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Aspergillus spp. are important fungi because of the high mortality associated with aspergillosis. However, we have only three classes of anti-aspergillus drugs available; and only two drugs, itraconazole (ITCZ) and voriconazole (VRCZ), are available as oral agents in Japan. If azole-resistant Aspergillus causes chronic aspergillosis, we cannot treat such patients with oral azoles on an outpatient basis. As is the case with foreign countries, we found the existence of azole-resistant Aspergillus fumigatus in clinical settings in Japan. Resistance was attributed to mutations of the target protein (CYP51A). Additionally, we also found that long-term itraconazole treatment induced G54 substitution in CYP51A, causing itraconazole-resistance. Although there are few resistant Aspergillus strains existing in Japan now, we have to continue to find such resistant isolates, which are spreading worldwide.

The in vitro activity of ravuconazole (RVCZ) was compared with those of itraconazole (ITCZ) and terbinafine (TBF) against 73 dermatophyte isolates and 18 Candida spp. isolates recovered from patients with dermatomycosis at 4 dermatological clinics in Japan in 2011. The dermatophyte isolates consisted of Trichophyton rubrum (n=51), Trichophyton mentagrophytes (n=20 : these strains were not identified by molecular phylogenetic analysis.), Trichophyton tonsurans (n=1), and Microsporum canis (n=1). The Candida spp. isolates comprised C. albicans (n=11), C. parapsilosis (n=5), C. guilliermondii (n=1), and C. pseudohaemulonii (n=1). RVCZ was highly active against all dermatophytes and all Candida spp. : the geometric mean (GM) MICs for T. rubrum and T. mentagrophytes were 0.035 μg/mL and MICs for T. tonsurans and M. canis were ≤ 0.03 μg/mL, and GM MICs for C. albicans and C. parapsilosis were ≤ 0.03 μg/mL and MICs for C. guilliermondii and C. pseudohaemulonii were 0.25 and ≤ 0.03 μg/mL, respectively. Compared to RVCZ, ITCZ showed similar anti-dermatophytic and anti-Candida activities, while TBF had a slightly higher anti-dermatophytic but a markedly lower anti-Candida activity. These results suggest that RVCZ is a potential candidate systemic antifungal therapy against onychomycosis and other dermatomycoses that are refractory to topical antifungal therapy.

To evaluate the currently used Japanese Society for Medical Mycology (JSMM) method for testing the azole susceptibility of yeasts, the activities of fluconazole and itraconazole were tested against recently collected clinical isolates of Candida spp. (n=946) and compared with the National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 microdilution reference method. Favorable correlation with the M27-A2 method was not seen for isolates of C. albicans, C. tropicalis or other Candida spp., particularly their trailing-growth isolates. However, the degree of correlation and agreement of MIC values were markedly improved when testing was performed by the modified JSMM method in which the end-point to be read was changed from IC80 (for the current JSMM method) to IC50. These results suggest that there is an urgent need to revise the current JSMM method.

This article reviews the in vitro metabolic and the in vivo pharmacokinetic drug-drug interactions with antifungal drugs, including fluconazole, itraconazole, micafungin, miconazole, and voriconazole. In the in vitro interaction studies, the effects of antifungal drugs on specific activities of cytochrome P450s (CYPs), including CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4, in human liver microsomes are compared to predict the possibility of drug interactions in vivo. Fluconazole, micafungin, and voriconazole have lower inhibitory effects on CYP3A4 activities than itraconazole and miconazole, and IC(50) and/or K(i) values against CYP2C9 and CYP2C19 activities are the lowest for miconazole, followed by voriconazole and fluconazole. In in vivo pharmacokinetic studies, it is well known that itraconazole is a potent clinically important inhibitor of the clearance of CYP3A4 substrates, and fluconazole and voriconazole are reported to increase the blood or plasma concentrations of not only midazolam and cyclosporine (CYP3A4 substrates) but also of phenytoin (CYP2C9 substrate) and/or omeprazole (CYP2C19/CYP3A4 substrate). On the other hand, no inhibition of CYP activities except for CYP3A4 activity by micafungin is observed in vitro, and the blood concentrations of cyclosporine and tacrolimus are not affected by coadministration of micafungin in vivo, suggesting that micafungin would not cause clinically significant interactions with drugs that are metabolized by CYPs via the inhibition of metabolism. Miconazole is a potent inhibitor of all CYPs investigated in vitro, although there are few detailed studies on the clinical significance of this except for CYP2C9. Therefore the differential effects of these antifungal drugs on CYP activities must be considered in the choice of antifungal drugs in patients receiving other drugs.

We have examined the antifungal activities of the available antifungal agents including micafungin (MCFG), one of the echinocandin antifungal group, against 92 yeast-like fungi isolated at our hospital during a 3-month period from November 2002 to February 2003. Determination of the antifungal susceptibility was conducted in conformity with the Standards of the Japanese Society for Medical Mycology. The MIC 80% of the antifungal agents against 4 fungi species including C. albicans (55 strains), C. tropicalis (20 strains), C. glabrata (8 strains), C. krusei (5 strains) were as follows; MCFG: 0.03-0.125 microgram/ml, amphotericin-B: 0.125-0.25 microgram/ml, 5-fluorocytosine: 0.125-16 micrograms/ml, itraconazole: 0.25-2 micrograms/ml, fluconazole: 0.5-32 micrograms/ml. The isolation rate of the drug-resistant fungi was 20% for the fluconazole (FLCZ)-resistant C. tropicalis and 33% when including the susceptible dose dependent (S-DD) class. The rate was 5% for FLCZ-resistant strains of C. albicans and 11% when including the S-DD class. However, MCFG was shown to have an excellent antifungal activity against those azole-resistant strains of Candida species. An analysis of the randomly amplified polymorphic DNA pattern (RAPD) was carried out to assess the fingerprinting of the azole-resistant strains. The results demonstrated a common pattern in 3 of the 6 strains of C. tropicalis that showed MIC of > or = 16 micrograms/ml for fluconazole, while all of the 6 strains of C. albicans demonstrated their respective patterns.

Olopatadine hydrochloride (olopatadine), a novel antiallergic agent, is effective in the treatment of allergic rhinitis, chronic urticaria, eczema and dermatitis. It has been reported that terfenadine and astemizole cause side effects on the circulatory system such as QT prolongation followed by serious ventricular arrhythmias (torsades de pointes). To investigate the possibility of QT prolongation, we used both conscious normal dogs and hypokalemia-anesthetized dogs under two conditions: 1) olopatadine used alone and 2) olopatadine used in combination with itraconazole, the CYP3A4-inhibiting antifungal agent, in the present investigation. The group treated with terfenadine alone (30 mg/kg, p.o.) and the group treated with a combination of terfenadine (10 mg/kg, p.o.) and itraconazole (100 mg/kg, p.o.) had a significantly prolonged QT interval. On the other hand, the group treated with olopatadine alone (30 mg/kg, p.o.) and the group treated with a combination of olopatadine (30 mg/kg, p.o.) and itraconazole (100 mg/kg, p.o.) did not show any significant changes in QT interval. Moreover, olopatadine (1 and 5 mg/kg, i.v.) did not influence the QT interval in hypokalemia-anesthetized dogs. These results suggest that there is very little possibility of QT prolongation as a result of clinically used olopatadine.

We showed a digoxin-itraconazole interaction in three patients in whom digoxin serum concentrations were increased. Their electrocardiograms revealed arrhythmias such as ventricular premature contraction, atrioventricular block, and ST depression. The elimination half-life of digoxin in case 3 patient who continued itraconazole therapy was 8.4 days, which was estimated by nonlinear least squares method from the serum concentrations of digoxin versus time curve. In order to evaluate the influence of itraconazole on pharmacokinetic parameters of digoxin, we estimated digoxin clearance by the Bayesian method using the population pharmacokinetic parameters in Japanese patients. During the concomitant use of itraconazole and digoxin, the digoxin clearance in all patients decreased to 50.5 +/- 8.8% (mean +/- S.D.) of the clearance without itraconazole. When digoxin and itraconazole are used concomitantly, careful monitoring of digoxin serum concentrations is necessary. Based on our results of digoxin clearance evaluation, the dose of digoxin should be reduced to 50% of original dose after itraconazole is started, and digoxin serum concentration might be controlled at the same level before the concomitant use.

In this study we investigated the antifungal susceptibility of 285 strains of Candida albicans isolates at Kinki University Hospital from March 1995 to December 1996. The antifungal agents tested were fluconazole, miconazole, intraconazole, amphotericin B and flucytosine. The susceptibility testing were performed according to the broth microdilution method standardized by National Committee for Clinical Laboratory Standards (M27-T). Most isolates of C. albicans showed relatively a low MIC value and the MIC90S were calculated at 1 microgram/ml; fluconazole, 0.125 microgram/mg; miconazole, 0.06 microgram/ml; itraconazole, 1 microgram/ml; amphotericin B, 0.25 microgram/ml; flucytosine. There was only one strain that showed high resistance against fluconazole and it showed cross-resistance against miconazole and itraconazole. There were two flucytosine resistant strains. The MICs of amphotericin B were tightly clustered and resistant strain were not observed.

To clarify the involvement of cytochromeP450 (CYP) 3A4 in the metabolism of haloperidol and bromperidol in humans, the effects of itraconazole, a potent inhibitor of CYP3A4, on steady-state plasma concentrations of both drugs and their reduced metabolites were investigated using 21 schizophrenic patients. Patients treated with haloperidol 12 or 24 mg/day (n = 13) or bromperidol 12 or 24 mg/day (n = 8) for at least 2 weeks were then given itraconazole 200 mg/day for 7 days. Blood samplings were performed before administration, 1 week during itraconazole coadministration and 1 week after its discontinuation together with clinical assessments. Plasma concentrations of haloperidol and bromperidol and their reduced metabolites were significantly higher during itraconazole treatment (P < 0.01). Deterioration in the neurological side effects of haloperidol was observed during itraconazole coadministration. Thus, this study suggests that itraconazole inhibits the metabolism of haloperidol and bromperidol, and that CYP3A4 is involved in the metabolism of both drugs.

Effects of itraconazole (ITCZ) on anti-Candida activity of a macrophage (MP)-neutrophil axis were examined in vitro. Murine peritoneal MP, stimulated by an enterococcal preparation FK-23, released tumor necrosis factor a (TNF) into the culture medium in 20 hr-culture period. This level of TNF production by the stimulated MP, which were measured by a bioassay or immunological assay, was heightened by co-stimulation with 0.5 mg/ml of ITCZ. Supernatants obtained from the culture medium of FK-23 and ITCZ-treated MP not only contained about 10 ng/ml of TNF but also had the capacity to enhance anti-Candida activity of murine neutrophils. This enhancement was blocked by pretreatment of the MP-supernatant with anti-TNF antibody. Observation by scanning electron microscope suggested that cell walls of the candida mycelia, the growth of which was inhibited by the neutrophils in the presence of either the MP-supernatant or TNF, sustained similar structural damage. These results suggest that ITCZ augments anti-Candida activity of neutrophils through enhanced production of TNF by MP in vitro.

The combined effects of lysozyme and various antifungal agents were investigated by microbroth dilution method against Candida albicans in vitro. Synergistic anti-Candida activity was observed between egg white lysozyme and itraconazole, clotrimazole, miconazole and lanoconazole. Similar, although not as clear, combination anti-Candida activities were seen in the cases of ketoconazole, bifonazole, amphotericin B and nystatin. Anti-Candida activity of fluconazole was not affected by the addition of lysozyme, however. Physiological roles of this combination effect in anti-Candida therapy by azole antifungals were discussed.

The susceptibilities of clinical isolates of Candida spp. to fluconazole (FLCZ), itraconazole (ITCZ) and ketoconazole (KCZ) were determined by a new method, E-test (AB BIODISK, Sweden) and compared with those obtained by the microbroth dilution method. The MIC values of the 3 azoles obtained by the two methods were, in general, in good agreement. While MICs of FLCZ and KCZ demonstrated higher essential agreement rates and better correlation by both methods, MIC values of ITCZ by the E-test had a tendency to be higher than those of the microbroth dilution method in azole resistant strains. Potential usability of E-test as a broth dilution method for susceptibility tests for clinically isolated Candida spp. including azole resistant strains was discussed.

A new colorimetric microdilution antifungal susceptibility testing using an air-dried microplate containing serially diluted antifungal agents and an oxidation-reduction dye, sodium resazurin, as a color indicator to detect the growth of yeasts was evaluated at multiple sites. Six ATCC reference strains described in the National Committee for Clinical Laboratory Standards (NCCLS) M27-T, two quality control and four reference strains, were repeatedly tested against five antifungal agents, amphotericin B(AMPH), flucytosine (5-FC), fluconazole (FCZ), miconazole (MCZ) and itraconazole (ITZ) at 17 different laboratories. Of 1,521 MIC determinations, 1,393 (91.6%) were within the acceptable limits described in the M27-T, ranging from 23.8% to 100%. However, significant discrepancies from the M27-T limits were noted in some combinations, such as FCZ against C. albicans ATCC 90028. In addition, the MIC limits for MCZ and ITZ against the six reference strains were determined. A total of 1,645 clinical isolates of yeasts were tested against five antifungal agents. Most isolates of C. albicans were highly susceptible to the five agents with MIC90 ranging from 0.03 microgram/ml (MCZ) to 1.0 microgram/ml (AMPH). The MICs against AMPH and 5-FC were tightly clustered, except with C. krusei and Trichosporon spp. Whereas, the distribution of MICs for FCZ, MCZ and ITZ had a wide range from < or = 0.03 microgram/ml to > 64 micrograms/ml. From these results, it can be concluded that our colorimetric broth microdilution antifungal susceptibility test is reliable, easy-to-perform and a suitable alternative for the determination of the MICs in clinical microbiology laboratories.