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Antimicrob Agents Chemother. 2016 November; 60(11): 6916–6919.
Published online 2016 October 21. Prepublished online 2016 August 29. doi:  10.1128/AAC.01193-16
PMCID: PMC5075107

Potent Activities of Novel Imidazoles Lanoconazole and Luliconazole against a Collection of Azole-Resistant and -Susceptible Aspergillus fumigatus Strains

Abstract

A collection of azole-susceptible (n = 141) and azole-resistant (n = 27) Aspergillus fumigatus isolates was tested against seven antifungal drugs, including the new imidazoles lanoconazole and luliconazole. The luliconazole and lanoconazole MIC90 values for the azole-susceptible strains were 0.001 μg/ml and 0.008 μg/ml, and those for the azole-resistant strains were 0.016 μg/ml and 0.032 μg/ml.

TEXT

Invasive aspergillosis caused by Aspergillus fumigatus is a difficult-to-diagnose, life-threatening opportunistic fungal infection associated with significant morbidity and mortality (1). Survival rates improved significantly after the introduction of triazole antifungal agents such as voriconazole (2,4). However, triazole-resistant Aspergillus fumigatus strains are emerging worldwide due to long-term triazole therapy or, more commonly, are selected in the environment through exposure to azole fungicides (4,7). Recently, a new antifungal agent, isavuconazole, was shown to be noninferior to voriconazole for treatment of infections caused by Aspergillus species (8). However, cross-resistance exists for azole-resistant Aspergillus. Luliconazole, a topically related compound of lanoconazole, has been approved by the FDA for topical treatment of tinea cruris, tinea corporis, and tinea pedis. Luliconazole had neither clastogenic or mutagenic effects in genotoxicity tests, and no effect on fertility or reproductive function was noted. Luliconazole affects ergosterol biosynthesis by inhibiting the azole target protein lanosterol 14α-demethylase (cyp51A), which is the key enzyme that catalyzes the oxidative removal of the 14α-methyl group of lanosterol to give 14-15-desaturated intermediates in ergosterol biosynthesis (9). Recently, in vitro antifungal susceptibility testing of lanoconazole and luliconazole demonstrated potent efficacy against Trichophyton rubrum and Epidermophyton floccosum (9,14). In addition, animal studies and small human series suggested that luliconazole and lanoconazole are effective in treating dermatophytosis and onychomycosis (15, 16). Only limited data on the in vitro activity of lanoconazole and luliconazole against Aspergillus species are available. Therefore, the aim of the present study was to investigate the in vitro activity of these two new imidazoles and five comparators against a large collection of azole-susceptible and -resistant A. fumigatus strains with various point mutations from clinical and environmental sources. A total of 168 well-characterized A. fumigatus strains from the culture collection of the Invasive Fungi Research Center (IFRC) were included. Azole-susceptible (n = 141) and -resistant (n = 27) strains originated from nail, sputum, bronchoalveolar lavage, sinus discharge, and skin biopsy samples. Environmental samples came from soil and air samples. Most of the azole-resistant A. fumigatus strains (n = 10) harbored a leucine-to-histidine substitution at codon 98, along with a 34-bp tandem repeat in the cyp51A promoter region, but TR46/Y121F/T289 (n = 2) and other point mutations (n = 8) such as G54, M220, G138C, and G432C were also included. Resistant isolates without mutations in cyp51A were also included (n = 7). MICs were determined based on CLSI M38-A2 (17). Concentration ranges of 0.001 to 1 μg/ml for luliconazole (Nihon Nohyaku Co, Osaka, Japan) and lanoconazole (Nihon Nohyaku Co.), 0.016 to 16 μg/ml for itraconazole (Janssen, Beerse, Belgium), voriconazole (Pfizer, Sandwich, United Kingdom), and amphotericin B (Bristol-Myers-Squib, Woerden, The Netherlands), and 0.008 to 8 μg/ml for posaconazole (Merck Sharp & Dohme BV, Haarlem, The Netherlands) and caspofungin (Merck Sharp & Dohme BV) were used. Stock solutions were prepared in dimethyl sulfoxide. Conidial suspensions were prepared by scraping the surface of fungal colonies with a sterile cotton swab moistened with physiological saline solution containing 0.05% Tween 40 and were adjusted to optical densities ranging from 0.09 to 0.11 (0.5 × 106 to 3.1 × 106 CFU/ml) measured at 530 nm. Inoculum suspensions, including mostly nongerminated conidia, were diluted 1:50 in RPMI 1640 medium, and the final inoculum in assay wells was between 0.4 × 104 and 5 ×104 CFU/ml. Microdilution trays were incubated at 35°C for 48 h. MICs were determined visually as the lowest concentration which provided complete inhibition of growth, while minimum effective concentrations (MECs [caspofungin only]) were determined microscopically as the lowest concentration of drug promoting the growth of small, round, compact hyphae relative to the appearance of the filamentous forms seen in the control wells. Candida krusei (ATCC 6258) and Paecilomyces variotii (ATCC 3630) were included as quality controls (17). All tests were performed in duplicate, and differences of the mean values were determined by using Student's t test with the statistical SPSS package (version 7.0). P values of <0.05 were considered statistically significant. Table 1 summarizes the in vitro susceptibility of 168 susceptible and resistant A. fumigatus isolates. The novel imidazoles luliconazole and lanoconazole demonstrated potent activity against all A. fumigatus isolates, in comparison to voriconazole, itraconazole, and posaconazole. MICs of lanoconazole and luliconazole against all A. fumigatus isolates ranged from <0.001 to 0.5 μg/ml and from <0.001 to 0.016 μg/ml, respectively, compared to 0.064 to >16 μg/ml for itraconazole, 0.064 to >16 μg/ml for voriconazole, and 0.008 to 8 μg/ml for posaconazole. The lanoconazole and luliconazole geometric mean (GM) MICs against all isolates were 0.0024 μg/ml and 0.0012 μg/ml, respectively, while those of the other agents were as follows: itraconazole, 0.4243 μg/ml; voriconazole, 0.2555 μg/ml; posaconazole, 0.0968 μg/ml; caspofungin, 0.0535 μg/ml. Basically, the GM MIC value of luliconazole against all A. fumigatus isolates was 2 log2 dilutions lower than that of lanoconazole. However, no statistically significant (P > 0.05) differences in the lanoconazole and luliconazole susceptibility patterns were detected between strains. MICs of luliconazole and lanoconazole for the resistant isolates with various point mutations in the cyp51A gene were approximately similar to those of the susceptible isolates, but strains with TR46/Y121F/T289 mutations showed less susceptibility, with a 4-log2-dilution step compared to the other resistant strains harboring TR34/L98H, G54, M220, G138C, and G432C (Table 2).

TABLE 1
In vitro susceptibility of 168 Aspergillus fumigatus isolates to seven antifungal agentsa
TABLE 2
In vitro activity of seven antifungal drugs against triazole-resistant Aspergillus fumigatus isolates with different point mutationsa

In previous studies, we demonstrated that the prevalence of azole-resistant A. fumigatus with predominant TR34/L98H mutations in the cyp51A gene in Iran has increased remarkably from 3.3% to 6.6% (6, 7). Treatment regimens of Aspergillus infections with triazole agents are associated with a poor outcome when azole resistance is involved (18). In this study, molecularly identified strains of A. fumigatus strains from both clinical and environmental sources were subjected to antifungal susceptibility testing with newer imidazoles.

Recently, several studies have shown high in vitro and in vivo efficacy of luliconazole against a limited number of dermatophytes and other agents causative of onychomycosis (11,14, 19, 20). In addition, in the present study, lanoconazole and luliconazole showed potent activity against the wild-type strain as well as against azole-resistant mutants of A. fumigatus, but high MIC values for lanoconazole against two isolates with TR46/Y121F/T289 were observed. While the most common TR34/L98H mutation confers resistance to all azoles, TR46/Y121F/T289A confers resistance to voriconazole and isavuconazole but shows a variable influence on the MICs of itraconazole and posaconazole. The point mutations G54 and M220 in cyp51A induce resistance mainly to itraconazole and posaconazole (21). Only limited data are available on the effect and related toxicity of systemic use of luliconazole and lanoconazole. Oral luliconazole therapy in a murine model of invasive aspergillosis was superior to that of itraconazole, and intravenous luliconazole appeared to be highly effective in comparison with intravenous amphotericin B (22). In this study, 90% of the animals stayed alive when 2.5 mg/kg of body weight/day of luliconazole was used whereas only 30% of animals survived when amphotericin B (5 mg/kg/day) was used (22).

The current study demonstrated that the in vitro antifungal activities of luliconazole and lanoconazole against susceptible and resistant A. fumigatus isolates are apparently superior to those of polyenes, other azoles, and echinocandins. Clinical effectiveness in the treatment of Aspergillus infection and pharmacodynamic assessment and development of epidemiologic cutoff values (ECVs)/breakpoints remain to be established. In conclusion, we suggest that these two new imidazoles are promising candidates for treatment of invasive aspergillosis caused by either azole-susceptible or -resistant isolates.

ACKNOWLEDGMENTS

This study was supported financially by a grant from the School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran (no. 1814), which we gratefully acknowledge.

We are grateful to Iman Haghani for excellent technical assistance and help with antifungal susceptibility testing.

J.F.M. received grants from Astellas, Basilea, and Merck. He has been a consultant to Astellas and Merck and has received speaker's fees from Gilead Sciences, Merck, Pfizer, and United Medical. The rest of us have no conflicts of interest to declare.

REFERENCES

1. Lestrade PP, Meis JF, Arends JP, van der Beek MT, de Brauwer E, van Dijk K, de Greeff SC, Haas PJ, Hodiamont CJ, Kuijper EJ, Leenstra T, Muller AE, Oude Lashof AM, Rijnders BJ, Roelofsen E, Rozemeijer W, Tersmette M, Terveer EM, Verduin CM, Wolfhagen MJ, Melchers WJ, Verweij PE 2016. Diagnosis and management of aspergillosis in the Netherlands: a national survey. Mycoses 59:101–107. doi:.10.1111/myc.12440 [PubMed] [Cross Ref]
2. Patterson TF, Thompson GR III, Denning DW, Fishman JA, Hadley S, Herbrecht R, Kontoyiannis DP, Marr KA, Morrison VA, Nguyen MH, Segal BH, Steinbach WJ, Stevens DA, Walsh TJ, Wingard JR, Young JA, Bennett JE 2016. Practice guidelines for the diagnosis and management of aspergillosis: 2016 update by the Infectious Diseases Society of America. Clin Infect Dis 63:e1–e60. doi:.10.1093/cid/ciw326 [PMC free article] [PubMed] [Cross Ref]
3. Heinz WJ, Egerer G, Lellek H, Boehme A, Greiner J 2013. Posaconazole after previous antifungal therapy with voriconazole for therapy of invasive aspergillus disease, a retrospective analysis. Mycoses 56:304–310. doi:.10.1111/myc.12023 [PubMed] [Cross Ref]
4. Verweij PE, Chowdhary A, Melchers WJ, Meis JF 2016. Azole resistance in Aspergillus fumigatus: can we retain the clinical use of mold-active antifungal azoles? Clin Infect Dis 62:362–368. doi:.10.1093/cid/civ885 [PMC free article] [PubMed] [Cross Ref]
5. Chowdhary A, Kathuria S, Xu J, Meis JF 2013. Emergence of azole-resistant Aspergillus fumigatus strains due to agricultural azole use creates an increasing threat to human health. PLoS Pathog 9:e1003633. doi:.10.1371/journal.ppat.1003633 [PMC free article] [PubMed] [Cross Ref]
6. Badali H, Vaezi A, Haghani I, Yazdanparast SA, Hedayati MT, Mousavi B, Ansari S, Hagen F, Meis JF, Chowdhary A 2013. Environmental study of azole-resistant Aspergillus fumigatus with TR34/L98H mutations in the cyp51A gene in Iran. Mycoses 56:659–663. doi:.10.1111/myc.12089 [PubMed] [Cross Ref]
7. Nabili M, Shokohi T, Moazeni M, Khodavaisy S, Aliyali M, Badiee P, Zarrinfar H, Hagen F, Badali H 2016. High prevalence of clinical and environmental triazole-resistant Aspergillus fumigatus in Iran: is it a challenging issue? J Med Microbiol 65:468–475. doi:.10.1099/jmm.0.000255 [PubMed] [Cross Ref]
8. Maertens JA, Raad II, Marr KA, Patterson TF, Kontoyiannis DP, Cornely OA, Bow EJ, Rahav G, Neofytos D, Aoun M, Baddley JW, Giladi M, Heinz WJ, Herbrecht R, Hope W, Karthaus M, Lee DG, Lortholary O, Morrison VA, Oren I, Selleslag D, Shoham S, Thompson GR III, Lee M, Maher RM, Schmitt-Hoffmann AH, Zeiher B, Ullmann AJ 2016. Isavuconazole versus voriconazole for primary treatment of invasive mould disease caused by Aspergillus and other filamentous fungi (SECURE): a phase 3, randomised-controlled, non-inferiority trial. Lancet 387:760–769. doi:.10.1016/S0140-6736(15)01159-9 [PubMed] [Cross Ref]
9. Khanna D, Bharti S 2014. Luliconazole for the treatment of fungal infections: an evidence-based review. Core Evid 9:113–124. [PMC free article] [PubMed]
10. Scher RK, Nakamura N, Tavakkol A 2014. Luliconazole: a review of a new antifungal agent for the topical treatment of onychomycosis. Mycoses 57:389–393. doi:.10.1111/myc.12168 [PubMed] [Cross Ref]
11. Baghi N, Shokohi T, Badali H, Makimura K, Rezaei-Matehkolaei A, Abdollahi M, Didehdar M, Haghani I, Abastabar M 2016. In vitro activity of new azoles luliconazole and lanoconazole compared with ten other antifungal drugs against clinical dermatophyte isolates. Med Mycol 54:757–763. doi:.10.1093/mmy/myw016 [PubMed] [Cross Ref]
12. Koga H, Nanjoh Y, Makimura K, Tsuboi R 2009. In vitro antifungal activities of luliconazole, a new topical imidazole. Med Mycol 47:640–647. doi:.10.1080/13693780802541518 [PubMed] [Cross Ref]
13. Koga H, Tsuji Y, Inoue K, Kanai K, Majima T, Kasai T, Uchida K, Yamaguchi H 2006. In vitro antifungal activity of luliconazole against clinical isolates from patients with dermatomycoses. J Infect Chemother 12:163–165. doi:.10.1007/s10156-006-0440-4 [PubMed] [Cross Ref]
14. Wiederhold NP, Fothergill AW, McCarthy DI, Tavakkol A 2014. Luliconazole demonstrates potent in vitro activity against dermatophytes recovered from patients with onychomycosis. Antimicrob Agents Chemother 58:3553–3555. doi:.10.1128/AAC.02706-13 [PMC free article] [PubMed] [Cross Ref]
15. Ghannoum M, Long L, Kim H, Cirino A, Miller A, Mallefet P 2010. Efficacy of terbinafine compared to lanoconazole and luliconazole in the topical treatment of dermatophytosis in a guinea pig model. Med Mycol 48:491–497. doi:.10.3109/13693780903373811 [PubMed] [Cross Ref]
16. Jarratt M, Jones T, Kempers S, Rich P, Morton K, Nakamura N, Tavakkol A 2013. Luliconazole for the treatment of interdigital tinea pedis: a double-blind, vehicle-controlled study. Cutis 91:203–210. [PubMed]
17. Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard M38-A2. Clinical and Laboratory Standards Institute, Wayne, PA.
18. van der Linden J, Snelders E, Kampinga GA, Rijnders B, Mattsson E, Debets-Ossenkopp YJ, Kuijper EJ, Van Tiel FH, Melchers WJ, Verweij PE 2011. Clinical implications of azole resistance in Aspergillus fumigatus, The Netherlands, 2007–2009. Emerg Infect Dis 17:1846–1854. doi:.10.3201/eid1710.110226 [PMC free article] [PubMed] [Cross Ref]
19. Uchida K, Nishiyama Y, Yamaguchi H 2004. In vitro antifungal activity of luliconazole (NND-502), a novel imidazole antifungal agent. J Infect Chemother 10:216–219. doi:.10.1007/s10156-004-0327-1 [PubMed] [Cross Ref]
20. Gupta AK, Daigle D 2016. A critical appraisal of once-daily topical luliconazole for the treatment of superficial fungal infections. Infect Drug Resist 9:1–6. doi:.10.2147/IDR.S61998 [PMC free article] [PubMed] [Cross Ref]
21. Meis JF, Chowdhary A, Rhodes JL, Fisher MC, Verweij PE 2016. Clinical implications of globally emerging azole resistance in Aspergillus fumigatus. Philos Trans R Soc Lond B Biol Sci doi:.10.1098/rstb.2015.0460 [Cross Ref]
22. Niwano Y, Kuzuhara N, Goto Y, Munechika Y, Kodama H, Kanai K, Yoshida M, Miyazaki T, Yamaguchi H 1999. Efficacy of NND-502, a novel imidazole antimycotic agent, in experimental models of Candida albicans and Aspergillus fumigatus infections. Int J Antimicrob Agents 12:221–228. doi:.10.1016/S0924-8579(99)00076-X [PubMed] [Cross Ref]

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