Abstract
The activity of CS-758 (R-120758), a new triazole antifungal agent, was evaluated and compared with those of fluconazole, itraconazole, and amphotericin B in vitro and with those of fluconazole and itraconazole in vivo. CS-758 exhibited potent in vitro activity against clinically important fungi. The activity of CS-758 against Candida spp. was superior to that of fluconazole and comparable or superior to those of itraconazole and amphotericin B. CS-758 retained potent activity against Candida albicans strains with low levels of susceptibility to fluconazole (fluconazole MIC, 4 to 32 μg/ml). Against Aspergillus spp. and Cryptococcus neoformans, the activity of CS-758 was at least fourfold superior to those of the other drugs tested. CS-758 also exhibited potent in vivo activity against murine systemic infections caused by C. albicans, C. neoformans, Aspergillus fumigatus, and Aspergillus flavus. The 50% effective doses against these infections were 0.41 to 5.0 mg/kg of body weight. These results suggest that CS-758 may be useful in the treatment of candidiasis, cryptococcosis, and aspergillosis.
The risk of opportunistic infections is greatly increasing in patients who are immunocompromised due to cancer chemotherapy, organ or bone marrow transplantation, or human immunodeficiency virus infection (7). Candida albicans is the organism most often associated with both mucosal and hematogenously disseminated infections (2, 4, 16); and recently, other Candida spp. such as Candida glabrata, Candida tropicalis, and Candida krusei have emerged as clinically important pathogens (3, 4, 15). Aspergillus spp. and Cryptococcus neoformans are also important pathogens which cause serious infections (5, 6, 8, 11).
Triazole antifungal agents such as fluconazole (FLC) or itraconazole (ITC) are now widely used for the treatment of fungal infections due to their broad spectra of activity and improved safety profile compared to those of other antifungal agents such as flucytosine and amphotericin B (AMB) (1, 7, 18). CS-758 (R-120758; Fig. 1) is a new triazole antifungal agent that is under development. Here, we report on the in vitro antifungal activity of CS-758 against clinically important fungi and the in vivo activity of CS-758 against systemic infections caused by C. albicans, C. neoformans, Aspergillus fumigatus, and Aspergillus flavus in mice.
FIG. 1.
Chemical structure of CS-758.
(This research was presented at the 40th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 17 to 20 September 2000 [Y. Kamai, T. Harasaki, T. Fukuoka, S. Ohya, K. Uchida, H. Yamaguchi, and S. Kuwahara, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1088, 2000; Y. Kamai, T. Harasaki, T. Fukuoka, S. Ohya, H. Yasuda, K. Uchida, H. Yamaguchi, and S. Kuwahara, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1091, 2000].)
MATERIALS AND METHODS
Antifungal agents.
CS-758 was synthesized by Sankyo Co., Ltd., for both in vitro and in vivo studies. For the in vitro study, FLC and ITC were extracted from commercial preparations purchased from Pfizer Pharmaceuticals, Inc. (Tokyo, Japan), and Janssen Kyowa Co., Ltd. (Tokyo, Japan), respectively. AMB was obtained commercially from Sigma Chemical Co. (St. Louis, Mo.). All the drugs for the in vitro study were dissolved in dimethyl sulfoxide (10, 12, 13, 14, 19, 21). Commercial preparations of FLC and ITC were used for the in vivo study. All the drugs for the in vivo study were suspended in 0.5% sodium carboxymethyl cellulose.
Organisms.
A total of 401 strains of 13 fungal species were used for the in vitro study. Yeast (Candida spp., Trichosporon asahii, and C. neoformans) and Aspergillus spp. were cultured on Sabouraud dextrose agar (SDA; Eiken Chemical Co., Ltd., Tokyo, Japan) and potato dextrose agar (PDA; Eiken Chemical Co., Ltd.), respectively. The following strains were used for the in vivo study: C. albicans SANK51486, C. neoformans TIMM1855, C. neoformans TIMM0362, A. fumigatus SANK10569, A. fumigatus TIMM1776, and A. flavus SANK18497. C. albicans was cultured on SDA and was then inoculated into YPG medium, which consisted of 0.5% yeast extract (Difco Laboratories, Detroit, Mich.), 1% peptone (Difco Laboratories), and 2% glucose (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and incubated at 35°C overnight with shaking. C. neoformans was cultured on SDA at 35°C for 48 to 72 h. Aspergillus spp. were cultured on PDA at 30°C for 1 week.
Antifungal susceptibility testing.
The MICs for Candida spp. and T. asahii were determined by a broth microdilution method based on the NCCLS method outlined in documents M27-T (12) and M27-A (13), with slight modifications (10, 21). RPMI 1640 medium buffered to pH 7.0 with 3-(N-morpholino)propanesulfonic acid (MOPS) was used as the culture medium, and the inoculum size was 1 × 103 to 5 × 103 cells/ml. The microdilution plates inoculated with fungi were incubated at 35°C, and the turbidity of the growth control wells was observed every 24 h. The end point was determined visually when the growth control wells gave a turbidity of approximately 0.2 at a wavelength of 630 nm. After the plates were shaken, 80% inhibition standard wells were prepared by diluting 40 μl of the contents of the growth control well with 160 μl of the culture medium. The MICs of AMB were defined as the lowest concentrations that inhibited visible growth. The MICs of the triazole agents were defined as the lowest concentrations that inhibited 80% of the growth as determined by comparison with the growth in the 80% inhibition standard wells. The MICs for C. neoformans were determined by a broth microdilution method based on the method outlined in NCCLS document M27-T (12). Yeast nitrogen base buffered to pH 7.0 with MOPS was used as the culture medium, and the inoculum size was 1 × 104 to 5 × 104 cells/ml. The plates were incubated at 35°C for 48 h, and the end point was determined spectrophotometrically on the basis of the absorbance at a wavelength of 485 nm. The MICs of AMB were defined as the lowest concentrations which completely inhibited growth. The MICs of the triazole agents were defined as the lowest concentrations which inhibited 50% of the growth compared with the growth for the growth control. The MICs for Aspergillus spp. were determined by the broth microdilution method with an oxidation-reduction indicator (14, 19). RPMI 1640 medium buffered to pH 7.0 with MOPS containing 10% Alamar Blue (BioSource International, Inc., Camarillo, Calif.) was used as the culture medium, and the inoculum size was 1.0 × 104 cells/ml. The plates were incubated at 30°C until the color of the growth control changed to red, and the MICs were determined on the basis of the absorbance at a wavelength of 570 nm. The MICs of all drugs tested were defined as the lowest concentrations which inhibited 80% of the growth compared with the growth for the growth control. The MICs at which 50% of the strains were inhibited (MIC50s) and MIC90s were calculated for all species except those for which too few strains (six or fewer strains) could be tested.
Animals.
Specific-pathogen-free male ddY mice (age, 4 weeks) were purchased from Japan SLC, Inc. (Shizuoka, Japan), and were used for the present study after an acclimation period of approximately 1 week. Mice were given food and water ad libitum. All experiments with animals were carried out according to the guidelines provided by the Institutional Animal Care and Use Committee of Sankyo Co., Ltd.
Systemic infections.
For C. albicans, a fungal suspension was prepared with sterile saline. The mice were inoculated with 5.0 × 106 cells via the tail vein. The drugs were administered orally once daily for 10 days starting at 4 h postinoculation at doses of 0.39, 1.6, and 6.3 mg/kg of body weight. For C. neoformans, a fungal suspension was prepared with sterile saline. The mice were inoculated with 1.0 × 106 and 5.0 × 106 cells of C. neoformans TIMM1855 and TIMM0362, respectively, via the tail vein. The drugs were administered orally once daily for 10 days starting at 24 h postinoculation at doses of 1.6, 6.3, and 25 mg/kg for CS-758 and 6.3, 25, and 100 mg/kg for FLC and ITC. For A. fumigatus and A. flavus, conidial suspensions were prepared with sterile saline containing 0.05% Tween 80. The mice were inoculated with 5.0 × 106 cells via the tail vein. The drugs were administered orally once daily for 10 days starting at 24 h postinoculation at doses of 0.39, 1.6, and 6.3 mg/kg for CS-758 and 6.3, 25, and 100 mg/kg for ITC. In all experiments, each group contained 10 mice, and the control group received 0.2 ml of 0.5% sodium carboxymethyl cellulose per day.
Statistical analysis.
The 50% effective doses (ED50s) and 95% confidence limits were calculated by the probit method from the survival rates on day 15 for mice infected with C. albicans, A. fumigatus, and A. flavus. For C. neoformans, the ED50s were calculated from the survival rates on day 25 because in preliminary experiments we found that the ED50s among the drugs could not be compared on day 15.
RESULTS
In vitro antifungal activity.
Table 1 shows the MIC ranges, MIC50s, and MIC90s for the yeast strains tested. A total of 139 strains of C. albicans were susceptible to FLC (MICs, ≤0.5 μg/ml). CS-758 exhibited potent activity against yeasts (MIC range, ≤0.008 to 1 μg/ml). Among the drugs tested CS-758 exhibited the lowest MIC90s for C. albicans, C. tropicalis, C. parapsilosis, and C. neoformans; these were at least fourfold superior to those of the reference drugs. The MIC90s of CS-758 for C. glabrata and C. krusei were equal to or superior to those of the other drugs tested. CS-758 also exhibited potent activity against the other yeasts tested.
TABLE 1.
In vitro antifungal activities of CS-758 against yeasts
| Organism (no. of strains) and drug | MIC (μg/ml)
|
||
|---|---|---|---|
| Range | 50% | 90% | |
| Candida albicansa (139) | |||
| CS-758 | ≤0.008–0.016 | ≤0.008 | ≤0.008 |
| FLC | ≤0.06–0.5 | 0.12 | 0.25 |
| ITC | ≤0.008–0.03 | 0.016 | 0.03 |
| AMB | 0.12–1 | 0.25 | 0.5 |
| Candida glabrata (73) | |||
| CS-758 | ≤0.008–1 | 0.12 | 0.25 |
| FLC | 0.5–64 | 4 | 8 |
| ITC | 0.06–1 | 0.25 | 0.5 |
| AMB | 0.06–0.5 | 0.25 | 0.25 |
| Candida tropicalis (43) | |||
| CS-758 | ≤0.008–0.06 | ≤0.008 | 0.016 |
| FLC | ≤0.12–4 | 0.5 | 0.5 |
| ITC | ≤0.008–0.12 | 0.03 | 0.06 |
| AMB | 0.12–0.5 | 0.25 | 0.25 |
| Candida parapsilosis (29) | |||
| CS-758 | ≤0.008–0.25 | 0.016 | 0.06 |
| FLC | 0.5–32 | 1 | 8 |
| ITC | 0.03–0.5 | 0.12 | 0.5 |
| AMB | ≤0.03–2 | 0.5 | 1 |
| Candida krusei (15) | |||
| CS-758 | 0.03–0.25 | 0.12 | 0.25 |
| FLC | 2–64 | 32 | 64 |
| ITC | 0.06–0.25 | 0.12 | 0.25 |
| AMB | 0.25–1 | 0.5 | 1 |
| Candida guilliermondii (4) | |||
| CS-758 | 0.03–0.06 | ||
| FLC | 2–4 | ||
| ITC | 0.25–0.5 | ||
| AMB | 0.12–1 | ||
| Candida kefyr (3) | |||
| CS-758 | ≤0.008 | ||
| FLC | 0.25–1 | ||
| ITC | 0.06 | ||
| AMB | 0.25 | ||
| Trichosporon asahii (6) | |||
| CS-758 | 0.03 | ||
| FLC | 4–8 | ||
| ITC | 0.25–0.5 | ||
| AMB | 0.12–2 | ||
| Cryptococcus neoformans (22) | |||
| CS-758 | ≤0.008–0.016 | ≤0.008 | ≤0.008 |
| FLC | 1–16 | 4 | 8 |
| ITC | 0.016–0.12 | 0.016 | 0.06 |
| AMB | 0.12–0.25 | 0.25 | 0.25 |
FLC MIC, ≤0.5 μg/ml.
Table 2 shows the MICs for C. albicans strains with low levels of susceptibility to FLC (FLC MICs, ≥4 μg/ml). Although the CS-758 MICs were high (4 to >16 μg/ml) for highly resistant strains (FLC MICs, ≥256 μg/ml), as were those of ITC, CS-758 exhibited potent activity against other strains (FLC MICs, 4 to 32 μg/ml), with an MIC range of 0.016 to 0.5 μg/ml.
TABLE 2.
In vitro antifungal activities of CS-758 against C. albicans strainsa
| Strain | MIC (μg/ml)
|
|||
|---|---|---|---|---|
| CS-758 | FLC | ITC | AMB | |
| CAL-0013 | 0.016 | 4 | 0.03 | 0.25 |
| CAL-0066 | 0.03 | 4 | 0.12 | 0.5 |
| CAL-0148 | 0.016 | 8 | 0.03 | 0.25 |
| CAL-0008 | 0.5 | 16 | 0.5 | 0.5 |
| CAL-0012 | 0.06 | 16 | 0.25 | 0.25 |
| CAL-0015 | 0.03 | 16 | 0.25 | 0.25 |
| CAL-0014 | 0.06 | 32 | 0.12 | 0.5 |
| CAL-0097b | 4 | 256 | 4 | 0.5 |
| CAL-0076b | 8 | 512 | 8 | 0.5 |
| CAL-0002b | 8 | >512 | 8 | 0.5 |
| CAL-0145b | >16 | >512 | >16 | 1 |
FLC MIC, ≥ 4 μg/ml.
For these strains, MICs were determined again, and the results of the second test are provided here.
Table 3 shows the in vitro activities of the drugs tested against Aspergillus spp. CS-758 exhibited potent activity, with an MIC range of ≤0.016 to 0.12 μg/ml. When the MIC90s for A. fumigatus and A. flavus were compared, CS-758 was found to be the most potent drug tested.
TABLE 3.
In vitro antifungal activities of CS-758 against Aspergillus spp.
| Organism (no. of strains) and drug | MIC (μg/ml)
|
||
|---|---|---|---|
| Range | 50% | 90% | |
| Aspergillus fumigatus (29) | |||
| CS-758 | ≤0.016–0.12 | 0.03 | 0.06 |
| ITC | 0.03–0.25 | 0.12 | 0.25 |
| AMB | 0.5–2 | 1 | 2 |
| Aspergillus flavus (19) | |||
| CS-758 | ≤0.016–0.06 | 0.06 | 0.06 |
| ITC | 0.03–0.25 | 0.12 | 0.25 |
| AMB | 0.5–2 | 2 | 2 |
| Aspergillus niger (5) | |||
| CS-758 | 0.03–0.06 | ||
| ITC | 0.12–0.5 | ||
| AMB | 0.25–0.5 | ||
| Aspergillus terreus (3) | |||
| CS-758 | 0.03 | ||
| ITC | 0.03–0.12 | ||
| AMB | 2 | ||
Efficacy against systemic infections.
The efficacy of CS-758 was evaluated against murine systemic infections, and the results are shown in Table 4. Against infections caused by C. albicans, CS-758 exhibited a strong efficacy, with an ED50 of 0.41 mg/kg, which was comparable to that of FLC and superior to that of ITC. Against two C. neoformans strains, CS-758 exhibited the strongest efficacies, with ED50s of 3.1 and 5.0 mg/kg, respectively. CS-758 also exhibited strong efficacies against A. fumigatus and A. flavus, with ED50s of 2.4 to 3.1 mg/kg, which made it more potent than ITC.
TABLE 4.
Efficacies of CS-758 against murine systemic infections
| Pathogen and drug | MIC (μg/ml) | ED50 (mg/kg) | 95% confidence limit (lower, upper mg/kg) |
|---|---|---|---|
| Candida albicans SANK51486 | |||
| CS-758 | ≤0.008 | 0.41 | NCa |
| FLC | 0.12 | 0.43 | NC |
| ITC | 0.016 | >6.3 | NC |
| Cryptococcus neoformans TIMM1855 | |||
| CS-758 | ≤0.008 | 3.1 | 1.9, 5.1 |
| FLC | 4 | 45 | 22, 140 |
| ITC | 0.016 | >100 | NC |
| Cryptococcus neoformans TIMM0362 | |||
| CS-758 | ≤0.008 | 5.0 | NC |
| FLC | 8 | 50 | 31, 82 |
| ITC | 0.016 | >100 | NC |
| Aspergillus fumigatus SANK10569 | |||
| CS-758 | 0.03 | 2.7 | 1.6, 4.7 |
| ITC | 0.12 | 29 | 14, 61 |
| Aspergillus fumigatus TIMM1776 | |||
| CS-758 | ≤0.016 | 2.4 | 1.3, 4.3 |
| ITC | 0.12 | 91 | NC |
| Aspergillus flavus SANK18497 | |||
| CS-758 | 0.06 | 3.1 | NC |
| 1 ITC | 0.12 | 50 | 31, 82 |
NC, not calculable.
DISCUSSION
In the present study, CS-758, a new triazole antifungal agent, was shown to have broad-spectrum and potent activities against clinically important fungi compared with those of FLC, ITC, and AMB. Recently, the increase in the numbers of FLC-resistant C. albicans isolates has become a clinical problem (9, 17). In the present study, we separated C. albicans strains which exhibited reduced susceptibilities to FLC (FLC MICs, ≥4 μg/ml) from strains which were susceptible to FLC (FLC MICs, ≤0.5 μg/ml). According to NCCLS document M27-A (13), the interpretive breakpoints for FLC are as follows: susceptible, ≤8 μg/ml; susceptible-dose dependent, 16 to 32 μg/ml; and resistant, ≥64 μg/ml. Our results showed that FLC-resistant strains exhibit cross-resistance to CS-758 as well as ITC. However, CS-758 retained potent activity against FLC-susceptible and FLC-susceptible-dose dependent strains. It is known that various mechanisms are responsible for resistance to FLC, for example, mutations in the drug target gene, ERG11, and elevated levels of expression of a multiple drug efflux pump such as MDR1, CDR1, or CDR2 (20). Although we have no information about the mechanisms of resistance for the resistant strains used in the present study, one possible mechanism is the activation of CDR proteins because they are known to pump out both FLC and ITC (20).
As for the in vivo activity, CS-758 exhibited strong efficacies against murine systemic infections caused by C. albicans, C. neoformans, A. fumigatus, and A. flavus and was the most potent among the drugs tested under the experimental conditions used in the present study.
In addition to the potent activities of CS-758 both in vitro and in vivo, preliminary pharmacokinetic and metabolic analyses demonstrated the good oral absorption of CS-758 and the wide distribution of CS-758 in tissue (T. Shibayama, N. Kikuchi, Y. Matsushita, K. Kawai, A. J. John, T. Hirota, and S. Kuwahara, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1090, 2000), and preliminary safety studies have indicated that CS-758 is expected to be highly tolerable (N. Maeda, T. Hosokawa, A. Hyogo, S. Manabe, and S. Kuwahara, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 1092, 2000). These results warrant the development of CS-758 as a therapeutic agent in humans.
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