Abstract
TAK-456 is a novel oral triazole compound with potent and broad-spectrum in vitro antifungal activity and strong in vivo efficacy against Candida albicans and Aspergillus fumigatus. TAK-456 inhibited sterol synthesis of C. albicans and A. fumigatus by 50% at 3 to 11 ng/ml. TAK-456 showed strong in vitro activity against clinical isolates of Candida spp., Aspergillus spp., and Cryptococcus neoformans, except for Candida glabrata. The MICs at which 90% of the isolates tested were inhibited byTAK-456, fluconazole, itraconazole, voriconazole, and amphotericin B were 0.25, 4, 0.5, 0.13, and 0.5 μg/ml, respectively, for clinical isolates of C. albicans and 1, >64, 0.5, 0.5, and 0.5 μg/ml, respectively, for clinical isolates of A. fumigatus. Therapeutic activities of TAK-456 and reference triazoles against systemic lethal infections caused by C. albicans and A. fumigatus in mice were investigated by orally administering drugs once daily for 5 days, and efficacies of the compounds were evaluated by the prolongation of survival. In normal mice, TAK-456 and fluconazole were effective against infection caused by fluconazole-susceptible C. albicans at a dose of 1 mg/kg. In transiently neutropenic mice, therapeutic activity of TAK-456 at 1 mg/kg of body weight against infection with the same strain was stronger than those at 1 mg/kg of fluconazole. TAK-456 was effective against infections with two strains of fluconazole-resistant C. albicans at a dose of 10 mg/kg. TAK-456 also expressed activities similar to or higher than those of itraconazole against the infections caused by two strains of A. fumigatus in neutropenic mice at a dose of 10 mg/kg. These results suggest that TAK-456 is a promising candidate for development for the treatment of candidiasis and aspergillosis in humans.
Invasive mycoses are life-threatening infections for the immunocompromised patients receiving immunosuppressive chemotherapy (9, 22, 25, 28). The major opportunistic pathogens are Candida albicans and Aspergillus fumigatus (6, 18, 22). Although triazole antifungal agents such as fluconazole and itraconazole are used to treat mycoses (5, 8), their clinical efficacies are still limited, especially against A. fumigatus (10, 13, 23). Moreover, an increase of azole-resistant C. albicans (1, 7, 20) and non-C. albicans Candida species (19, 27), which are refractory to the repeated treatment with fluconazole, is also a big problem. Amphotericin B is active against deep-seated mycoses, to which triazole antifungal agents are ineffective, but clinical use of amphotericin B is limited because of its severe toxicity (10). Thus, development of a potent and safe antifungal agent against candidiasis caused by fluconazole-resistant strains as well as against aspergillosis is needed. In order to meet the above-mentioned need, we have searched for new triazole antifungal agents and selected TAK-456,1-[(1 R,2 R)-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-(1 H-1,2,4-tri-azol-1-yl) propyl]-3-[4-(1 H-1-tetrazolyl)phenyl]-2-imidazolidinone (Fig. 1). TAK-456 has broad antifungal activities against major pathogenic fungi, such as Candida spp., A. fumigatus, and Cryptococcus neoformans. This paper deals with the in vitro and in vivo antifungal activities of TAK-456.
FIG. 1.
Chemical structures of TAK-456 and reference azoles.
(Most of this work was presented at the 40th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 17 to 20 September 2000 [abstr. 1085 and 1086].)
MATERIALS AND METHODS
Organisms.
C. albicans TIMM1756 and A. fumigatus TIMM1728 were kindly supplied by H. Yamaguchi, Teikyo University School of Medicine, Tokyo, Japan. C. albicans CA383 and A. fumigatus 437 were generous gifts from K. Shimada, University of Tokyo, Institute of Medical Science, Tokyo, Japan, and A. Polak, Hoffman-La Roche, Basel, Switzerland, respectively. C. albicans S78941 was kindly provided by J. R. Perfect, Duke University Medical Center, Durham, N.C. The other fungal strains used in the study were distinct clinical isolates recently obtained from hospitals in the United States and Japan. Clinical isolates were obtained from invasive or mucosal infection.
Animals.
Five-to 6-week-old Crj:CDF1 female mice (Charles River Japan, Inc., Kanagawa, Japan) weighing 17 to 22 g were used. They were caged in groups of 10 and given food and water ad libitum. All procedures were performed in accordance with the standards for humane handling, care, and treatment of research animals and were approved by the Takeda Institutional Animal Ethics Committee (Approval No. E1-019).
Antifungal agents.
TAK-456 (12, 15) was synthesized at Takeda Chemical Industries, Ltd., Osaka, Japan. Voriconazole was prepared in our laboratory according to the method described in the patents (Pfizer Co., PCT Int. Appl. W0 9706160 A1, 20 February 1997). Fluconazole (Pfizer, Tokyo, Japan) and itraconazole (Janssen-Kyowa, Tokyo, Japan) were extracted from commercially available intravenous injection and capsules, respectively, in our laboratory. The purity of all azoles as determined by high-performance liquid chromatography was more than 99%. Amphotericin B was obtained commercially from Bristol-Myers Squibb K. K., Tokyo, Japan. The drugs were dissolved individually in dimethyl sulfoxide (Wako Pure Chemical Ltd., Osaka, Japan) for in vitro studies. For in vivo and pharmacokinetic studies, TAK-456 and fluconazole were suspended in 0.5% carboxymethyl cellulose (CMC; Wako Pure Chemical Ltd.). In order to enhance oral absorption of itraconazole in mice, it was dissolved in 60% 2-hydroxypropyl β-cyclodextrin (HPCD; Nihon Shokuhin Kako Ltd., Tokyo, Japan) at 25 mg per ml (11) and was diluted with 10% HPCD.
Ergosterol biosynthesis by cell extract.
Incorporation of [14C]mevalonic acid into ergosterol by cell extract was determined by previous method (4) with some modifications. Briefly, yeast cells of C. albicans and conidia of A. fumigatus were grown with shaking in Sabouraud dextrose broth (Difco Laboratories, Detroit, Mich.) until the late exponential phase. The extract was prepared by disrupting cells by vortexing with glass beads (diameter, 0.25 to 0.45 mm) using a Braun disintegrator (Braun GmbH, Melsungen, Germany). Cell extract (4 mg of protein per test tube) was incubated aerobically at 37°C for 60 min with 0.125 μCi (40 mCi/mmol) of [14C]mevalonic acid (Amersham, Tokyo, Japan) and an NADPH generator in 0.5 ml of 0.1 M potassium phosphate buffer (pH 7.4) in the presence of serially diluted test azole. After saponification at 80°C for 60 min with 1 ml of 2.7 M KOH in 90% (vol/vol) methanol, the nonsaponifiable lipids were extracted and separated by thin-layer chromatography. The thin-layer chromatography plate was then exposed to a Fuji film BAS III, and the incorporation of radioactivity into desmethylated sterol was measured by densitometry of the autoradiogram. Inhibitory activity of the azole against sterol synthesis was expressed in terms of the concentration giving a 50% reduction in the synthesis of desmethylated sterol, i.e., a 50% inhibitory concentration (IC50).
Antifungal susceptibility testing.
The MICs for Candida and Cryptococcus strains were determined by the broth microdilution method with RPMI 1640 medium (Gibco BRL Laboratories, Grand Island, N.Y.) according to the procedures of the National Committee for Clinical Laboratory Standards described in document M27-A (16), and those for Aspergillus strains were determined by the procedures described in document M38-P (17). Yeasts were grown on Sabouraud dextrose agar (Difco Laboratories) at 35°C for 24 to 48 h, and filamentous fungi were grown on potato dextrose agar (Eiken Chemical Co., Tokyo, Japan) at 35°C for 7 days. The wells were inoculated with 100 μl of the culture suspension diluted to a final inoculum of 5 × 102 to 2.5 × 103 cells/ml or 0.4 × 104 to 5 × 104 conidia/ml. Fungal growth was observed 48 h (72 h for C. neoformans) after incubation at 35°C. The MICs of the azoles were the lowest drug concentrations to give a prominent decrease in turbidity from that of the control fungal growth. The MICs of amphotericin B were the lowest drug concentrations at which there was an absence of visible fungal growth. The MIC50 and MIC90 were the concentrations of a drug required at which 50 and 90% of the clinical isolates tested were inhibited, respectively.
Preparation of inocula for in vivo study.
Infections were caused with fungal cells or conidia which had been stored at −80°C. Cells of C. albicans were grown overnight in Sabouraud dextrose broth at 30°C, washed twice with saline, suspended in 15% glycerol in Trypticase soy broth (Becton Dickinson, Cockeysville, Md.), and stored at −80°C until use. The cell counts in the stored suspension of C. albicans TIMM1756, CA383, and S78941 were 9.7 × 108, 1.1 × 109, and 2.6 × 109 CFU/ml, respectively. The cell suspension was thawed at room temperature and diluted with saline before infection.
A. fumigatus was grown on a Malt agar (Difco) plate for 10 days at 28°C. Conidia were collected with 0.1% Tween 80 (Sigma Chemical Co., St. Louis, Mo.) in saline, washed twice with 0.01% Tween 80 in saline, suspended in Trypticase soy broth containing 0.01% Tween 80 and 10% glycerol, and stored at −80°C. The numbers of conidia in the stored suspension of A. fumigatus TIMM1728 and 437 were 7.0 × 108 and 5.8 × 108 CFU/ml, respectively. The suspension of conidia was thawed at room temperature and was diluted with 0.01% Tween 80 in saline before infection.
Systemic candidiasis and aspergillosis.
Normal (5-week-old) and neutropenic (6-week-old) mice were used for candidiasis. To induce transient immunosuppression, mice were treated intraperitoneally with 200 mg of 5-fluorouracil (Kyowa Hakko Kogyo, Tokyo, Japan)/kg of body weight 4 days before infection. It has been reported that this treatment induced neutropenia from days 1 to 9 (24). The fungal cells or conidial suspension of 0.2 ml was inoculated into the tail veins of mice. A challenge dose of C. albicans TIMM1756 for normal mice was 5.3 × 105 CFU/mouse. Challenge doses of C. albicans TIMM1756, CA383, and S78941 for neutropenic mice were 3.9 × 104, 2.8 × 104, and 2.6 × 104 CFU/mouse, respectively. Six-week-old transient neutropenic mice were used for aspergillosis. Transient immunosuppression was induced by intraperitoneal treatment with 200 mg of cyclophosphamide (Shionogi, Osaka, Japan)/kg 4 days before infection. A. fumigatus conidia were inoculated into the tail veins of mice. Challenge doses of A. fumigatus TIMM1728 and 437 were 1.4 × 105 and 1.2 × 105 CFU/mouse, respectively.
Antifungal treatment.
Test compounds were administered orally in a volume of 0.01 ml per g using oral gavage, once daily for 5 days starting 2 h after the fungal challenge. Groups of 10 mice were used for each dose level of an antifungal agent. The mortality of mice in candidiasis and aspergillosis was recorded for 14 and 10 days, respectively, after infection. The MICs of TAK-456 and reference drugs against C. albicans TIMM1756, C. albicans S78941, and A. fumigatus 437 are shown in Table 1. The MICs of TAK-456, fluconazole, and itraconazole for C. albicans CA383 are 0.5, 32, and 0.5 μg/ml, respectively, and those of TAK-456, fluconazole, and itraconazole for A. fumigatus TIMM1728 are 0.25, >64, and 0.25 μg/ml, respectively.
TABLE 1.
Inhibitory effect of TAK-456, fluconazole, and itraconazole on ergosterol synthesis by cell extract of C. albicans and A. fumigatus
| Compound | IC50 (μg/ml)a | MIC (μg/ml)b |
|---|---|---|
| C. albicans TIMM1756 | ||
| TAK-456 | 0.003 | 0.016 |
| Fluconazole | 0.007 | 0.25 |
| Itraconazole | 0.004 | 0.03 |
| Voriconazole | 0.003 | 0.008 |
| S78941 | ||
| TAK-456 | 0.010 | 1 |
| Fluconazole | 1.44 | 64 |
| Itraconazole | 0.005 | 0.5 |
| Voriconazole | 0.025 | 0.5 |
| A. fumigatus 437 | ||
| TAK-456 | 0.011 | 1 |
| Fluconazole | 2.10 | >64 |
| Itraconazole | 0.012 | 0.25 |
| Voriconazole | 0.018 | 0.5 |
The concentration causing 50% inhibition of incorporation of [14C]mevalonic acid into the ergosterol fraction.
MICs were determined following the NCCLS broth microdilution procedure.
Pharmacokinetics.
TAK-456 and itraconazole were orally administered to normal (5-week-old) female CDF1 mice. To obtain blood, mice were sacrificed by bleeding from the axillary arteries and veins under ether anesthesia and plasma was obtained by centrifugation (3,000 × g) of the blood sample. For TAK-456, plasma samples were obtained from three mice each time at 0.5, 1, 2, 4, 6, 8, and 24 h after administration. For itraconazole, plasma samples were obtained from three mice at 1, 2, 4, 6, 8, and 24 h after administration. Levels of TAK-456 and itraconazole in plasma were measured by bioassay with some modifications of the method described previously (14). There was no major detectable bioactive metabolite of TAK-456 in the mouse plasma. C. albicans IFO1389 was grown on a glucose-peptone agar plate (Nihon Seiyaku, Tokyo, Japan) for 2 days at 28°C. Organisms were recovered by washing the plate with water and by adjusting the absorbance of the suspension to 0.8 at 660 nm. The cell suspension was diluted 200-fold with glucose-peptone agar medium, and 10 ml of the medium was poured into plates. After the solidification of the medium, 8-mm-diameter wells were bored. A standard curve was made with 0.025, 0.05, 0.1, 0.2, and 0.4 μg of drug/ml in normal mouse plasma. Plasma samples of mouse which could have included active substances at more than 0.4 μg/ml were serially twofold diluted with normal mouse plasma before the assay. Standard and sample solutions were placed in the wells in duplicate, and the agar plate contents were incubated for 40 h at 28°C. The inhibition zone was then measured, and the drug concentrations in plasma were calculated from the calibration curves of the standard. The correlation coefficient of the standard curve was > 0.99 for all assays. The limit of detection for TAK-456 and itraconazole by this method was 0.025 μg/ml. The mean residence time (MRT) was determined by the equation MRT = AUMC0-8/AUC0-8, where AUMC0-8 is the area under the first moment of the concentration-time curve and AUC0-8 is the area under the plasma concentration-time (zero moment) curve (29). AUMC0-8 and AUC0-8 were calculated by the trapezoidal method (30).
Statistical analysis.
The survival time of mice was analyzed by the log-rank test with Bonferroni's correction of multiple comparisons. Statistical significance was defined as P < 0.05.
RESULTS
Sterol biosynthesis inhibition.
Inhibitory activities of TAK-456, fluconazole, itraconazole, and voriconazole against sterol synthesis by cell extract of C. albicans and A. fumigatus were compared. All compounds showed almost the same strong inhibitory activities against sterol synthesis by C. albicans TIMM1756, which is a fluconazole-susceptible strain, with IC50s of 0.003 to 0.007 μg/ml (Table 1). In the inhibition of ergosterol synthesis by C. albicans S78941, which has altered CYP51 with 4 amino acid changes from sensitive C. albicans ATCC 90028 (3) and is highly resistant to fluconazole, TAK-456 was 100 times more active than fluconazole and was as active as itraconazole and voriconazole. TAK-456 as well as itraconazole and voriconazole showed strong inhibitory activity against sterol synthesis in an extract of A. fumigatus 437 and was 200 times more active than fluconazole.
In vitro antifungal activities.
In vitro antifungal activity of TAK-456 against clinical isolates of yeast and filamentous fungi was compared with those of several azoles and amphotericin B. The MICs of TAK-456 and reference drugs for clinical isolates of Candida species and C. neoformans are summarized in Table 2. TAK-456 showed potent activities against isolates of C. albicans, Candida krusei, Candida parapsilosis, and Candida tropicalis, with MIC90s of 2 μg/ml or less, which were similar to those of itraconazole and voriconazole and 8 to 64 times lower than that of fluconazole. On the other hand, the MIC90 of TAK-456 for Candida glabrata was 32 μg/ml. TAK-456 was less active than itraconazole and voriconazole against C. glabrata. The MIC90 of TAK-456 against C. neoformans was 0.25 μg/ml, and its antifungal activity was similar to that of itraconazole but slightly inferior to that of voriconazole. MIC90s of amphotericin B against all Candida species and C. neoformans were 0.5 to 1 μg/ml. The antifungal activities of TAK-456 and reference drugs against clinical isolates of Aspergillus species are shown in Table 3. MIC90s of TAK-456 against Aspergillus species were 0.5 to 1 μg/ml. The in vitro activity of TAK-456 was superior to that of fluconazole and comparable to those of itraconazole, voriconazole, and amphotericin B. These results indicate that TAK-456 inhibits the growth of clinical isolates of Candida spp., Aspergillus spp., and C. neoformans at the concentrations of 2 μg/ml or less with the exception of C. glabrata.
TABLE 2.
Susceptibilities of clinical isolates of Candida species and C. neoformans to TAK-456 and reference antifungal agents
| Organism (no. of strains) and antifungal agent | MIC range (μg/ml)a | MIC50 | MIC90 |
|---|---|---|---|
| C. albicans (105) | |||
| TAK-456 | 0.004-8 | 0.016 | 0.25 |
| Fluconazole | 0.06-32 | 0.25 | 4 |
| Itraconazole | 0.016-1 | 0.06 | 0.5 |
| Voriconazole | 0.004-1 | 0.016 | 0.13 |
| Amphotericin B | 0.25-0.5 | 0.25 | 0.5 |
| C. krusei (11) | |||
| TAK-456 | 0.25-4 | 1 | 1 |
| Fluconazole | 16->64 | 32 | 64 |
| Itraconazole | 0.25-1 | 0.5 | 0.5 |
| Voriconazole | 0.13-2 | 0.25 | 1 |
| Amphotericin B | 0.5-1 | 0.5 | 1 |
| C. parapsilosis (20) | |||
| TAK-456 | 0.008-0.13 | 0.03 | 0.06 |
| Fluconazole | 0.25-2 | 1 | 2 |
| Itraconazole | 0.06-0.25 | 0.13 | 0.25 |
| Voriconazole | 0.008-0.13 | 0.03 | 0.06 |
| Amphotericin B | 0.25-0.5 | 0.25 | 0.5 |
| C. tropicalis (21) | |||
| TAK-456 | 0.016-4 | 0.13 | 2 |
| Fluconazole | 0.25->64 | 1 | 16 |
| Itraconazole | 0.03-1 | 0.13 | 1 |
| Voriconazole | 0.03-4 | 0.13 | 2 |
| Amphotericin B | 0.25-0.5 | 0.25 | 0.5 |
| C. glabrata (40) | |||
| TAK-456 | 0.008-32 | 4 | 32 |
| Fluconazole | 0.25->64 | 8 | 64 |
| Itraconazole | 0.016-16 | 1 | 16 |
| Voriconazole | 0.004-8 | 0.5 | 4 |
| Amphotericin B | 0.25-0.5 | 0.25 | 0.5 |
| C. neoformans (14) | |||
| TAK-456 | 0.06-0.5 | 0.13 | 0.25 |
| Fluconazole | 1-4 | 2 | 4 |
| Itraconazole | 0.06-0.25 | 0.13 | 0.25 |
| Voriconazole | 0.016-0.06 | 0.03 | 0.06 |
| Amphotericin B | 0.25-0.5 | 0.25 | 0.5 |
MICs were determined following the NCCLS M27-A broth microdilution procedure.
TABLE 3.
Susceptibilities of clinical isolates of Aspergillus species to TAK-456 and reference antifungal agents
| Organism (no. of strains) and antifungal agent | MIC range (μg/ml)a | MIC50 | MIC90 |
|---|---|---|---|
| A. fumigatus (41) | |||
| TAK-456 | 0.13-1 | 0.5 | 1 |
| Fluconazole | >64 | >64 | >64 |
| Itraconazole | 0.13-0.5 | 0.25 | 0.5 |
| Voriconazole | 0.06-0.5 | 0.25 | 0.5 |
| Amphotericin B | 0.25-1 | 0.5 | 0.5 |
| Aspergillus flavus (12) | |||
| TAK-456 | 0.25-1 | 0.25 | 0.5 |
| Fluconazole | 64->64 | >64 | >64 |
| Itraconazole | 0.25-0.5 | 0.25 | 0.5 |
| Voriconazole | 0.25-1 | 0.25 | 0.5 |
| Amphotericin B | 0.25-1 | 0.5 | 1 |
| Aspergillus niger (9) | |||
| TAK-456 | 0.25-1 | ||
| Fluconazole | 64->64 | ||
| Itraconazole | 0.25-0.5 | ||
| Voriconazole | 0.06-0.5 | ||
| Amphotericin B | 0.13-0.5 |
MICs were determined following the NCCLS M38-P broth microdilution procedure.
In vivo efficacy in mice.
We compared protective effects of TAK-456 with those of fluconazole and itraconazole against lethal systemic infections in mice caused by C. albicans and A. fumigatus, since these fungi are the major pathogens which cause deep-seated mycoses in humans. Voriconazole was not used in the in vivo study, since autoinduction of metabolism was observed in mice after multiple oral administrations of the drug as reported by Jezequel et al. (S. G. Jezequel, M. Clark, S. Cole, K. E. Evans, and P. Wastall, Abstr. 35th Intersci. Conf. Antimicrob. Agents Chemother., abstr. F76, p. 126, 1995). The MICs of TAK-456 and reference drugs for strains used in the in vivo experiments are given in Table 1 and in the antifungal treatment section of Materials and Methods. Antifungal activities of TAK-456 against these strains were similar to those of itraconazole. In normal mice, TAK-456 and fluconazole were effective against infection caused by fluconazole-susceptible C. albicans TIMM1756 at a dose of 1 mg/kg (P < 0.01 versus control [Fig. 2A]). In neutropenic mice, 80% of mice infected with C. albicans TIMM1756 survived at the end of observation when treated with 1 mg/kg TAK-456 (P < 0.01 versus control [Fig. 2B]). Although prolongation of survival time was observed by treatment with 1 mg/kg fluconazole (P < 0.01 versus control), TAK-456 was more effective than fluconazole (P < 0.01). In infections caused by fluconazole-resistant C. albicans CA383 and S78941 in neutropenic mice, TAK-456 significantly (P < 0.01 and P < 0.05 versus control, respectively) prolonged the survival time of mice at a dose of 10 mg/kg (Fig. 3A and B). Fluconazole and itraconazole at the same dose were not effective against both infections. TAK-456 was more effective than itraconazole against CA383 infection (P < 0.05).
FIG. 2.
Effects of TAK-456 and fluconazole against systemic infection caused by C. albicans TIMM1756 in normal (A) and neutropenic (B) mice. TAK-456 and fluconazole were administered orally once a day for 5 days at a dose of 1 mg/kg. n = 10. Symbols: •, vehicle (CMC); ○, TAK-456; and ◊, fluconazole.
FIG. 3.
Effects of TAK-456 and reference azoles against systemic infection caused by fluconazole-resistant C. albicans CA383 (A) and C. albicans S78941 (B) in neutropenic mice. In both infections, 10 mg of TAK-456 and of reference triazoles per kg was administered orally once a day for 5 days. n = 10. Symbols: ×, vehicle (HPCD) for itraconazole; •, vehicle (CMC) for other azoles; ○, TAK-456; ◊, fluconazole; and □, itraconazole.
We next examined therapeutic activities of TAK-456 and reference azoles against systemic infections caused by two strains of A. fumigatus in neutropenic mice (Fig. 4A). In the infection with A. fumigatus TIMM1728, 100 and 70% of mice treated with 10 mg of TAK-456 and of itraconazole per kg, respectively, were alive at the end of observation (P < 0.01 versus control). TAK-456 also rescued all the mice infected with A. fumigatus 437 at a dose of 10 mg/kg (P < 0.01 versus control [Fig. 4B]). Although itraconazole at a dose of 10 mg/kg was also effective (P < 0.05 versus control), TAK-456 was more effective than itraconazole (P < 0.05). Fluconazole at a dose of 10 mg/kg was ineffective against both infections with A. fumigatus. Thus, TAK-456 showed excellent in vivo activities against systemic infections caused by two strains of A. fumigatus in neutropenic mice.
FIG. 4.
Effects of TAK-456 and reference azoles against systemic infection caused by A. fumigatus TIMM1728 (A) and A. fumigatus 437 (B) in neutropenic mice. In both infections, 10 mg of TAK-456 and of reference triazoles per kg was administered orally once a day for 5 days. n = 10. Symbols: ×, vehicle (HPCD) for itraconazole; •, vehicle (CMC) for other azoles; ○, TAK-456; ◊, fluconazole; and □, itraconazole.
Levels in plasma of TAK-456 and itraconazole in mice.
Levels in plasma of TAK-456 and itraconazole after single oral administration to normal mice at a dose of 10 mg/kg were measured by bioassay, and the results are summarized in Table 4. The maximum concentration of TAK-456 in plasma (Cmax) was 3.0 ± 0.091 μg/ml, which was obtained 1 h after administration, and the plasma level of TAK-456 was reduced to 0.96 ± 0.19 μg/ml at 8 h. The AUC0-8 of TAK-456 was 14 μg·h/ml, and the MRT was 3.3 h. Itraconazole showed pharmacokinetic parameters almost similar to those of TAK-456, with some exceptions. The Cmax of itraconazole was 1.9 ± 0.50 μg/ml 1 h postadministration, and it was slightly lower than that of TAK-456. The level of itraconazole in plasma gradually decreased until 8 h postadministration in comparison to that of TAK-456. Neither TAK-456 nor itraconazole was detected in mouse plasma 24 h after oral administration at 10 mg/kg.
TABLE 4.
Pharmacokinetic parameters after oral administration of TAK-456 and itraconazole to mice
| Compounda | Mean level in plasma (μg/ml)b at different times (h)
|
MRT (h) | AUC0-8 (μg · h/ml) | ||||||
|---|---|---|---|---|---|---|---|---|---|
| 0.5 | 1 | 2 | 4 | 6 | 8 | 24 | |||
| TAK-456 | 2.3 ± 0.19 | 3.0 ± 0.091 | 2.3 ± 0.22 | 1.8 ± 0.22 | 1.2 ± 0.090 | 0.96 ± 0.19 | <0.025 | 3.3 | 14 |
| Itraconazole | NTc | 1.9 ± 0.50 | 1.3 ± 0.39 | 1.6 ± 0.46 | 1.6 ± 0.27 | 1.4 ± 0.56 | <0.025 | 4.2 | 12 |
Test compounds were administered to mice at a dose of 10 mg/kg.
Values are the means ± standard deviations of results with three mice each.
NT, not tested.
DISCUSSION
TAK-456 showed strong in vitro antifungal activities against clinically isolated major opportunistic pathogens such as Aspergillus and Candida species, except C. glabrata. The in vitro antifungal activities of TAK-456 were superior to those of fluconazole and similar to those of itraconazole and voriconazole, except that the activities of TAK-456 against C. glabrata and C. neoformans were slightly inferior to those of voriconazole. Azole compounds are known to show antifungal activities by inhibiting ergosterol biosynthesis of fungal cells. As shown in Table 1, TAK-456 strongly inhibited ergosterol synthesis in cell extract of fluconazole-susceptible and -resistant C. albicans and A. fumigatus, as did itraconazole and voriconazole. The strong inhibitory activity of TAK-456 in the sterol synthesis was reflected in the strong in vitro antifungal activity against clinical isolates of fungi.
Triazoles exhibit their antifungal activities by inhibiting biosynthesis of ergosterol, which is a major component of fungal cell membrane. The target enzyme of triazoles in the process of ergosterol synthesis is a lanosterol 14α-demethylase (CYP51), a family of cytochrome P-450. In spite of the strong inhibitory activity of TAK-456, itraconazole, and voriconazole against ergosterol synthesis by C. albicans S78941, MICs of these compounds for the strains were higher than those for the fluconazole-susceptible C. albicans TIMM1756. We believe that increased drug efflux pumps of the azoles from the cell confer resistance for C. albicans S78941 against triazoles, as suggested previously by Sanglard et al. (21) and White (26).
To clarify the correlation between in vitro and in vivo activities of antimicrobial drugs is very important. It is well known that the clinical efficacy of triazoles may not always correlate with their in vitro antifungal activity (2). Accordingly, the in vivo efficacy of triazoles is much more important than their in vitro activity for predicting their clinical efficacy. TAK-456 showed good in vivo efficacies in lethal systemic infections with C. albicans and A. fumigatus, especially in neutropenic mice. For example, the therapeutic activity of TAK-456 against systemic infection with fluconazole-susceptible C. albicans in neutropenic mice was superior to that of fluconazole (P < 0.01), though TAK-456 and fluconazole showed similar effects against the infection with this strain in normal mice (Fig. 2). TAK-456 expressed superior in vivo activities against systemic infections caused by fluconazole-resistant C. albicans and A. fumigatus in neutropenic mice (Fig. 3 and 4). The excellent efficacies of TAK-456 in mycoses in neutropenic mice suggest a potent clinical efficacy of TAK-456, since deep-seated mycoses occur frequently in the immunocompromised patients with severe underlying diseases. Although we have not yet tested the in vivo activity of TAK-456 against the non-albicans Candida strains, it is expected that TAK-456 is active against systemic infections with these species, since TAK-456 showed similar in vitro activities to C. albicans and other Candida species.
Itraconazole possesses potent in vitro activity against fluconazole-resistant C. albicans and A. fumigatus (23). Although in vitro activities of TAK-456 against these strains were comparable to those of itraconazole, TAK-456 showed in vivo activity superior to that of itraconazole against some infections with fluconazole-resistant C. albicans and A. fumigatus at a dose of 10 mg/kg. As shown in Table 4, the Cmax of TAK-456 was slightly higher than that of itraconazole in mice after oral administration at 10 mg/kg. Therefore, in vivo activities of TAK-456 that were superior to those of itraconazole against these infections might be attributed to the higher levels in plasma of TAK-456 than of itraconazole. The possibility that the difference in the accumulation of TAK-456 and itraconazole in mouse plasma after repeated administration once daily for 5 days might affect their protective efficacies can be excluded, since none of these triazoles was detected in mouse plasma 24 h post administration at a dose of 10 mg/kg.
Toxicities of TAK-456 in animals including hepatotoxicity were similar or weaker than those of triazoles on the market (data not shown).
In conclusion, TAK-456 showed strong therapeutic activity against mouse models of systemic candidiasis and aspergillosis. Excellent in vitro and in vivo activities of TAK-456 against these clinically relevant fungal species suggest that TAK-456 is a promising candidate for development for the treatment of candidiasis and aspergillosis in humans.
Acknowledgments
We thank J. R. Perfect, Duke University Medical Center; S. G. Filler, Harbor-UCLA Medical Center, Los Angeles, Calif.; A. Polak, Hoffman-La Roche; H. Yamaguchi, Teikyo University School of Medicine; K. Shimada, University of Tokyo, Institute of Medical Science; and Y. Niki, Kawasaki Medical College, Okayama, Japan, for providing the clinical isolates of fungal strains.
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