In Vitro and In Vivo Assessment of Dermatophyte Acquired Resistance to Efinaconazole, a Novel Triazole Antifungal

Abstract

Efinaconazole is a novel triazole antifungal drug for the topical treatment of onychomycosis, a nail infection caused mainly by dermatophytes. We assessed the potential of efinaconazole to induce resistance in dermatophytes by continuous exposure of Trichophyton rubrum strains to efinaconazole in vitro (12 passages) and in a guinea pig onychomycosis model (8 weeks). There was no evidence of efinaconazole resistance development in the tested strains under the experimental conditions used.

TEXT

The development of antifungal resistance has become a major concern in the treatment of deep-seated mycoses, such as aspergillosis and candidiasis, as many treatment failures have been associated with reduced drug susceptibility of the causative pathogens (1, 2). In contrast, resistance has rarely been reported in dermatophyte infections (3), despite the extensive use of antifungals. The reason is unknown but may be due to the very high drug concentrations achieved at the infection site following topical administration, effectively killing the pathogens. The slow growth of dermatophytes may also restrict alterations in the molecular mechanisms responsible for drug resistance, such as target enzyme mutation or efflux pump overexpression, which have been reviewed in Candida and Aspergillus species (4).

In onychomycosis, a fungal nail infection caused mainly by dermatophytes, the nail barrier hampers topical drug delivery to the site of infection in the nail bed. With the possibility of subinhibitory drug concentrations reaching the infection site and the need for chronic treatment (5, 6), it is important to assess the risk of emerging drug resistance.

Efinaconazole, also known as KP-103, is a novel triazole antifungal drug used for topical treatment of onychomycosis. Efinaconazole showed potent in vitro antifungal activity against various pathogenic fungi, including dermatophytes, molds, and yeasts (7). In two phase III studies, 912 patients with mild to moderate distal and lateral subungual onychomycosis were treated with an efinaconazole 10% solution for 48 weeks. At the end of treatment and at follow-up visits, 13 isolates were recovered, and all were susceptible to efinaconazole (7, 8). However, considering the limited number of isolates and the possibility of reinfection with a different strain during the clinical trials, the potential of efinaconazole to induce resistance was further investigated under controlled laboratory conditions.

(This work was presented in part at the 53rd Interscience Conference on Antimicrobial Agents and Chemotherapy, Denver, CO, 10 to 13 September 2013, abstract M-248 [9].)

We evaluated the effect of continuous efinaconazole exposure on dermatophyte susceptibility under two experimental conditions: (i) in vitro, in which fungi were serially subcultured for 12 passages in the presence of subinhibitory concentrations of efinaconazole, and (ii) in a guinea pig model of onychomycosis, with topical efinaconazole treatment for 8 weeks. To our knowledge, this is the first report of an in vivo evaluation of antifungal resistance development using an animal model of dermatophyte infection.

We assessed the potential of efinaconazole to induce drug resistance in Trichophyton rubrum, the major causative pathogen of onychomycosis, performed according to a previously used approach involving serial passaging with a drug (10, 11). T. rubrum strains were obtained from the Medical Mycology Research Center, Chiba University (Chiba, Japan), and 6 strains were randomly selected for the study. The strains were all clinical isolates from Japanese patients with tinea pedis and were susceptible to all antifungals tested in this study. The strains were subcultured for 12 passages (2 weeks/passage) on potato dextrose agar plates containing serial dilutions of efinaconazole (synthesized at Kaken Pharmaceutical Co., Ltd.) or itraconazole (Sigma-Aldrich). At the end of each passage for each strain, the plate with the highest drug concentration in which spore formation was observed macroscopically was employed to prepare a conidial suspension for subculturing and susceptibility testing. The drug concentrations at which spores were collected were used in the subsequent passage and ranged from 0.0078 to 0.0315 μg/ml for efinaconazole and 0.0395 to 0.3582 μg/ml for itraconazole (geometric mean of n = 6 strains at each passage). MICs were determined according to the CLSI M38-A2 guideline (12), except for the use of Sabouraud dextrose broth due to the poor growth of strains in RPMI 1640 medium, which prevented reliable MIC readings. The MIC was determined visually as the lowest concentration of drug that inhibited growth by approximately 80% or more. After 12 passages, the efinaconazole MICs for all T. rubrum strains were comparable to prestudy MICs (Table 1; see Fig. S1 in the supplemental material for the MICs at each passage). Itraconazole did not induce drug resistance in these strains (Table 1), consistent with the rare reports in the literature of itraconazole-resistant dermatophytes. Using a similar methodology, it was found that efinaconazole did not increase the MIC in one Trichophyton mentagrophytes strain (data not shown).

TABLE 1.

MICs obtained for efinaconazole and commercially available antifungal drugs against Trichophyton rubrum strains prior to and after 12 passages in the presence of subinhibitory concentrations of either efinaconazole or itraconazole

Test substance	MIC range (μg/ml) for indicated antifungal (n = 6 strains):
	Pretreatment	After 12 passages with efinaconazole	After 12 passages with itraconazole
Efinaconazole	0.0020–0.016	0.0039–0.031	0.0039–0.031
Itraconazole	0.00050–0.0078	0.0010–0.016	0.0010–0.016
Terbinafine	0.0020	0.0020–0.0039	0.0020–0.0039
Amorolfine	0.0078–0.031	0.016–0.125	0.0078–0.031
Ciclopirox	1.0–2.0	0.5–2.0	1.0–2.0

Antifungal drug exposure is believed to induce a stress response in fungal cells, leading to decreased drug susceptibility via a variety of mechanisms, such as the induction of drug efflux pumps (13). To assess the effects of efinaconazole exposure on the susceptibility of T. rubrum to commercially available antifungal drugs, the MICs of itraconazole, terbinafine, amorolfine, and ciclopirox were also determined. There was no meaningful difference in the MIC ranges for all drugs tested relative to the pretreatment values (Table 1); similarly, no significant MIC range change was observed in itraconazole-treated strains. Thus, continuous exposure to efinaconazole or itraconazole did not affect the susceptibility of T. rubrum to other antifungal drugs.

Unlike in vitro culture conditions in rich medium, dermatophytes infecting tissues in vivo grow more slowly, often producing arthroconidia, which are associated with decreased susceptibility to antifungals (14). Therefore, an in vivo study in infected animal nails is expected to provide more clinically relevant data. Onychomycosis was produced in guinea pigs by inoculating T. mentagrophytes strain SM-110 to the interdigital and plantar skin and nails of both hind paws (procedures approved by the Institutional Animal Care and Use Committee of Kaken Pharmaceutical Co., Ltd.) (15). Four weeks after inoculation, the infected nails were treated topically with 30 μl/foot/day efinaconazole (5% [wt/vol]) or vehicle (ethanol-to-propylene glycol ratio, 4:1 [vol/vol]) for 8 weeks (n = 4 animals/group). One week after the final treatment, the nails were clipped and homogenized, and the number of CFU per foot (CFU/3 nails/foot) was determined. In the efinaconazole-treated group, the nail viable cell counts were significantly reduced (Fig. 1), suggesting that fungal cells were exposed to severe drug pressure during treatment. The MICs of efinaconazole and commercially available antifungal drugs were subsequently determined in randomly selected colonies (n = 12 colonies/group) and in the T. mentagrophytes SM-110 stock strain using the CLSI broth microdilution method (12). The MIC₉₀s of efinaconazole and other antifungal drugs against isolates from the efinaconazole-treated group were comparable to that of the vehicle-treated group and to the MIC of the stock strain (Table 2). These results were consistent with the absence of efinaconazole-resistant isolates in the phase III clinical trials (7).

FIG 1.

Therapeutic efficacy of efinaconazole in a guinea pig model of onychomycosis.

TABLE 2.

MICs obtained for efinaconazole and commercially available antifungal drugs against Trichophyton mentagrophytes isolates from guinea pigs treated with efinaconazole or vehicle for 8 weeks

T. mentagrophytes strain/group (no. of isolates)	MIC or MIC90 (range) (μg/ml) for:
	Efinaconazole	Terbinafine	Itraconazole	Amorolfine	Ciclopirox
SM-110, stock strain	0.0078	0.016	0.13	0.5	0.25
Vehicle-treated group (12)	0.016 (0.0078–0.016)	0.031 (0.0078–0.031)	0.13 (0.063–0.13)	0.5 (0.25–0.5)	0.25 (0.25–0.5)
Efinaconazole-treated group (12)	0.0078 (0.0039–0.016)	0.016 (0.0078–0.016)	0.13 (0.063–0.13)	0.5 (0.25–0.5)	0.25 (0.25–0.25)

In conclusion, no evidence of efinaconazole resistance was observed in the tested strains under the experimental conditions presented here, suggesting that efinaconazole has low potential to induce drug resistance in dermatophytes.