Effect of low-temperature degradation on the mechanical and microstructural properties of tooth-colored 3Y-TZP ceramics.

The aim of the present study was to evaluate the effects of low-temperature degradation (LTD) induced by autoclaving on the mechanical and microstructural properties of tooth-colored 3 mol% yttria-stabilized tetragonal zirconia polycrystals (3Y-TZP). In total, 162 disc-shaped 3Y-TZP specimens were prepared. Two-thirds of the specimens were shaded by either the infiltration or powder mixing methods while the remaining specimens were used without coloring. The specimens were autoclaved at 134 °C for 0, 10, and 100 h to induce LTD (n=18 for each group). Chemical compositions were analyzed with X-ray fluorescence spectroscopy. Biaxial flexural strength was measured using a piston-on-three-ball test. The surface fraction and penetration depth of the monoclinic phase were examined using X-ray diffraction and scanning electron microscopy, respectively. The tooth-colored 3Y-TZP specimens contained Fe2O3 and Er2O3 (infiltration technique), and Fe2O3 (powder mixing method) at concentrations of<0.5 wt%. The tooth-colored 3Y-TZP had higher strength than the non-colored material after 100 h of autoclaving. In terms of surface fraction and penetration depth, the generation of monoclinic phase was significantly lower in the tooth-colored 3Y-TZP than in the non-colored material. The tooth-colored 3Y-TZP possessed equivalent biaxial flexural strength to that of the non-colored material and higher resistance to LTD regardless of the coloring technique (infiltration technique or powder mixing method) when the coloring pigments were contained at concentrations used in the present study.

The influence of low-temperature degradation and cyclic loading on the fracture resistance of monolithic zirconia molar crowns.

The present study analyzed the kinetics of low-temperature degradation (LTD) in zirconia, and evaluated the influence of LTD and cyclic loading on the fracture resistance of monolithic zirconia molar crowns. Bar-shaped zirconia specimens were divided into nine groups and autoclaved at 134°C for 0-200h to induce LTD. The surface fraction and penetration depth of the monoclinic phase were examined using X-ray diffraction and scanning electron microscopy. Monolithic zirconia molar crowns were prepared for crown fracture testing. The crowns were autoclaved for 0-100h (n=6) and cemented to dies. Six crown-die samples that were not autoclaved and six samples that were autoclaved for 100h were subjected to cyclic loading with a load of 300N for 240,000 cycles. All samples were tested in a load-to-failure test. The monoclinic fraction on the surface increased with autoclaving time and reached a plateau after 50h. The depth of the monoclinic phase increased without reaching a plateau. The fracture load of the crowns significantly decreased from 5683N (SD: 342) to 3975N (SD: 194) after 100h of autoclaving. Cyclic loading did not significantly affect the fracture resistance of the crowns in all cases. Kinetic analysis showed no linear correlation between the surface fraction and depth of the monoclinic phase after 50h of autoclaving. Even though LTD increased the monoclinic phase, resulting in lower strength, the fracture resistance of the monolithic zirconia crowns was still sufficient to withstand the loading conditions in the molar regions.

Inhibitory or scavenging action of ketoconazole and ciclopiroxolamine against reactive oxygen species released by primed inflammatory cells.

BACKGROUND: Reactive oxygen species (ROS) released from inflammatory cells constitute one of the critical causative factors in inflammatory skin diseases such as seborrhoeic dermatitis and atopic dermatitis. OBJECTIVES: To investigate inhibitory effects of ketoconazole (KCZ) and ciclopiroxolamine (CPO), both of which have been used for the treatment of seborrhoeic dermatitis, on ROS released from inflammatory cells. METHODS: The methyl-Cypridina-luciferin analogue-dependent chemiluminescence method was employed for the detection of ROS production by phorbol 12-myristate 13-acetate (PMA)-stimulated inflammatory cells. Moreover, the radical scavenging activities of both agents were examined by using a hypoxanthine-xanthine oxidase system and the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH). NADPH oxidase activity was determined in particulate (membrane) fractions prepared from PMA-stimulated RAW 264 x 7 cells, a macrophage-like cell line. RESULTS: Both of these antifungal agents inhibited PMA-stimulated ROS production. However, only CPO significantly scavenged both ROS generated by the hypoxanthine-xanthine oxidase system and DPPH, and the scavenging activity of CPO seemed to act on ROS other than superoxide anions. Although KCZ inhibited PMA-stimulated ROS production, it did not show radical-scavenging activities. The inhibition of ROS production by KCZ is probably attributable to the inhibition of NADPH oxidase activity. CONCLUSIONS: The mechanism of the inhibitory action of KCZ against PMA-stimulated ROS production is distinct from that of CPO. Knowledge of the inhibitory or scavenging effects of both antifungal agents on ROS released from inflammatory cells may be useful in developing a therapeutic strategy for dermatitis.

Actin gene-targeted RT-PCR could be a useful method for evaluating in vitro fungicidal activity against dermatophytes.

This study examined the effects of the antifungal agents amorolfine hydrochloride (AMF) and bifonazole (BFZ) on actin mRNA expression determined by reverse transcription-polymerase chain reaction (RT-PCR) amplification, and the morphology of Trichophyton mentagrophytes. In AMF-treated cultures, the hyphal cells of T. mentagrophytes exhibited concentration- and/or time-dependent progressively degenerative morphological damage. Those cultures exhibiting severe and necrotic morphological changes (such as crushed, bent and flattened hyphal cells) did not express actin mRNA. In BFZ-treated cultures, similar morphological changes were seen in the hyphae of T. mentagrophytes, but these changes were milder than observed in the AMF-treated samples, and actin gene fragments were amplified in all of these samples. These results indicate that actin gene-targeted RT-PCR could be a useful tool for testing the susceptibility of dermatophytes to antifungal agents in vitro.

Comparison of in vitro antifungal activities of topical antimycotics launched in 1990s in Japan.

In vitro anti-dermatophyte, anti-Candida albicans and anti-Malassezia furfur activities of amorolfine hydrochloride (AMF), terbinafine hydrochloride (TBF), butenafine hydrochloride (BTF), neticonazole hydrochloride (NCZ) and ketoconazole (KCZ), all of which were introduced for the treatment of dermatomycoses in the 1990s in Japan, were compared. Although all of the test drugs are classified as an ergosterol biosynthesis inhibitor, the antifungal properties were found to be different. TBF and BTF exerted extremely potent antifungal activity against Trichophyton spp. but not against C. albicans and M. furfur, whilst KCZ and NCZ showed potent antifungal activity against C. albicans and M. furfur rather than Trichophyton spp. AMF exhibited potent antifungal activity against all of the fungal species tested. Fungicidal activities of these antifungal agents against T. rubrum were determined by using neutral red staining. The fungicidal potentialities correlated with those obtained in the in vitro susceptibility test as determined by MICs against dermatophytes. TBF, BTF and AMF exerted more potent fungicidal action than NCZ and KCZ.

Inhibition of sterol 14 alpha-demethylation of Candida albicans with NND-502, a novel optically active imidazole antimycotic agent.

To investigate the mode of action of the newly synthesized optically active imidazole compound, NND-502, (-)-(E)-[4-(2, 4-dichlorophenyl)-1,3-dithiolan-2-ylidene]-1-imidazolylacetonit rile, its effect on ergosterol biosynthesis in cell-free extracts of Candida albicans was examined and compared with that of the (S)-enantiomer of NND-502 in addition to lanoconazole and bifonazole, both of which are clinically used for the treatment of dermatomycoses. NND-502 was found to interfere with ergosterol biosynthesis by inhibition of sterol 14alpha-demethylase, while no interference due to the (S)-enantiomer of NND-502 was found, indicating that the stereochemical orientation of the 2, 4-dichlorophenyl group plays an important role in the interaction with the enzyme. In terms of drug concentration exerting 50% inhibition of ergosterol biosynthesis, NND-502 was 2.5 and 28 times more effective than that of lanoconazole and bifonazole, respectively.

Efficacy of NND-502, a novel imidazole antimycotic agent, in experimental models of Candida albicans and Aspergillus fumigatus infections.

In vitro and in vivo anti-Candida albicans and anti-Aspergillus fumigatus activities of NND-502, a new imidazole-antimycotic, were compared with those of fluconazole (FCZ), itraconazole (ITZ) and/or amphotericin B (AmB). NND-502 exhibited strong in vitro antifungal activity against both fungal species; its MIC against C. albicans was 1-4 times lower than that of FCZ, and its MIC against A. fumigatus was at least 60-2000 times lower than that of ITZ and AmB. In vivo antifungal treatments with each drug were initiated 1 h after inoculation in the experimental models, so that antifungal potential reflected prophylactic activity rather than therapeutic activity. The oral regimen of NND-502 in a murine model of systemic C. albicans infection was much less effective than that of FCZ. In vivo anti-A. fumigatus activity of oral NND-502 in a murine model of systemic infection was apparently superior to that of FCZ and ITZ in terms of prolonging survival. In addition to the murine model of systemic aspergillosis, intravenous NND-502 was shown to be highly effective in a rat model of pulmonary aspergillosis compared with intravenous AmB; 90% of animals survived at a dose of 2.5 mg/kg per day of NND-502 while only 30% of animals escaped death when 5 mg/kg per day of AmB was used. This potent efficacy of NND-502 was also confirmed in a sublethal challenge study in which the administration of the agent at a dose as low as 1.25 mg/kg per day resulted in the significant reduction of organisms in the lung; no comparable effect of AmB was found.

Fibroblast-migration in a wound model of ascorbic acid-supplemented three-dimensional culture system: the effects of cytokines and malotilate, a new wound healing stimulant, on cell-migration.

To assess the migratory response of fibroblasts in vitro, normal human dermal fibroblasts (NHDF) were cultured in the presence of L-ascorbic acid 2-phosphate to induce a multilayered structure. Round wounds were made by punching, and the migratory response was evaluated by counting the number of migrating cells in the wounded areas. Collagenase activity in the culture-medium was then measured. When the wound model was treated with bFGF, IL-1 alpha or PDGF, the migratory response was facilitated with increased collagenase secretion. In contrast, treatment with TGF-beta reduced the migratory response and collagenase secretion. Since the multilayered structure is rich in collagenous matrix, degradation of the matrix by secreted collagenase is probably necessary for the cells to migrate into the wounded areas. Furthermore, malotilate, which is now under development as an agent for wound therapy, facilitated the migratory response of NHDF with increased collagenase secretion in this wound model, suggesting that the wound healing effect of malotilate is in part attributable to stimulated migration of fibroblasts to wounded areas subsequent to extracellular matrix-degradation.

In vitro and in vivo antidermatophyte activities of NND-502, a novel optically active imidazole antimycotic agent.

In vitro and in vivo antidermatophyte activities of NND-502, a new imidazole antimycotic agent, were compared with those of two existing antifungal agents, lanoconazole and terbinafine. NND-502 exhibited strong in vitro antifungal activity against Trichophyton spp.; its MIC was 1 to 4 times lower than that of lanoconazole or terbinafine. In an in vivo study with a guinea pig model of tinea pedis, 7-day topical treatment with a 0.5% solution of NND-502 (dissolved in polyethylene glycol 400) was more effective than that with a 0.5% solution of either lanoconazole or terbinafine for eradicating fungi from the infected feet. When the duration of treatment was shortened to 3 days, a topical 1% solution of NND-502 achieved a complete mycological cure, while topical 1% solutions of lanoconazole and terbinafine did not.

Studies on the antifungal activity of the new imidazole antimycotic lanoconazole in infected sites. Distribution in the skin and in vitro activity in the presence of stratum corneum.

To answer the question why topically applied lanoconazole (CAS 101530-10-3, NND-318) is so highly effective in the treatment of dermatomycoses in both animal models and human patients, the antifungal activity of lanoconazole in infected sites was investigated. 1. Distribution of lanoconazole in rat skin: The distribution of lanoconazole within the dorsal skin of rats was examined by measurement of radioactivity and microscopic autoradiography. 24 h after dermal application of 14C-lanoconazole cream formulation, 83% of the total radioactivity in the skin was recovered from the stratum corneum, and thereafter the radioactivity decreased gradually up to 96 h. Metabolite analysis showed that more than 94% of the extractable radioactivity was lanoconazole itself after 24 and 48 h. Microautoradiograms of the skin also supported the radioactivity distribution. 2. In vitro antifungal activity in stratum corneum-containing medium: Candida albicans TIMM 2640 was incubated for 10 days at 27 degrees C in a vitamin-supplemented yeast carbon base medium containing 5 mg/ml of human stratum corneum and different concentrations of lanoconazole or bifonazole (CAS 60628-96-8, reference agent). Compared with the control culture, lanoconazole strongly inhibited fungal growth in a concentration dependent manner at concentration above 0.1 microgram/ml, resulting in a reduction of viable cell recovery to less than 50% after 10 days. The inhibitory activity of bifonazole was clearly weaker than that of lanoconazole. At concentrations of 0.1-10 micrograms/ml lanoconazole reduced keratinolytic acid proteinase activity in the culture-supernate to 40-70% of the control value, while bifonazole showed 50% reduction of the activity at a concentration of 10 micrograms/ml. These results indicate that lanoconazole is mainly distributed and retained in the stratum corneum after topical application where it exerts strong antifungal activity.

Monoclonal antibodies against pig ovarian follicular granulosa cells induce apoptotic cell death in cultured granulosa cells.

Two monoclonal antibodies capable of inducing granulosa cell apoptosis were produced against granulosa cells prepared from antral follicles of pig ovaries. The healthy follicles, 4-5 mm in diameter, were dissected from the ovaries of gilts, and then granulosa cells were isolated. BALB/c female mice were immunized with the isolated granulosa cells. Antibodies against the granulosa cells were detected by immunofluorescent staining using frozen ovarian sections. The isolated spleen cells prepared from immunized mice producing antibodies against the granulosa cells were fused with Sp2/O-Ag 14 mouse myeloma cells by standard hybridization techniques. Two hybridoma clones, PFG-1 and PFG-2, which produced specific IgM antibodies against granulosa cells were selected. Western blotting analysis revealed that PFG-1 and PFG-2 antibodies specifically recognized cell-membrane proteins with molecular weights of 55 and 70 kD and isoelectric points of 5.9 and 5.4, respectively. The monoclonal antibodies immunohistochemically reacted with granulosa cells of healthy follicles. When the isolated granulosa cells prepared from healthy follicles were cultured in medium containing 0.1 or 10 micrograms/m/PFG-1 or PFG-2 antibodies, respectively, the cells underwent apoptosis as determined by nuclear morphology, DNA electrophoresis and flow cytometric analysis. In conclusion, these two monoclonal antibodies against granulosa cells have cell-killing activity in cultured granulosa cells.

Wound healing effect of malotilate in rats.

The wound healing effect of malotilate (CAS 59937-28-9, NKK-105) was investigated by using an excisional skin-wound model produced on the back of normal and healing-impaired (induced by prednisolone pretreatment) rats. The rapid decrease in the square measure of wound areas and the improvement in the histological evaluation clarified that 0.3% and 1% cream preparations of malotilate were obviously effective in accelerating spontaneous healing in the normal rats. The accelerative effect of malotilate cream preparations was likely superior to that of an ointment containing 5% deproteinized calf blood extract used as a reference agent. The same effect was also observed in the healing-impaired rats. The histological findings revealed that a thicker and more cellular granulation tissue, which in turn created an adequate bed for rapid re-epithelization, was formed in the malotilate-treated animals. Acceleration of granulation tissue formation by malotilate was also supported by the cotton pellet implantation method. It is concluded from these results that malotilate seems to be a promising agent for topical wound therapy.

Wound healing effect of the new imidazole antimycotic lanoconazole in rats.

The wound healing effect of cream preparations of lanoconazole (CAS 101530-10-3, NND-318), an antimycotic imidazole compound, was examined using an excisional open skin-wound model produced on the back of rats. The rapid decrease in the size of wounded areas showed that 0.5% and 1% lanoconazole creams accelerated spontaneous healing. The effectiveness was almost similar to that of an ointment containing 5% deproteinized calf blood extract (DCBE), a wound healing agent on the market. In contrast, neither 1% clotrimazole cream nor 1% bifonazole cream, both of which are imidazole antimycotics, showed an accelerative effect. The wound healing effect of lanoconazole was further confirmed by histological evaluation; a thicker and more cellular granulation tissue was formed, and epidermal regeneration was more stimulated by lanoconazole than by non-treatment or vehicle alone. The effect of lanoconazole on the formation of granulation tissue in rats was also studied using a cotton pellet implantation method. Lanoconazole accelerated the formation of this tissue in terms of dry weight in a dose dependent manner (0.5-4 mg/pellet), and collagen content and angiogenesis also increased in the stimulated tissue, indicating that these accompany the compound-induced acceleration of tissue formation. These results suggest that lanoconazole has a distinctive wound healing effect which is a feature no other imidazole antimycotic is known to possess to date.

Short-term topical therapy of experimental tinea pedis in guinea pigs with lanoconazole, a new imidazole antimycotic agent.

The therapeutic efficacy of short-term treatment with a 1% cream of lanoconazole, a new imidazole antimycotic agent, in comparison with that of a 1% cream of terbinafine was evaluated in the guinea pig model of tinea pedis. Each agent was topically applied once a day for 3 or 7 consecutive days, starting on day 10 postinfection, and a culture study was conducted on day 5 after the last treatment with each agent. The 1% cream of lanoconazole was as highly effective as the 1% cream of terbinafine in terms of eradicating the fungi from the infected feet.

[Studies on the in vitro antifungal activity of lanoconazole, a new topical antimycotic, against Trichophyton spp].

In vitro studies with a new imidazole antifungal agent lanoconazole (LCZ) were carried out to confirm its fungicidal property of the anti-dermatophytic activity and unlikeliness of dermatophytes to acquire secondary resistance to this drug. Minimal cidal concentration (MCC) values of LCZ against 6 strains each of Trichophyton mentagrophytes and T. rubrum determined by the cellophane membrane method were in a range of 0.063 to 0.5 micrograms/ml, and much lower than those of bifonazole (16-32 micrograms/ml). The LCZ-sensitivity of both of the two testing strains of T. mentagrophytes did not decrease to less than one-fourth of the initial level during 15 subcultures on LCZ-containing agar. These results suggest that LCZ potently inhibits dermatophytes in a fungicidal manner and that secondary resistance to LCZ is not easily developed in the dermatophytes tested.

[In vitro antifungal activities of lanoconazole against stock cultures and clinical isolates of Candida albicans].

In vitro antifungal activities of lanoconazole (LCZ) against 9 stock cultures and 10 clinical isolates of Candida albicans were determined using three different testing media, Sabouraud's glucose broth (SGB), Sabouraud's glucose agar (SGA) and casitone agar (CA). MIC values of LCZ against both the stock cultures and clinical isolates measured on CA distributed in a range of 0.63-5 micrograms/ml. The values were 8-64-fold lower than those obtained in SGB and on SGA. MIC ranges and the geometric mean MIC values of LCZ against stock cultures were virtually the same as those against clinical isolates, no matter which assay techniques were used. These results suggest that there is no difference in the LCZ susceptibility between stock cultures and clinical isolates of C. albicans, although anti-Candida activity of LCZ was determined to be greater on CA than that in SGB or on SGA.

[Therapeutic efficacy of lanoconazole ointment in guinea pig model of tinea corporis, a comparative study with ointment and cream preparations].

The therapeutic efficacy of ointment and cream preparations of lanoconazole in a guinea pig model of tinea corporis was compared on the basis of degrees of improvement in local symptoms and negative culture rates. When infected animals were treated once daily with 0.25%, 0.5% and 1% lanoconazole ointments, significant improvement of the symptoms and mycological curative effects were observed as compared to those of non-treated control and vehicle-treated control groups of animals. Particularly, in animals treated with 0.5 or 1% lanoconazole ointment, no fungus was recovered from any infected loci. Comparing these results with those obtained with comparable concentrations of lanoconazole cream, no significant difference was found. These studies, therefore, suggested that ointment and cream preparations of lanoconazole on topical application would show basically equivalent therapeutic efficacy in the tinea corporis model.

Therapeutic efficacy of lanoconazole, a new imidazole antimycotic agent, for experimental cutaneous candidiasis in guinea pigs.

The therapeutic efficacy of 1% cream and 1% solution of lanoconazole, a new imidazole antimycotic agent, in the model of cutaneous candidiasis in prednisolone-treated guinea pigs was evaluated in comparison with that of comparable formulations of bifonazole. Each preparation was topically applied once a day for 3 consecutive days, starting on the fifth day postinfection, and quantitative culture study wsa conducted on the ninth day postinfection. Both formulations of lanoconazole were much more highly effective in terms of eradication of fungi than the bifonazole formulations.

[Therapeutic efficacy of lanoconazole ointment in guinea pig model of tinea pedis, in comparison with that of cream preparations].

The therapeutic efficacy of ointment and cream preparations of lanoconazole in a guinea pig model of tinea pedis was compared. When infected animals were treated once daily with 0.25% and 0.5% lanoconazole ointments, 7 of 10 and 8 of 10 infected feet became culture-negative, respectively. In animals treated with 1% lanoconazole ointment, fungus was not recovered from any infected foot. Comparing these results with those of culture study in animals which were treated with comparable concentrations of lanoconazole cream, no significant differences were found. These studies, therefore, suggested that ointment and cream preparations of lanoconazole on topical application in the tinea pedis model would show basically equivalent therapeutic efficacy.

Improvement of cardiac function and myocardial energy metabolism of rats with chronic heart failure by long-term coenzyme Q10 treatment.

The effects of long-term treatment with coenzyme Q10 (CoQ10) on myocardial energy metabolism of rats with chronic heart failure (CHF) were examined. Left coronary artery ligation resulted in decreases in blood pressure, left ventricular developed pressure, the first derivative of left ventricular developed pressure, cardiac output and stroke volume indices and caused an increase in left ventricular end-diastolic pressure 12 weeks after the operation. Significant decreases in adenosine-5'-triphosphate, creatine phosphate, creatine and inorganic phosphate contents and the mitochondrial oxygen consumption rate of the viable left and right ventricles were detected in the CHF rat. Oral administration of 5 mg/kg/day CoQ10 for 12 weeks attenuated the changes in the first derivative of left ventricular developed pressure, cardiac output and stroke volume indices of the CHF rat but did not significantly improve the survival of CHF animals. The developed infarct area was approximately 40% of the whole left ventricle, irrespective of treatment with or without CoQ10. There was no reversal in the decreased myocardial CoQ9 and CoQ10 contents of the CHF rat after treatment with exogenous CoQ10. In the right ventricle of CoQ10-treated animals, a significant recovery of creatine, inorganic phosphate and mitochondrial oxygen consumption rate, and a small restoration of creatine phosphate but not of adenosine-5'-triphosphate, were observed, which suggests an appreciable recovery of energy-producing ability in the right ventricle. In contrast, a significant restoration of tissue creatine and inorganic phosphate, but not of other variables, was detected in the left ventricle.(ABSTRACT TRUNCATED AT 250 WORDS)