In Vitro Activities of Miltefosine and Two Novel Antifungal Biscationic Salts against a Panel of 77 Dermatophytes

Abstract

The susceptibilities of 77 dermatophytes to miltefosine (MI), 1,12-bis(4-pentylpyridinium)dodecane (PYR), 1,12-bis(tributylammonium)dodecane (AM), itraconazole (ITC), terbinafine (TRB), and butenafine (BTF) were compared. Geometric mean MICs of TRB, BTF, ITC, MI, PYR, and AM were 0.039, 0.059, 1.718, 0.671, 6.006, and 4.771 μg/ml, respectively. MI was more active than ITC (P < 0.001).

Dermatophytoses are an important public health problem (5, 9). Despite therapy, relapse is frequent and disease persists in up to 25% of cases (5, 26). Furthermore, resistance to and clinical failures with itraconazole (ITC) and terbinafine (TRB), the two oral agents commonly used to treat chronic and severe disease, have been reported previously (11, 16).

Our approach to the evaluation of new antidermatophyte compounds arose from the finding that extracellular phospholipase B produced by the fungus Cryptococcus neoformans is important for fungal survival and disease dissemination (2, 21, 23, 27). Moreover, we have demonstrated previously that novel inhibitors of cryptococcal phospholipase B exhibit broad-spectrum antifungal activity (8, 19, 31). One of these, the alkylphosphocholine miltefosine (MI), is of particular interest because it is already licensed (Impavido; Aeterna Zentaris, Frankfurt, Germany) for the treatment of visceral leishmaniasis in many countries (28, 29). Since dermatophytes also produce phospholipase(s) (3, 15), we compared the in vitro activities of MI and two biscationic salts (Fig. 1) against common dermatophytes with those of established antifungal agents by using the Clinical and Laboratory Standards Institute (CLSI) M38-A method (17) with recently determined optimal test conditions (7, 24, 25). MICs of test agents for Trichophyton rubrum as measured by using unfiltered (hyphae and microconidia) and filtered (microconidia) inocula were also compared.

FIG. 1.

Structures of three investigational antifungal compounds.

Seventy-seven isolates (nine species) were studied. These included 74 clinical strains (19 T. rubrum, 17 Trichophyton mentagrophytes, 15 Trichophyton tonsurans, 3 Trichophyton soudanense, 3 Trichophyton violaceum, 5 Epidermophyton floccosum, 5 Microsporum canis, 5 Microsporum gypseum, and 2 Microsporum cookei isolates) and 3 American Type Culture Collection (Rockville, MD) strains (T. rubrum ATCC 28188, T. mentagrophytes ATCC 28185, and T. tonsurans ATCC 28942). Isolates were stored as suspensions in sterile water at 25°C until required. Prior to testing, each isolate was subcultured onto potato dextrose agar to ensure purity and growth. Quality control strains Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were included with each test run as described previously (18).

MI was obtained from Cayman Chemical Company (Ann Arbor, MI). Details of the synthesis of 1,12-bis(tributylammonium)dodecane dichloride (AM) have been published previously (19), and those of the synthesis of 1,12-bis(4-pentylpyridinium)dodecane dichloride (PYR) are presently unpublished (details are available upon request). All three compounds were prepared as 128-μg/ml stock solutions in RPMI 1640 medium (Sigma-Aldrich, St Louis, MO), providing a final concentration range of 64 to 0.125 μg/ml (17). ITC (Janssen Research Foundation, Beerse, Belgium) was dissolved in 100% dimethyl sulfoxide, and TRB (Novartis, North Ryde, Australia) and butenafine (BTF; Sequoia Research Products, Oxford, United Kingdom) were dissolved in 95% dimethyl sulfoxide-5% Tween 80 (vol/vol) (11). Stock solution concentrations of the three antifungal drugs (1,600 μg/ml) were diluted in accordance with CLSI M38-A methodology (17) to yield final concentration ranges of 16 to 0.031 μg/ml for ITC and 2 to 0.0039 μg/ml for TRB and BTF.

Stock inoculum suspensions were prepared from 7- to 10-day-old cultures grown on potato dextrose agar slants at 28°C. The suspensions were adjusted to an optical density ranging from 0.9 to 1.1 McFarland (70 to 72% transmittance) and then diluted 1:50 in RPMI 1640 medium to obtain a final inoculum concentration of 0.3 × 10⁴ to 6.4 × 10⁴ CFU/ml (17). For T. rubrum isolates, a suspension consisting only of microconidia (final cell number, 2.5 × 10⁴ to 5 × 10⁴ CFU/ml as verified with a hemacytometer) was additionally prepared (25). Broth microdilution tests were prepared in accordance with the CLSI M38-A protocol (17). Plates were incubated at 28°C and read visually after 4 and 7 days of incubation. Each isolate was tested in duplicate in two independent experiments. For ITC, the MIC was defined as the lowest concentration showing 80% inhibition. For the other agents, the MIC was the lowest concentration showing 100% growth inhibition (concentrations producing 80 and 100% growth inhibition were identical). Geometric mean (GM) MICs for the different genera were compared by using the Kruskal-Wallis test or the Mann-Whitney U test with SPPS version 14.0 software (SPPS Inc., Chicago, IL). P values of <0.05 were statistically significant.

MICs were determined at 7 days since the growth of some Trichophyton strains was insufficient at 4 days; all isolates produced good growth after 6 to 7 days. ITC MICs for quality control Candida strains were within previously published limits (18). Table 1 summarizes the susceptibility data for the 77 isolates. MI demonstrated good antidermatophyte activity, with a significantly lower GM MIC than ITC (0.671 versus 1.718 μg/ml; P < 0.001); MICs of MI at which 50% and 90% of the isolates were inhibited were fourfold lower than those of ITC (Table 1)_. Also notable was the narrow range of MI MICs (0.25 to 2.0 μg/ml). ITC MICs for 15 strains (19.5%) were ≥4 μg/ml. PYR and AM were 100- to 200-fold less potent than the most active agents, TRB and BTF.

TABLE 1.

In vitro activity of three investigational compounds in comparison with three established antifungal agents against nine dermatophyte species

Species (no. of isolates tested)	Compound	MIC (μg/ml)
		Range	GM	50%a	90%b
T. rubrum (20)	ITC	0.25-4	1.955	2	4
	TRB	0.008-0.063	0.035	0.031	0.063
	BTF	0.008-0.125	0.050	0.063	0.125
	MI	0.25-2	0.547	0.5	1
	PYR	4-16	10.41	8	16
	AM	2-8	5.876	4	8
T. mentagrophytes (18)	ITC	0.125-4	1.682	2	4
	TRB	0.008-0.5	0.030	0.031	0.063
	BTF	0.008-1	0.063	0.063	0.125
	MI	0.25-2	1.133	1	2
	PYR	2-32	9.007	8	16
	AM	2-8	4.609	4	8
T. tonsurans (16)	ITC	0.25-4	1.645	2	4
	TRB	0.008-0.063	0.030	0.031	0.063
	BTF	0.008-0.063	0.040	0.031	0.063
	MI	0.25-1	0.398	0.25	1
	PYR	1-8	2.802	2	8
	AM	2-8	3.366	4	4
T. soudanense (3)	ITC	1-4	3.420
	TRB	0.031-0.063	0.039
	BTF	0.031-0.125	0.059
	MI	0.25-1	0.52
	PYR	8-16	14.54
	AM	4-8	6.604
T. violaceum (3)	ITC	2-8	3.302
	TRB	0.063	0.063
	BTF	0.063	0.063
	MI	0.25-0.5	0.328
	PYR	1-32	8.963
	AM	2-4	2.885
All Trichophyton spp. (60)	ITC	0.125-8	1.993	2	4
	TRB	0.008-0.5	0.044	0.031	0.063
	BTF	0.008-1	0.074	0.063	0.125
	MI	0.25-2	0.652	0.5	1
	PYR	1-32	6.981	8	16
	AM	2-8	4.467	4	8
E. floccosum (5)	ITC	0.063-0.5	0.226
	TRB	0.031-0.125	0.059
	BTF	0.063-0.125	0.081
	MI	0.25-1	0.548
	PYR	2-16	7.635
	AM	4-8	6.0
M. canis (5)	ITC	0.25-8	1.230
	TRB	0.031-0.125	0.083
	BTF	0.063-0.25	0.101
	MI	0.25-2	0.555
	PYR	1-4	2.954
	AM	4	4
M. gypseum (5)	ITC	1-8	3.680
	TRB	0.031-0.125	0.065
	BTF	0.063-0.25	0.084
	MI	0.5-1	0.842
	PYR	1-4	2.408
	AM	4-8	6
M. cookei (2)	ITC	4-8	6
	TRB	0.063-0.125	0.077
	BTF	0.125-0.25	0.153
	MI	0.5-1	0.866
	PYR	0.5-2	1.061
	AM	4-16	8.485
All Microsporum spp. (12)	ITC	0.25-8	2.633	4	8
	TRB	0.031-0.125	0.075	0.063	0.125
	BTF	0.063-0.25	0.106	0.125	0.125
	MI	0.25-2	0.750	1	1
	PYR	0.5-4	2.244	2	4
	AM	4-16	5.246	4	8
All isolates (77)	ITC	0.063-8	1.718	2	4
	TRB	0.008-0.5	0.039	0.031	0.063
	BTF	0.008-1	0.059	0.063	0.125
	MI	0.25-2	0.617	0.5	1
	PYR	0.5-32	6.006	8	16
	AM	2-16	4.771	4	8

^aMIC at which 50% of the isolates were inhibited.

^bMIC at which 90% of the isolates were inhibited.

GM MICs for species of different genera varied significantly except for those of MI and AM (P < 0.001; data not shown). This variation was most evident with ITC, which was most active against E. floccosum (GM MIC for E. floccosum, 0.226 μg/ml, versus 1.993 μg/ml for Trichophyton spp. [P = 0.002] and 2.633 μg/ml for Microsporum spp. [P = 0.002]) (Table 1). Unlike those of ITC, MI MICs demonstrated little inter- or intraspecies variation (Table 1). Drug MICs for T. rubrum were within one dilution when this organism was tested by using filtered and unfiltered inocula; inoculum sizes were similar (data not shown).

Although TRB and BTF remain the most active drugs (this study; 7, 13, 24), cost is often a factor in dictating prescription choice. Furthermore, BTF is available only as a topical formulation and is unsuitable for use in treating onychomycosis.

This study provides the first indication that MI has good activity against clinically important dermatophytes. Indeed, its MIC range (Table 1) was comparable to those of the azoles (7, 13). Not only was the overall GM MIC of MI significantly lower than that of ITC (P > 0.001), but the MICs of MI at which 90% of the Trichophyton and Microsporum spp. strains were inhibited were also lower. Given that mean ITC concentrations in plasma range from 0.8 to 1.5 μg/ml (4), it is possible that the relatively high ITC MICs (≥4 μg/ml) noted for nearly 20% of the isolates may be associated with clinical failures and/or relapses. Conversely, serum MI concentrations achieved in rat models of infection (44.8 μg/ml) (14, 30) and patients with leishmaniasis (median, >20 μg/ml) (1) significantly exceed the observed MIC of MI at which 90% of the isolates tested were inhibited (1 μg/ml). We also noted, in contrast to findings of previous studies (7, 13), that ITC MICs for species of different genera varied. The reasons for these differences are unknown. Further study is required to determine if these differences are sufficient to caution against prescribing ITC for the treatment of infections with certain Microsporum spp. (Table 1). AM and PYR showed less promise as antidermatophyte agents, having higher MICs than MI. However, as MICs of both compounds for Candida, Cryptococcus, and other molds range from 0.6 to 13.4 μg/ml (our unpublished results), we are continuing to evaluate these simple salts as potential topical antifungal agents.

The ideal incubation temperature and time for susceptibility testing of dermatophytes remain incompletely resolved. Although others have proposed higher incubation temperatures (35 or 37°C) (10, 20, 22), we conducted susceptibility testing at 28°C. Adequate growth with clear MIC end points was observed in all instances. Regarding incubation times, 4 days was insufficient for adequate growth for some Trichophyton strains, in contrast to previous reports (6, 10, 12); 7 days was necessary to obtain reproducible MICs. In one study, the separation of hyphae from conidial structures influenced MICs (25). Our results do not support this observation for T. rubrum.

In conclusion, MI is as active as ITC against common dermatophytes and has a broader spectrum. The results of our study suggest that MI and related alkylphosphocholines could be exploited for the development of novel antifungal drugs.