 and 1.56 micrograms/ml) and yeasts of genus Candida (67 strains; MIC range, 3.13 to greater than 100 micrograms/ml).

Effects of butenafine hydrochloride, a new benzylamine derivative, on experimental dermatophytosis in guinea pigs.

Butenafine hydrochloride, N-4-tert-butylbenzyl-N-methyl-1-naphthalenemethylamine hydrochloride (butenafine), is a novel antifungal agent of the class of benzylamine derivatives. Butenafine was investigated for its activity against guinea pig dermatophytosis caused by Trichophyton mentagrophytes or Microsporum canis in comparison with those of naftifine, tolnaftate, clotrimazole, and bifonazole. Topical butenafine showed excellent efficacy against dermatophytosis when it was applied once daily, and the effect was superior to those of all four reference drugs. When applied once at 24 or 48 h before infection, the drug exhibited excellent prophylactic efficacy against experimental T. mentagrophytes infection. The concentrations of butenafine in animal skin at 24 and 48 h after application of 0.2 ml of a 1% solution were several hundred times higher than those required to kill T. mentagrophytes and M. canis. The good efficacy of butenafine against dermatophytosis may be attributable to its fungicidal activity and long retention in the skin after topical application.

Effects of butenafine hydrochloride, a new benzylamine derivative, on experimental tinea pedis in guinea pigs.

Butenafine is a new antifungal benzylamine. The efficacy of butenafine was investigated in an experimental tinea pedis model in guinea pigs, which is pathologically similar to natural infections in humans. Butenafine (0.1 ml) in 0.2 to 1.0% solutions was applied to the site of infection. Treatment was started on day 10 postinfection and was continued for 20 days. Butenafine applied once daily exhibited excellent dose-related therapeutic efficacy. The efficacy of butenafine was significantly superior to those tolnaftate, clotrimazole, and bifonazole.

Malignant ameloblastoma with pulmonary metastasis and hypercalcemia. Report of an autopsy case and review of the literature.

A case of malignant ameloblastoma with hypercalcemia in a 67-year-old Japanese woman is presented. The tumor of the maxilla was removed and diagnosed as a follicular ameloblastoma. The tumor recurred in the lower orbita-zygoma region, and multiple tumors of the lungs and hypercalcemia were detected eight months after the second operation. The recurrent tumor resembled the primary tumor but was less well differentiated. Autopsy revealed widespread lung metastasis of the malignant ameloblastoma, nephrocalcinosis, and sigmoid colon cancer. Histologic examination showed the metastatic ameloblastoma to be composed of nests and strands of basaloid and spindle-shaped cells surrounded by columnar cells arranged in palisade formation, with focal areas of squamous differentiation and occasional cystic degeneration. Only two cases of malignant ameloblastoma with hypercalcemia have previously been reported. This is the first case of malignant ameloblastoma with hypercalcemia and sigmoid colon cancer. In addition, prostaglandin E2 assay revealed that ameloblastoma produces prostaglandin E2, which results in hypercalcemia.

[Combination therapy of radiation and schizophyllan (SPG) in C3H mouse squamous-cell carcinoma NR-S1].

The efficacy of schizophyllan, a beta-1, 3-D-glucan with a beta-1, 6-linked D-glucose residue isolated from Schizophyllum commune Fries, in combination with local radiation therapy was investigated using C3H mouse squamous-cell carcinoma NR-S1. Male C3H/He mice were implanted with 10(5) cells of NR-S1 tumor on day 0. On day 9 after implantation, mice received electron irradiation with a dose of 5,500 rads. Intramuscular injection of SPG (1.0 mg/kg and 5.0 mg/kg) was then started from the day following irradiation and repeated 15 times at one-day intervals. The antitumor effect was measured by the tumor size and survival time. Significant tumor growth suppression and prolongation of life-span were observed in the group given radiation and SPG (5.0 mg/kg) in comparison with the group which received radiation alone. In histopathological examination of irradiated tumor sites, stromal reactions accompanied by a much greater degree of cellular infiltration consisting of mainly lymphocytes were observed in the group given combined radiation and SPG (5.0 mg/kg).

Purification, physicochemical characterization, and antitumor activity of a cancer-associated human serum protein that is increased by treatment with schizophyllan, an antitumor polysaccharide.

A serum protein was purified from normal human sera by several steps of purification, and tentatively designated as cancer-associated serum protein (CAP-135), since the content of the protein was remarkably decreased in cancer patients. The purified CAP-135 has an approximate molecular weight of 135,000 daltons, and is composed of three subunits of 78,000, 34,500 and 24,800 daltons. CAP-135 showed an isoelectric point of pH 5.5-5.8 and contained a small amount (1.38%) of neutral sugar. CAP-135 is assumed to be modified complement C3 on the basis that it reacted only with anticomplement C3 in immunodiffusion assay, and one of its subunits had a molecular weight similar to that of the beta-chain of human complement C3. The ip injection of CAP-135 apparently inhibited the growth of sarcoma-180 implanted into the groin of Jc1:ICR female mice. The present results indicate that CAP-135 may be of diagnostic value.

[Antitumor activity of schizophyllan (SPG) against syngeneic ACI/N rat tumor, AMC-60 fibrosarcoma and BC-47 bladder cancer].

The antitumor activity of schizophyllan (SPG) against syngeneic ACI/N rat tumor, AMC-60 fibrosarcoma and BC-47 bladder cancer was investigated. Intramuscular injection of SPG caused a marked suppression of AMC-60 tumor growth. Results obtained with cytotoxicity tests in vitro and lymphoblastogeneic response in vitro suggest the association of enhanced macrophage cytostasis and high reactivity of lymphoid cells with the resistance of the host to AMC-60 tumor. Moreover, SPG showed pronounced antitumor activity against BC-47 bladder cancer. The highest therapeutic effectiveness was obtained when SPG injection was started at an advanced stage of the tumor, 4 out of 15 rats treated being completely cured of cancer. Cytotoxicity tests in vitro and neutralization tests in vivo indicated the important role of cytotoxic lymphocytes as well as activated macrophages in the host defence mechanism against BC-47 cancer. This, taken together with the fact that the rats cured completely by SPG therapy acquired transplantation resistance to BC-47 cancer, shows that SPG may promote tumor immunity in the host.

Special toxicology--physical dependence potential, antigenicity and mutagenicity--of mabuterol.

The physical dependence potency of dl-1- (4-amino-3-chloro-5-trifluoromethyl-phenyl)-2-tert.-butyl-amino-ethanol hydrochloride (mabuterol) was tested in male rats dosed with 20, 100 and weekly increasing doses of 20, 40, 60, 80 and 100 mg/kg over 7 weeks. The results were compared with those after the same increasing dosages of morphine. 10 males were used per group. The effects of the antagonist levallorphan on drug dependency were also studied. No withdrawal signs were observed in any mabuterol-treated groups. No antigenic potency of mabuterol was found in a series of studies on guinea pigs and rats. In a series of mutagenicity studies (Ames-test, DNA repair test and micronucleus test) it could be demonstrated that mabuterol is not a mutagen.

Cooperative role of T lymphocytes and macrophages in anti-tumor activity of mice pretreated with schizophyllan (SPG).

Anti-tumor activity was studied in mice injected with Schizophyllan (SPG), a glucan produced by Schizophyllum commune Fries. SPG-injected mice rejected subcutaneously inoculated sarcoma-180 and the anti-tumor activity was shown to be mediated by host spleen and lymph node cells. The anti-tumor activity lasted as long as 60 days after the SPG administration as assessed by transfer of cells into normal recipients. Cells involved in anti-tumor activity were shown to be T lymphocytes since anti-tumor activity was diminished when lymph node cells from SPG-treated mice were treated with anti-Thy-1.2 sera plus complement, whereas cells passed through a nylon wool column retained the activity. Macrophages were also shown to be involved since administration of carrageenan or trypan blue into the host decreased the inhibition ratio of tumor growth. It was concluded that anti-tumor activity in SPG-treated mice was mediated by the cooperation of T lymphocytes and macrophages, thus the impairment of either function decreased anti-tumor activity.

[Experimental study on immunochemotherapy using schizophyllan].

Immunochemotherapy with schizophyllan (SPG) combined with chemotherapeutic agents was evaluated in two syngeneic tumor-C3H/He mouse systems. The administration of SPG alone caused a significant growth inhibition of MM 46 mammary carcinoma, the highest therapeutic effect being obtained by SPG given at the advanced stage of tumors. When combined with neocarzinostatin given 7-times every other day the simultaneous administration of SPG produced an optimal response to that therapy. In X-5563 plasmacytoma, SPG alone was ineffective. However, when combined with mitomycin C given with 1 to 5-days interval, the concurrent administration of SPG prolonged significantly the life-span of the tumor-bearing mice. These results indicated that the simultaneous administration of SPG and antitumor drugs may be a useful application method for immunochemotherapy of experimental tumors.