In-vitro comparative activity of UR-9825, itraconazole and fluconazole against clinical isolates of Candida spp.

Gema Ramos, Manuel Cuenca-Estrella, Araceli Monzón and Juan L. Rodríguez-Tudela*

Unidad de Micología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
UR-9825 is a new broad-spectrum triazole antifungal agent with a good pharmacokinetic profile and excellent bioavailability. It shows high in-vitro activity and efficacy in models of systemic candidosis in rats and rabbits, comparing favourably with fluconazole. The purpose of this study was to evaluate the in-vitro activity of UR-9825 and to compare it with that of fluconazole and itraconazole against 283 clinical isolates of Candida spp. UR-9825 was more potent against Candida spp. than both fluconazole and itraconazole, even against some Candida albicans and Candida krusei isolates with decreased susceptibility to fluconazole (MIC 16 mg/L).


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Newer azoles such as fluconazole and itraconazole are frequently used in the treatment of fungal infections caused by Candida spp. Nevertheless, fluconazole lacks activity against certain Candida spp. 1 and itraconazole has frequent drug interactions and poor absorption after oral administration, although the latter can be improved by its new oral formulation. 1 There is, therefore, a clear need for new drugs to improve the treatment of fungal infections.

UR-9825 is a novel broad-spectrum triazole agent, with a quinazolinone nucleus. In general, these compounds display higher in-vitro activity against filamentous fungi and have shorter plasma half-lives. The substitution of a halogen radical at the 7-position of the quinazolinone ring produces the most potent products in vitro. In this way, the 7-Cl derivative (1R, 2R)-7-chloro-3-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-triazol-1-) propyl quinazolin-4(3H)-one (UR-9825), was selected from a series of derivatives because of its high in-vitro activity and good pharmacokinetic profile. UR-9825 was obtained by a new enantioselective synthesis using Evans' chiral auxiliaries (seven steps, 40% overall yield). The other three stereoisomers have reduced or no activity. 2,3

Previous in-vitro studies with UR-9825 have shown that this antifungal agent is more potent than fluconazole, itraconazole and other azole agents against Candida spp. and other fungal species. 3 In vivo, UR-9825 showed efficacy in models of systemic candidosis in rats and rabbits. In a murine model, UR-9825 displayed an activity comparable with fluconazole when given for 5 days at 5 mg/kg (po, bid). 4 Finally, excellent protection was obtained with the compound in an immunocompromised rat model of disseminated aspergillosis. This new triazole showed good bioavailability in different animal species and low toxicity when administered to rats at 250 mg/kg qd or 100 mg/kg bid for 28 days. 2,3

In this study, the in-vitro antifungal activity of UR-9825 against clinical isolates of Candida spp. was assessed in comparison with that of fluconazole and itraconazole.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antifungal drugs

The three antifungal agents used in the susceptibility testing procedure were UR-9825 (J. Uriach Cía., Barcelona, Spain), fluconazole (Pfizer, Madrid, Spain) and itraconazole (Janssen Farmacéutica, Madrid, Spain). They were supplied in the form of assay powders obtained from the manufacturers.

Stock solutions of 1600 mg/L were prepared in 100% dimethyl sulphoxide (DMSO; Sigma Aldrich Química, Madrid, Spain). All solutions of antifungal agents were diluted with RPMI–2% glucose. The final concentrations of the antifungal agents ranged from 0.0002 to 128 mg/L for UR-9825, 0.015 to 128 mg/L for fluconazole and 0.015 to 8 mg/L for itraconazole. The plates contained two-fold serial dilutions of the antifungal drugs and two drug-free medium wells for sterility and growth controls.

Yeast isolates

A total of 283 Candida spp.isolates were recovered from clinical samples over 6 months at 47 different Spanish hospitals. The isolates tested were Candida albicans (131 strains), Candida parapsilosis (62 strains), Candida tropicalis (36 strains), Candida glabrata (34 strains), Candida krusei (12 strains) and Candida guilliermondii (eight strains). Each strain represented a unique isolate from a patient and was sent to our laboratory for identification or antifungal susceptibility testing. The majority of the isolates, 155, were obtained from blood cultures, 58 from oropharyngeal exudate, 20 from vaginal samples and 50 from other specimens. Isolates were identified using routine microbiological techniques. The reproducibility of the susceptibility testing technique was assessed by comparing the results of 50 consecutive determinations of the MICs of antifungal agents for two reference organisms (quality control strains), C. parapsilosis(ATCC 22019) and C. krusei(ATCC 6258). 5

Determination of MICs

The in-vitro susceptibility testing method employed was a microdilution one following the guidelines published by the NCCLS, 5 with minor modifications. 6 The susceptibility testing medium was RPMI 1640 with L-glutamine (Sigma Aldrich Química) buffered to pH 7 with 0.165 M morpholinepropanesulphonic acid (MOPS, Sigma Aldrich Química) and 10 M NaOH, and supplemented with glucose 18 g/L (RPMI–2% glucose). 6

The spectrophotometric method was used to prepare starting inocula of 10 6 cfu/mL. Sterile plastic microtitration plates each containing 96 flat-bottomed wells were inoculated with this dilution with an automatic pipettor programmed to dispense 10 µL into each well (final inocula 10 5 cfu/mL). The plates were incubated at 35°C for 24 h in a humid atmosphere. Spectrophotometric readings were performed with a Labsystems IEMS Reader MF (Labsystems Oy, Barcelona, Spain) at 540 nm. The MIC was determined as the lowest concentration of antifungal agent giving rise to an inhibition of growth >=50% of that of the drug-free control (MIC 50%).

The NCCLS has proposed 64 mg/L as the breakpoint for fluconazole resistance, and strains showing 16–32 mg/L have been defined as susceptible-dose dependent. 5 We have proposed 16 mg/L as the resistance breakpoint in strains causing oropharyngeal candidosis in AIDS patients by correlation of in-vitro and in-vivo studies. 7 In this study, we defined 16 mg/L as the breakpoint of decreased susceptibility to fluconazole.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Table I summarizes the distribution of MICs of fluconazole, itraconazole and UR-9825 for the 283 clinical isolates of Candida spp.


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Table I. Susceptibility of Candida spp. isolates to fluconazole, itraconazole and UR-9825 (n = 283)
 
Among these isolates, a total of 39 strains (nine C. albicans, three C. tropicalis, 15 C. glabrata and 12 C. krusei) had decreased susceptibility to fluconazole (MIC 16 mg/L). Table II shows the MIC50 and MIC90 values of UR-9825 and of itraconazole for these isolates. MIC50 and MIC90 of UR-9825 and of itraconazole were <=0.0002 and 0.03 mg/L for C. albicans, 0.06 and 1 mg/L for C. glabrata, <=0.0002 and 0.06 mg/L for C. krusei, respectively. The most highly resistant strains were C. tropicalis strains (both MIC50, >=8.0 and MIC90, 64 mg/L).


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Table II. Candida spp. isolates with MICs of fluconazole >=16 mg/L, and their comparison with MICs of itraconazole and UR-9825 (n = 39)
 
The MIC values obtained for the control organisms varied by no more than one three-fold dilution. For C. krusei (ATCC 6258) the MICs of UR-9825 were between 0.0019 and 0.0078 mg/L; for C. parapsilosis (ATCC 22019), between 0.0002 and 0.00048 mg/L.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the past decade, there has been a noticeable shift in the Candida spp. causing infection toward those other than C. albicans.1The introduction of two new antifungal agents (fluconazole and itraconazole) has permitted treatment of invasive mycoses and represent alternatives to amphotericin B for some indications. However, a serious problem is the emerging resistance of the organisms to these antifungals, and the relative resistance of certain non-albicans species to fluconazole. 8,9 These problems stimulate the development of new antifungal agents.

UR-9825 is a new broad-spectrum triazole antifungal agent with excellent bioavailability.

The present study shows the in-vitro susceptibility to fluconazole, itraconazole and UR-9825 of 244 fluconazole-susceptible and 39 fluconazole-resistant clinical isolates of Candida spp.

These results support and extend findings reported previously. 2,3 Like Bartroli et al .,we found that UR-9825 was more active than fluconazole against all Candida spp. isolates tested. In addition, the spectrum of activity was better than that of itraconazole. However, some isolates of Candida spp. that were less susceptible to fluconazole (MIC > 16 mg/L) were also less susceptible to UR-9825. We found that for isolates for which fluconazole MICs were high, itraconazole and UR-9825 MICs were proportionally higher than those for fluconazole-susceptible isolates (MIC <= 8 mg/L), which might indicate cross-resistance. Nevertheless, the MIC50 of UR-9825 for C. albicansand C. krusei fluconazole-resistant isolates was <= 0.0002 mg/L, confirming that UR-9825 has excellent activity against C. albicansand C. krusei, even against some isolates with decreased susceptibility to fluconazole.

It can be concluded from in-vitro data presented in this report that UR-9825 is more potent against Candida spp. than both fluconazole and itraconazole, even against some C. albicansand C. krusei isolates with decreased susceptibility to fluconazole. Nevertheless isolates of Candida spp. with high MICs of fluconazole and itraconazole also have proportionally higher MICs of UR-9825. More comprehensive studies are needed for this new triazole, in order to establish the translation of this in-vitro activity into clinical efficacy.


    Acknowledgments
 
We thank J. Uriach Cía, Pfizer and Janssen Farmacéutica for supplying the antifungal powders. This work was supported in part by grant 96/0598 from the Fondo de Investigaciones Sanitarias. M. Cuenca-Estrella is a Fellow of the Fondo de Investigaciones Sanitarias (grant 98/5040).


    Notes
 
* Corresponding author: Tel: +34-91-5097961; Fax: 34-91-5097966 Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Edwards, J. E., Bodley, G. P., Bowden, R. A., Buchner, T., DePauw, B. E., Filler, S. G. et al.(1997). International conference for the development of a consensus on the management and prevention of severe candidal infections. Clinical Infectious Diseases 25, 43–59.

2 . Bartroli, J., Turmo, E., Algueró, M., Boncompte, M., Vericat, L., Conte, L., Ramis, J. et al. (1997). UR-9825: A new triazole derivative with potent broad-spectrum antifungal activity. In Abstracts of the Thirty-Seventh Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 1997. Abstract E-67, p. 125. American Society for Microbiology, Washington, DC.

3 . Bartroli, J., Turmo, E., Algueró, M., Boncompte, E., Vericat, M. L., Conte, L. et al. (1998). New azole antifungals. 3. Synthesis and antifungal activity of 3-substituted-4(3H)-quinazolinones. Journal of Medical Chemistry 41, 1869–82.

4 . Vericat, M. L., Algueró, M., Merlos, M., Pérez, L. L., Araño, A. & Forn, J. (1997). UR-9825, a novel broad spectrum triazole for treatment of fungal infections: oral activity in systemic mycoses. In Final Programme and Abstract Book Trends in Invasive Fungal Infections 4., Barcelona, Spain, 1997.>Abstract P-94, p. 142. Imedex, The Netherlands.

5 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard M27-A. NCCLS, Villanova, PA.

6 . Rodríguez-Tudela, J. L. & Martínez-Suárez, J. V. (1994). Improved medium for fluconazole susceptibility testing of Candida albicans. Antimicrobial Agents and Chemotherapy 38, 45–8.

7 . Rodríguez-Tudela, J. L., Martínez-Suárez, J. V., Dronda, F., Laguna, F., Chaves, F. & Valencia, E. (1995). Correlation of in-vitro susceptibility test results with clinical response: a study of azole therapy in AIDS patients. Journal of Antimicrobial Chemotherapy 35, 793–804.

8 . Barry, A. L. & Brown, S. D. (1996). In-vitro studies of two triazole antifungal agents (voriconazole [UK-109,496] and fluconazole) against Candida species. Antimicrobial Agents and Chemotherapy 40, 1948–9.

9 . McGinnis, M. R., Pasarell, L., Sutton, D. A., Fothergill, A. W., Cooper, C. R. & Rinaldi, M. G. (1997). In-vitro evaluation of voriconazole against some clinically important fungi. Antimicrobial Agents and Chemotherapy 41, 1832–4.

Received 3 August 1998; returned 29 October 1998; revised 18 January 1999; accepted 14 February 1999