ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2009, p. 4540–4541 0066-4804/09/$08.00⫹0 doi:10.1128/AAC.00565-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 53, No. 10
Efficacy of Albaconazole against Candida albicans in a Vaginitis Model䌤 Gloria M. Gonza´lez,1* Efre´n Robledo,1 Elvira Garza-Gonza´lez,1 Mariana Elizondo,1 and J. Gerardo Gonza´lez2 Departamento de Microbiología, Facultad de Medicina, Universidad Auto ´noma de Nuevo Leo ´n, Monterrey, Nuevo Leo ´n, Me´xico,1 and Hospital Universitario, Facultad de Medicina, Universidad Auto ´noma de Nuevo Leo ´n, Monterrey, Nuevo Leo ´n, Me´xico2 Received 27 April 2009/Returned for modification 29 May 2009/Accepted 17 July 2009
The efficacy of albaconazole (ABC) was evaluated using a murine model of vaginal Candida albicans infection. Both ABC and fluconazole (FLC) were effective in reducing the fungal load from vaginas of infected mice; however, ABC demonstrated encouraging activities against an FLC-resistant strain, with trends toward superiority over FLC in some treatment groups. shaker. C. albicans was harvested by centrifugation, and the cells were counted in a hemocytometer. Quantitative cultures by serial dilution were used to confirm the inoculum size. Five-week-old BALB/c mice (weight, 18 g) were purchased from Harlan Mexico. A total of 10 mice were randomly assigned to each treatment or control group. Mice were housed five per cage and were provided food and water ad libitum. All animal research procedures were approved by the ethics committee of our university. Care, maintenance, and handling of the animals were in accordance with the Mexican government license conditions for animal experimentation. Two confirmatory animal studies were performed. A previously described model of vaginal candidosis was utilized (1). Three days prior to infection and on day 4 postchallenge, mice were given 0.5 mg of estradiol valerate (Delestrogen, King Pharmaceuticals) subcutaneously to maintain pseudoestrus. On the day of infection, the mice were anesthetized intraperitoneally with 80 mg/kg of body weight ketamine hydrochloride and then inoculated intravaginally with 20 l of a suspension of 2 ⫻ 108/ml CFU of C. albicans isolate 03-2718. The vaginal cavity of each mouse was swabbed (prior to treatment) 1 day after inoculation with C. albicans to ensure infection was consistently distributed among animals (10). Mice were swabbed on day 6 to evaluate the early treatment effects. Each alginate swab was placed in 0.9 ml sterile saline; serial 10-fold dilutions were made, and 100 l was placed on Sabouraud dextrose agar supplemented with 0.5% (wt/vol) chloramphenicol to quantify the CFU/ml. ABC (Stiefel Laboratories, Inc., Coral Gables, FL) and FLC (Pfizer, Inc., New York, NY) were obtained in powder form from the manufacturers. ABC was prepared fresh daily and dissolved in 0.2% carboxymethylcellulose (low viscosity) with 1% Tween 80 (Sigma-Aldrich, St. Louis, MO). FLC was dissolved in distilled water. ABC was given by gavage once or twice daily at 5, 10, 20, or 40 mg/kg orally on days 1 through 5 postinoculation. FLC was administered by oral gavage once or twice daily at 20 mg/kg of body weight. All treatments were given in a volume of 0.2 ml. Control mice were infected but received no active treatment; one group received 0.2% carboxymethylcellulose with 1% Tween 80 and another sterile distilled water as vehicle controls.
Vulvovaginal candidosis is a common infection among healthy women and is caused by members of the genus Candida, predominately Candida albicans (12). Current treatments for Candida vulvovaginitis include a wide range of intravaginal azole preparations, usually administered over several days. No evidence exists to suggest that any one formulation results in a better cure rate; overall cure rates for topical azoles range from 80% to 90% (13). Recent reports on the comparative efficacies of local versus oral treatments of uncomplicated vulvovaginal candidosis found no significant difference between either treatment modality (8); however, many patients prefer the convenience of oral administration. Oral fluconazole (FLC), a triazole, with its marked in vitro activity against Candida species, clinical efficacy, and ease of administration, is a convenient alternative to topical therapy in a single-dose regimen (11, 14) and is the most-prescribed treatment for vulvovaginal candidosis. Albaconazole (ABC) (formerly UR-9825) is also a triazole antifungal molecule, under development by Stiefel Laboratories, Inc. (Coral Gables, FL). ABC has potent and broadspectrum antifungal activity, with good in vitro activity against yeast (4, 6, 9), dermatophytes (3), and some filamentous and opportunistic fungi, such as Aspergillus spp., Paecilomyces spp., and Chaetomium spp. (2). ABC also has activity against the protozoon Trypanosoma cruzi (15). In the present study, we compared the effectiveness of ABC and FLC for the treatment of experimental C. albicans vaginitis caused by an FLC-resistant isolate. C. albicans isolate 03-2718 has been used previously in our laboratory for studies of vaginal candidosis (5). The 48-h MICs of ABC and FLC were 0.5 g/ml and ⬎64 g/ml, respectively. This indicates that in vitro, the isolate is resistant to FLC (7). The yeast was grown in sterile bottles, each containing 100 ml of RPMI-1640 with L-glutamine (Hardy Diagnostics, Santa Maria, CA), and incubated for 24 h at 35°C on a gyratory * Corresponding author. Mailing address: Facultad de Medicina, Universidad Auto ´noma de Nuevo Leo ´n, Departamento de Microbiología, Madero y Dr. E. A. Pequen ˜o s/n, Colonia Mitras Centro, Monterrey, Nuevo Leo ´n, Me´xico 64460. Phone: (5281) 83294157. Fax: (5281) 83485477. E-mail:
[email protected]. 䌤 Published ahead of print on 27 July 2009. 4540
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ALBACONAZOLE IN A MURINE MODEL OF VAGINAL CANDIDOSIS
TABLE 1. Recovery of C. albicans strain 03-2718 from vaginas of mice treated orally with ABC or FLC Mean log10 CFU (range) on day postinoculationb:
Vehicle or drug (dose 关mg/kg兴 and frequency)a
1
6
Control 关CMC兴 (BID) Control 关water兴 (BID) ABC (5 OD) ABC (5 BID) ABC (10 OD) ABC (10 BID) ABC (20 OD) ABC (20 BID) ABC (40 OD) ABC (40 BID) FLC (20 OD) FLC (20 BID)
5.91 (5.09–6.88) 5.18 (4.41–7.10) 5.06 (3.94–6.36) 5.46 (4.79–6.11) 5.07 (4.10–6.07) 5.43 (4.88–6.90) 5.13 (4.70–6.35) 5.48 (4.98–7.95) 5.62 (3.88–6.96) 5.79 (4.65–6.44) 5.51 (3.82–6.81) 5.90 (4.80–6.99)
5.34 (4.86–6.91) 5.16 (4.68–6.87) 4.99 (3.96–6.62) 4.82 (4.01–6.23) 4.33 (3.88–6.02) 3.83 (2.42–4.64)* 3.81 (2.22–4.27)* 3.34 (2.02–4.09)* 3.13 (1.96–4.00)* 2.24 (1.14–3.14)* 4.51 (2.83–5.97)* 3.74 (2.90–4.70)*
a b
CMC, carboxymethylcellulose; OD, once a day; BID, twice a day. ⴱ, P ⬍ 0.05 vs. result for control.
Comparisons were performed using the Mann-Whitney U test, with significance set at a P value of ⬍0.05. The effects of ABC were compared with those of FLC for treatment of experimental vaginitis induced by an FLC-resistant isolate of C. albicans. The infection was evaluated 6 days after vaginal inoculation. The drug treatments were administered orally either once or twice daily for five consecutive days (days 1 to 5 postinoculation). Compared with results for vehicle-treated control mice, mice given doses of ABC at ⱖ20 mg/kg once daily greatly reduced vaginal loads of C. albicans (Table 1) (P ⬍ 0.01 to 0.0001). In addition, twice-daily administration of ABC at ⱖ10 mg/kg resulted in significant reductions of the vaginal load of C. albicans compared to results for single-dose administration. Oral administration with FLC at 20 mg/kg given once or twice a day significantly reduced the vaginal load of C. albicans (P ⬍ 0.008 to 0.001). A significant difference was found between the group that received ABC at 20 mg/kg once daily versus mice which received FLC at 20 mg/kg once daily (P ⫽ 0.02). However, there was no statistically significant difference in the fungal burden with the dose of 20 mg/kg twice daily of ABC or FLC (P ⫽ 0.07). Sterilization of the vaginal cavity was not observed with either the ABC or FLC regimen. ABC may therefore have a place in the treatment of vulvovaginal candidosis based on data from this in vivo study, which indicate that ABC has relevant activity and an overall greater
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therapeutic effect against azole-resistant Candida spp. To our knowledge, this is the first report evaluating the efficacy of ABC against C. albicans in a murine model of vaginal infection. Further studies are needed to confirm the clinical relevance of these data. We declare that we have no conflict of interest. We thank Timothy Raczniak for his helpful suggestions on the manuscript. REFERENCES 1. Calderon, L., R. Williams, M. Martinez, K. V. Clemons, and D. A. Stevens. 2003. Genetic susceptibility to vaginal candidiasis. Med. Mycol. 41:143–147. 2. Capilla, J., M. Ortoneda, F. J. Pastor, and J. Guarro. 2001. In vitro antifungal activities of the new triazole UR-9825 against clinically important filamentous fungi. Antimicrob. Agents Chemother. 45:2635–2637. 3. Ferna ´ndez-Torres, B., A. J. Carrillo, E. Martín, A. del Palacio, M. K. Moore, A. Valverde, M. Serrano, and J. Guarro. 2001. In vitro activities of 10 antifungal drugs against 508 dermatophyte strains. Antimicrob. Agents Chemother. 45:2524–2528. 4. Garau, M., M. Pereiro, and A. del Palacio. 2003. In vitro susceptibilities of Malassezia species to a new triazole, albaconazole (UR-9825), and other antifungal compounds. Antimicrob. Agents Chemother. 47:2342–2344. 5. Gonza ´lez, G. M., E. Robledo, D. Saldívar, G. Gonza ´lez, F. Bosques, and E. Garza. 2007. Therapeutic efficacy of posaconazole against isolates of Candida albicans with different susceptibilities to fluconazole in a vaginal model. Med. Mycol. 45:221–224. 6. Miller, J. L., W. A. Schell, E. A. Wills, D. L. Toffaletti, M. Boyce, D. K. Bejamin, J. Bartroli, and J. R. Perfect. 2004. In vitro and in vivo efficacies of the new triazole albaconazole against Cryptococcus neoformans. Antimicrob. Agents Chemother. 48:384–387. 7. National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard M27-A2, 2nd ed. National Committee for Clinical Laboratory Standards, Wayne, PA. 8. Nurbhai, M., J. Grimshaw, M. Watson, C. Bond, J. Mollison, and A. Ludbrook. 2007. Oral versus intravaginal imidazole and triazole anti-fungal agents for the treatment of uncomplicated vulvovaginal candidiasis (thrush). Cochrane Database Syst. Rev. 4:CD002845. 9. Ramos, G., M. Cuenca-Estrella, A. Monzo ´n, and J. L. Rodríguez-Tudela. 1999. In vitro comparative activity of UR-9825, itraconazole and fluconazole against clinical isolates of Candida spp. J. Antimicrob. Chemother. 44:283– 286. 10. Ryley, J. F., and S. McGregor. 1986. Quantification of vaginal Candida albicans infections in rodents. J. Med. Vet. Mycol. 24:455–460. 11. Slavin, M. B., G. I. Benrubi, R. Parker, C. R. Griffin, and M. J. Magee. 1992. Single dose oral fluconazole vs intravaginal terconazole in treatment of Candida vaginitis. Comparison and pilot study. J. Fla. Med. Assoc. 79:693– 696. 12. Sobel, J. D. 1993. Genital candidiasis, p. 225–245. In G. P. Bodey (ed.), Candidiasis: pathogenesis, diagnosis and treatment. Raven Press Ltd., New York, NY. 13. Sobel, J. D. 2007. Vulvovaginal candidosis. Lancet 369:1961–1971. 14. Tooley, P. J. 1990. Treatment of vaginal candidosis—a UK patient survey. 1989. Br. J. Clin. Pract. 71(Suppl.):S73–S76. 15. Urbina, J. A., R. Lira, G. Visbal, and J. Bartroli. 2000. In vitro antiproliferative effects and mechanism of action of the new triazole derivative UR9825 against the protozoan parasite Trypanosoma (Schizotrypanum) cruzi. Antimicrob. Agents Chemother. 44:2498–2502.
