The in vitro antifungal activity and spectrum of micafungin (MCFG) were compared with those of amphotericin-B (AMPH-B), fluconazole (FLCZ) and itraconazole (ITCZ) using a broth microdilution method as specified by the National Committee for Clinical Laboratory Standards (NCCLS) document M 27-A. MCFG exhibited broad-spectrum activity against clinically important pathogens including Candida species and Aspergillus species, and its MIC₉₀ levels against C. albicans (including FLCZ-resistant C. albicans), C. tropicalis, C. glabrata, C. krusei and Aspergillus species were ≤ 0.125μg/mL, which were lower than those for the other antifungal agents tested. The MIC₉₀ levels of MCFG against C. parapsilosis and C. guilliermondii were 4 and 2μg/mL, respectively, which were comparable to or higher than those for the other antifungal agents tested. MCFG exhibited concentration-independent fungicidal activity at concentrations higher than the MIC against most Candida species. In contrast, the MFCs of MCFG against A. fumigatus isolates were much higher than the MICs of the other agents, indicating that its action is fungistatic against this species. MCFG showed moderate to weak activity against most dematiaceous fungi and had no activity against Cryptococcus neoformans, Trichosporon species, Fusarium solani, Pseudallesheria boydii and zygomycetes. Although MCFG showed potent activity against the mycelial form of dimorphic fungi, it had weak or no activity against their yeast-like form. Neither the pH of the test medium nor the inoculum size greatly affected the MIC values of MCFG, while addition of human serum or human serum albumin increased the MIC values against Candida species and A. fumigatus. In experiments on resistance induction, the MIC of MCFG for C. albicans was not significantly changed; indicating that there is a low probability of MCFG-induced resistance.

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This report describes the anti-fungal mechanism of action of micafungin (MCFG) against Candida albicans and Aspergillus fumigatus using enzymological and morphological techniques. MCFG inhibits 1, 3-β-D-glucan synthesis derived from C. albicans ATCC 90028 and A. fumigatus TIMM 0063 in a concentration dependent manner. Inhibition kinetics between substrate and inhibitor were noncompetitive. MCFG is not active against chitin or mannan synthesis derived from C. albicans ATCC 90028 with both having a 50% inhibitory concentration (IC₅₀) over 100μg/mL. MCFG is also not active against deoxyribonucleic acid, ribonucleic acid or protein synthesis in C. albicans ATCC 90028 (IC₅₀ s were both over 100μg/mL). On differential-interference contrast micrographs and transmission electron micrographs of drug-challenged cells, abnormal cell wall structures were observed. These abnormalities included: thin cell walls, abnormal septum formation, split inhibition of daughter cells and lysis of the C. albicans ATCC 90028 yeast cells; inhibition of pseudohyphae extensions, swelling and abnormal extension at the tips of pseudohyphae in C. albicans FP 633; and inhibition of germination and hyphae extension, swelling and abnormal extension at the tip cells of hyphae in A. fumigatus TIMM 0063. These results suggest that the anti-fungal mechanism of action against C. albicans and A. fumigatus is inhibition of 1, 3-β-D-glucan synthesis.

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The efficacy of micafungin (MCFG), a novel water-soluble lipopeptide, was evaluated in mouse models of disseminated candidiasis and aspergillosis, and was compared with that of amphotericin B (AMPH-B), fl uconazole (FLCZ) and itraconazole (ITCZ). In the candidiasis model in mice with granulocytopenia induced by cyclophosphamide, MCFG significantly prolonged the survival of mice infected intravenously with Candida albicans at doses of 0.125mg/kg or higher (P<0.01). In candidiasis and aspergillosis caused by C. albicans, Candida glabrate, Candida tropicalis, Candida krusei, Candida guilliermondii and Aspergillus fumigatus, MCFG exhibited ED₅₀ s in the range of 0.14-1.61 mg/kg. These data were comparable or inferior to those of AMPH-B, but superior to FLCZ and ITCZ. In disseminated candidiasis in mice immunosuppressed by cyclophosphamide, hydrocortisone or 5-fluorouracil, the ED₅₀ s of MCFG were 0.14-0.33mg/kg, superior to ITCZ and FLCZ, but inferior to AMPH-B. In a target organ kidney assay, a single injection of MCFG at a doses of 0.5 or 1.0mg/kg significantly reduced the yeast viable cell counts in the kidney 24 hours after treatment compared to the yeast counts before treatment, with an efficacy comparable to AMPH-B. These results indicate that MCFG is a potent parenteral administered therapeutic agent for disseminated candidiasis and aspergillosis in immunosuppressed mice. The efficacy of MCFG was superior to that of FLCZ and ITCZ, but comparable or slightly inferior to that of AMPH-B.

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The efficacy of micafungin (MCFG), a novel water-soluble lipopeptide, was evaluated in mouse models of pulmonary aspergillosis, and compared with that of amphotericin B (AMPH-B), fluconazole (FLCZ) and itraconazole (ITCZ). In pulmonary aspergillosis in mice immunosuppressed by cyclophosphamide, MCFG significantly prolonged the survival of mice infected intranasaly with Aspergillus fumigatus at doses of 0.5 and 1mg/kg (P<0.0125). In mice with pulmonary aspergillosis caused by A. fumigatus, MCFG exhibited 50% effective doses (ED_50s) in the range of 0.26 to 0.45mg/kg 15 days after infection, which is comparable to the efficacy of AMPH-B (ED_50S; 0.25 to 0.46mg/kg), and superior to FLCZ and ITCZ. The ED₅₀ of MCFG was comparable to that of AMPH-B in mice immunosuppressed by 5-fluorouracil, however, it was 4.1 times inferior to that of AMPH-B in mice immunosuppressed by hydrocortisone. When treatment with MCFG was initiated 1 day after infection, the ED₅₀ of MCFG was 1.21mg/kg, which was 2.4 times inferior to that of AMPH-B and 3.8 times inferior to that of MCFG if initiated 1.5 hours after infection. These results indicate that MCFG may be a potent parenteral administered antifungal agent for pulmonary aspergillosis, with efficacy comparable or slightly inferior to that of AMPH-B, but superior to that of FLCZ and ITCZ.

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The activity of micafungin (MCFG), a novel water-soluble lipopeptide, was evaluated in a disseminated candidiasis and pulmonary aspergillosis target organ assay to determine the minimum effective plasma concentration. Immunosuppressed mice were challenged intravenously with Candida albicans or intranasally with Aspergillus fumigatus and treated with various concentrations of MCFG infused continuously via a subcutaneously implanted pump. Efficacy was evaluated on the basis of a comparison of the mean log₁₀ CFU in kidney or lung in groups treated with MCFG and in control groups, 5 days post infection. Plasma concentration of MCFG included the active metabolite M 1 (catechol form) and M 2 (methoxy form). As plasma concentrations of MCFG 5 days post-infection were lower than that 1 day post-infection, the minimum effective plasma concentration had a range between the plasma concentration 5 days post-infection and the calculated plasma concentration 1 day post-infection. The estimated minimum effective plasma concentrations of MCFG were 0.16-0.26 μg/mL and 0.55-0.80 μg/mL in mouse candidiasis and aspergillosis, respectively.

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The therapeutic efficacy of micafungin against a murine model of oropharyngeal candidiasis was evaluated. Oropharyngeal candidiasis was induced in congenitally immunodeficient N: NIH-bg-nu-xid BR mice by oral inoculation of Candida albicans cells for 4 days. The mice were intravenously administered micafungin, fluconazole, or saline alone from 13 to 23 days after initial inoculation, twice daily. Therapeutic effect was evaluated as reduction in tissue colony count and histopathology in the tongue 1 day and 8 days after the end of treatment. From the initial treatment to 8 days after the end of treatment, saline-treated control mice displayed 10⁴ to 10⁵ C. albicans in the tongue. Histopathological examination revealed continuous infection of C. albicans pseudohyphae in keratinized mucosal layers of the tongue throughout the experiments. In addition, this infection was accompanied by infiltration of inflammatory cells after the end of treatment. In this mouse model, micafungin showed therapeutic efficacy at 2 mg/kg or higher 1 day after the end of treatment, as demonstrated by a significant reduction in viable colony count and repaired normal morphology of the tongue. After an 8 day non-treatment interval, 5 mg/kg or higher doses were still effective in terms of both colony count and histopathology, although increased counts and re-infected tongue mucosa were observed in mice treated with 2mg/kg micafungin. The efficacy of micafungin at 5mg/kg or higher was comparable to that of fluconazole at 20 mg/kg. These results suggest that micafungin shows an eradicative effect at a lower dose than fluconazole in this mouse model, therefore it has potential usefulness as a therapeutic drug with a low incidence of relapse.

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The antifungal activities of the M 1, M 2 and M 5 micafungin (MCFG) metabolites were evaluated by MIC using broth supplemented with/without serum, and assessed in a murine disseminated candidiasis model. The MICs of M 1 (0.0625 to 4μg/mL) were 4 to 16 times greater than MCFG against Candida species, Saccharomyces cerevisiae and Aspergillus species. Against Cryptococcus neoformans and Trichosporon cutaneum, M1 displayed MICs of 16μg/mL, whereas for MCFG the MICs were over 64μg /mL. The MICs of M2 were comparable to these of MCFG against all strains used this study. The MICs of M5 were over 128 times greater than for MCFG against Candida and Aspergillus species. MIC values for the M 1 and M 2 metabolites against C. albicans FP 633 in a serum-supplemented broth were comparable to those of MCFG. The therapeutic effects of the M 1 and M 2 metabolites against murine candidiasis induced by C. albicans FP 633 were comparable to that of MCFG, with similar ED₅₀ values. These results suggest that the therapeutic effect of treatment of experimental fungal infection with MCFG reflects not only the antifungal activity of the parent MCFG, but also those of the primary metabolites, M 1 and M 2.

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In vitro interactions between micafungin (MCFG) and amphotericin B (AMPH-B), itraconazole (ITCZ) and fluconazole (FLCZ) were evaluated using a checkerboard method based on the standard broth microdilution method M 27-A recommended by the NCCLS. When MCFG was combined with AMPH-B, ITCZ and FLCZ, additive interaction was observed for 41%, 85% and 85% of Candida albicans isolates, respectively, and either synergistic or additive interaction was observed for 67%, 87% and 13% of Aspergillus fumigatus isolates, respectively. An excellent interaction was observed for Cryptococcus neoformans when MCFG was combined with AMPH-B, which was synergistic for 67% and additive for 33 % of isolates tested. Antagonism was observed only in the MCFG-ITCZ combination for 83% of C. neoformans. For the purpose of in vivo validation of the in vitro interaction of MCFG and AMPH-B, we evaluated the efficacy of combination therapy of the 2 drugs against a mouse model of pulmonary aspergillosis induced by A. fumigatus IFM 40836, against which the combination yielded an additive interaction in vitro. Combination therapy with MCFG (2 mg/kg) and AMPH-B (0.5 mg/kg) produced a significant decrease in fungal colony count in the lung compared to not only the control group but also to either dose alone 6 days after infection. The combination also strongly suppressed histopathologically determined pulmonary lesion, hyphal elongation and neutrophil infiltration. Furthermore, combination therapy of MCFG (1 mg/kg) and AMPH-B (0.25 mg/kg) was more effective than both MCFG alone (2mg/kg) and AMPH-B alone (0.5 mg/kg), which were both double the dose used in the combination treatment. These results suggest that the interaction of MCFG and AMPH-B in this pulmonary aspergillosis model was synergistic. In conclusion, MCFG showed an excellent interaction with AMPH-B both in vitro and in vivo, suggesting that combination therapy with these 2 drugs might have utility for the treatment of severe deep-seated fungal infections. In addition, MCFG may have clinical usefulness in combination therapy with other commercially available antifungal agents.

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A sensitive and specific HPLC method has been developed and validated for a new antifungal drug micafungin (MCFG) and its metabolites, M1 (catechol form) and M2 (methoxy form) in human plasma. The assay employs protein precipitation with acetonitrile for human plasma, to which phosphoric acid is added for the purpose of stabilization of MCFG, and utilizes fluorescence detection of MCFG and its metabolites. The method provides a linear response from a quantification limit of 0.05 to 25μg/mL for MCFG and 0.05 to 10μg/mL for M1 and M2 using 50μL of plasma. The intra-assay and inter-assay precision and accuracy were shown to be acceptable for the pharmacokinetic study. Furthermore, in the matrix for HPLC analysis, MCFG, M1 and M2 are stable for over 24 h at room temperature. The stability study showed that MCFG, M1 and M2 are stable for up to 72 h at 4°C in blood, and in plasma up to 2 h at room temperature and 6 h under ice-cold conditions. Additionally, these compounds are stable in plasmafor up to 21 day at-20°C and 413 day at-80°C, and can be frozen and thawed 4 times without loss of stability.

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The pharmacokinetics of micafungin (MCFG) were evaluated in male mice, male and female rats, and male dogs after intravenous bolus and infusion over 1 h of MCFG at doses from 0.32 mg/kg to 3.2 mg/kg. Plasma concentrations of MCFG decreased biexponentially in all animals tested. After doses of MCFG in the range 0.32-3.2 mg/kg, the respective total body clearances, volumes of distribution at steady state, and elimination half-lives, were 0.92-1.01 mL/min/kg, 0.38-0.49 L/kg and 5.34-5.71 h for mice, 1.04-1.50 mL/min/kg, 0.42-0.56 L/kg and 3.96-5.05 h for rats, and 0.59-0.78 mL/min/kg, 0.23-0.25 L/kg and 4.24-5.43 h for dogs. The pharmacokinetics of MCFG after intravenous dosing in mice, rats and dogs exhibited a linear profile in the range of 0.32 mg/kg to 3.2 mg/kg. Additionally, the pharmacokinetics of MCFG at a dose level of 1 mg/kg did not differ between dosing methods in rats and dogs, nor between the sexes in rats. The serum protein binding of MCFG exceeded 99% in mice, rats, dogs and humans.

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In this study, distribution and excretion after intravenous dosing of [¹⁴C] micafungin (1mg/kg) to rats and in vitro serum protein binding and distribution to blood cells in mouse, rat, dog and human were investigated.
1. The concentration of radioactivity in plasma at 5 min, which was the first observation point, was 3, 396 ng eq./mL and decreased triexponentially. At 24 h, the concentration had decreased to approximately 12.4% of that at 5 min, and thereafter it decreased with a half-time of 39.3 h. The blood to plasma radioactivity concentration ratios were in the range of 0.80 to 1.00 for up to 7 day after dosing, and were 1.00 or more thereafter.
2. The radioactivity was widely distributed immediately after dosing. The highest radioactivity concentrations were observed in the lungs at 5 min and were 1.86 times higher than that in plasma, followed by the kidneys (1.09). The relative radioactivity concentrations in brain, eyeball, white fat and testis were less than 0.08 of that in plasma, and those in other tissues were in the range of 0.17 to 0.86. The radioactivity concentrations in all tissues examined at 24 h had decreased compared with those at 5 min or 6 h, and decreased almost in parallel with plasma radioactivity concentrations from 72 h with the exception of concentrations for white fat.
3. Up to 240 h after intravenous dosing, 83.5% and 14.4% of the radioactivity had been excreted in feces and urine, respectively. At 240 h, 2.8% and 0.3% of the radioactivity were detected in carcass and gut, respectively. In the expired air, no radioactivity was detected up to 72h.
4. Up to 48 h after intravenous dosing, 43.9%, 13.2% and 8.3% of the radioactivity had been excreted in bile, urine and feces, respectively. At 48 h, 32.2% and 4.4% of radioactivity were detected in carcass and gut, respectively.

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The metabolism of micafungin (MCFG), a new echinocandin-like lipopeptide antifungal agent, was evaluated in rats and dogs, and the following results were obtained.
1. After an intravenous administration of ¹⁴C labeled MCFG to rats and dogs, MCFG and M 5 were mainly observed in plasma samples, and M 1 and M 2, which have antifungal activity, were barely detectable. The main compound observed was M 5 in urine samples and MCFG in feces and bile samples.
2. The relative ratios of M 1 and M 2 to the total radioactivity in lung, liver, spleen and kidney samples were higher than that in the plasma samples. In the liver sample at 24 hours after administration, M 1, M 2 and MCFG were 26.9%, 22.8% and 8.9% of total radioactivity, respectively.
3. From the analysis of plasma, urine, feces and bile samples, the catechol form of MCFG (M 1), methylated form of M 1 (M 2), open-ring form (M.3) and hydroxyl form at the side chain (M 5) were estimated.

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The in vitro drug interactions of micafungin (MCFG), a new echinocandin-like lipopeptide antifungal agent, were evaluated using human serum and human liver microsomes and the following results were obtained.
1. The percent of MCFG bound to human serum proteins was as high as 99.74%. Warfarin, diazepam, salicylic acid and methotrexate did not affect the protein binding of MCFG.
2. Based on the results at the concentrations ranging from 0.1 to 1 mmol/L (130-1, 300 μg/mL) of MCFG, the binding constant (K_D) of MCFG at the bilirubin binding site was calculated to be 2.0×10³ L/mol, indicating that MCFG has a lower affinity to the bilirubin binding site than salicylic acid (5.0×10³L/mol) or sulfisoxazole (1.4×10⁴L/mol).
3. M 5 and M 13 formation activities significantly correlated with the activities of coumarin 7-hydroxylase and testosterone 6, β-hydroxylase. M 13 formation activity also significantly correlated with the activities of tolbutamide methyl-hydroxylase and S-mephenytoin 4'-hydroxylase.
4. The metabolism of MCFG was inhibited by tranylcypromine and ketoconazole (KCZ). The 50% inhibitory concentration (IC₅₀) of cyclosporin A, tacrolimus and KCZ for the metabolic activity of MCFG was > 100, > 100 and 6.2 μmol/L, respectively.
5. The IC₅₀ of MCFG, fluconazole (FLCZ) and KCZ for the metabolic activity of terfenadine was 67.7, >100 and 0.46 μmol/L, respectively, and 24.9, 0.12 and 44.2 μmol/L for astemizole. The inhibition constant of MCFG, FLCZ and KCZ for the metabolic activity of nifedipine was 17.3, 0.012 and 10.7μmol/L, respectively.
6. The IC₅₀ of MCFG, caspofungin acetate and KCZ for the metabolic activity of cyclosporin A was 31, 39 and 0.14 μmol/L, respectively.

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The safety and pharmacokinetics of micafungin (MCFG) were examined using 36 healthy male volunteers both in single and repeated administration studies. In the single administration study, 2.5, 5, 12.5, 25 and 50mg of MCFG were given intravenously with continuous infusion pump over a 2-hour period. In the repeated administration study, 25mg of MCFG or placebo was given intravenously once daily for seven days with continuous infusion pump over a 1-hour period. The results were as follows:
1) MCFG was well tolerated with no drug-related adverse events observed.
2) Mean plasma concentrations of unchanged drug reached maximum at the end of administration, and declined in a bi-exponential manner afterwards.
3) There were no differences among dose groups in elimination rate constant (β), elimination halflife (t_1/2), volume of distribution at steady state (Vd_ss) and total clearance (CL_t). Area under the plasma concentration-time curve from time zero to infinity (AUC_0-∞) and maximum plasma concentration (C_max) increased in proportion to the dose. Consequently, the pharmacokinetics of MCFG were considered to be linear over the dose range studied.
4) The plasma concentrations of unchanged drug reached a steady state by day 4 in the repeated administration study with no accumulation observed.
5) The metabolites M 1 (catechol form) and M 2 (methoxy form), that are found in rats and dogs, were hardly detected. Urinary excretion was extremely low for the unchanged drug, and for metabolites M 1 and M2.

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The pharmacokinetics of micafungin (MCFG) were studied in 10 elderly volunteers (aged 66-78 years) and 10 non-elderly volunteers (aged 20-24 years) all of whom were healthy males. MCFG was given intravenously at a single administration of 50 mg with continuous infusion pump over a 1-hour period. The plasma concentrations of unchanged drug declined in a bi-exponential way both in elderly and non-elderly subjects after cessation of infusion. In the elderly and non-elderly, respectively, these values were found: maximum plasma concentration (C_max) of unchanged drug, 4.97±0.60 (mean±S.D.) and 4.95±0.56, ug/mL; elimination half-life (t_1/2), 14.9±1.0 and 15.2±0.9 h; volume of distribution at steady state (Vd_ss), 0.239±0.027 and 0.228±0.016 L/kg; total clearance (CL_t), 12.0±1.7 and 11.1±1.2 mL/h/kg; plasma protein binding rate, 99.85±0.01 and 99.85±0.02%. Since there were no significant differences in pharmacokinetic parameters or the plasma protein binding rate, it was concluded that the pharmacokinetics of MCFG were similar in both elderly and non-elderly subjects.

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The pharmacokinetics of micafungin (MCFG) were examined using 30 healthy male volunteers both in single and repeated administration studies. In the single administration studies, 25, 50, 75 and 150mg of MCFG were given intravenously using a continuous infusion pump. In the single administration studies, plasma concentrations of unchanged drug declined in a bi-exponential way after cessation of infusion. There were no differences between dosage groups in elimination half-life (t_1/2), volume of distribution at steady state (Vd_ss), total clearance (CL_t) and other pharmacokinetic parameters. The meantS.D. of t_1/2, Vd_ss, and CL_t were 13.9±1.0 h, 0.228±0.016L/kg and 0.197±0.018ml/min/kg, respectively. The area under the plasma concentration-time curve from time zero to infinity (AUC_0-∞) of unchanged drug increased proportionally to the dose of MCFG in a linear fashion. Urinary recovery of MCFG was less than 1% of the administered dose, showing virtually no urinary excretion. In the repeated administration studies, 75mg of MCFG was given intravenously with continuous infusion pump once daily for 7 days. C_max of unchanged drug during repeated administration reached a steady state by day 4. The t_1/2 and AUC_0-24h during repeated administration were about the same as those obtained in the single administration studies. Unchanged drug was minimally excreted into the urine, showing a urinary recovery of less than 1% of the administered dose. The percent of unchanged drug bound to plasma protein did not differ between day 1 (99.83±0.01%) and day 7 (99.82±0.01%). The drug was thus concluded to exhibit linear pharmacokinetics during repeated administration as well. Metabolite M 1 (catechol form) and M 2 (methoxy form), which are found in rats and dogs, were also studied. In the single administration studies, M 1 was detected in plasma at dose levels of 50mg or more. On the other hand, M 1 was detected from day 2 in plasma, and its concentration reached a constant level on day 7, at 0.297±0.039 to 0.307±0.042μg/ml, during repeated administration at a dose level of 75mg. Although metabolite M 1 was detected in plasma in the repeated administration studies, its concentration was less than 1/5 of the trough concentration of unchanged drug. In addition, M 1 showed very little urinary excretion, with a urinary recovery of only 0.15±0.02% of the administered dose. The amount of metabolite M 2 was less than the quantifiable limit in plasma and urine in most cases.

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