Activity of amorolfine or ciclopirox in combination with terbinafine against pathogenic fungi in onychomycosis—Results of an in vitro investigation

Onychomycoses are difficult‐to‐treat fungal infections with high relapse rates. Combining oral and topical antifungal drugs is associated with higher success rates. Additive or synergistic modes of action are expected to enhance treatment success rates.


| INTRODUC TI ON
Onychomycoses are a group of common fungal infections with increasing incidence, affecting approximately 12% of the German population. 1 The disease may cause nail destruction and deformity, pain and walking difficulties, secondary infections and social embarrassment.Fungal pathogens include dermatophytes, yeasts, and moulds.Despite significant therapeutic improvements, patients with onychomycosis still fail treatment for various reasons.Successful therapy of fungal nail infections is challenging due to the need for long-term therapies and high rates of treatment failures and relapses. 2ctors impacting the success rates of antifungal therapy, particularly if single drugs are used, include involvement of proximal nail plate and matrix, subungual dermatophytoma, higher patient age, history of previous onychomycosis, slow nail growth, inadequate nail care and poor adherence.
While in clinical practice the impression prevails that the overwhelming majority of onychomycoses is caused by dermatophytes such as Trichophyton rubrum and T. interdigitale, clinicoepidemiological studies indicate that moulds and Candida spp.are involved in 20%-30% of cases. 3,4This notion has major implications for the choice of the antifungal regimen, particularly given the widespread practice of empirical treatment without prior definitive identification of the pathogen.
Non-dermatophytes are often inadequately covered by the antifungal agents used for oral therapy in onychomycoses.For example, in the case of Scopolariopsis brevicaulis, the most common mould causing onychomycosis, terbinafine exhibits minimal inhibitory concentrations (MICs) that are considerably higher than those observed against dermatophytes. 4r these reasons, combining systemic therapy (mostly terbinafine or itraconazole) with another broadly effective topical antifungal drug applied as nail lacquer has been shown to increase mycological and clinical cure rates. 58][9][10] In this study, we investigated in vitro additive and synergistic effects of amorolfine and ciclopirox tested in combination with terbinafine against clinical isolates from onychomycosis.

| MATERIAL S AND ME THODS
Fungal isolates of the species T. rubrum, T. interdigitale and S. brevicaulis collected from infected nail specimens of onychomycosis patients were retrieved from the strain collection of the Dermatological University Clinic Tuebingen, Germany.An American Type Culture Collection (ATCC) strain of T. rubrum with known antifungal susceptibility patterns (resistant [MYA-4438]) was used as a reference control.Susceptibility testing was performed in U-shaped 96-well polystyrene plates according to the microdilution methodology M38A2 of the Clinical Laboratory Standards Institute (CLSI) with read-out of fungal growth after 5 days of incubation. 11orolfine hydrochloride, terbinafine hydrochloride (both obtained from Sigma Aldrich), and ciclopirox olamine 12,13 (obtained from Cayman Chemicals US) were dissolved in dimethyl sulfoxide (DMSO), diluted in RPMI-1640 (obtained from Sigma Aldrich) cell culture medium, buffered with 0.165 mol/L morpholinium propane sulfonate (MOPS) and containing DMSO at a final concentration of 1%, in 96-well polystyrene cell culture plates.Antifungal drug concentrations ranged from 0.0019 to 2 mg/L.
From pathogen suspensions containing standardised numbers of fungal cells, individual wells were inoculated with 1 × 10 3 through 3 × 10 3 colony forming units of precultured fungi per millilitre (cfu/ mL) and incubated at 35°C for 5 days.Growth was determined visually using an inverted reading mirror.
Based on this methodology and the measured MIC 99.9% values, sub-inhibitory and sub-fungicidal concentrations of amorolfine, ciclopirox and terbinafine were tested in combinations at increasing dilutions to probe for additive or synergistic effects.The checkerboard method described by Gupta et al. (2003)  14 was used to test all combinations of concentrations (2-fold steps) of two antifungal agents from 0.0019 to 2.0 mg/L, and buffer as negative control.All experiments were repeated in three independent sets (triplicates).

Synergistic activity was observed for amorolfine plus terbinaf-
ine in 1 of 18 experiments with Trichophyton spp., versus none for ciclopirox plus terbinafine.Additive activity was the most common effect as seen in 12 of 18 experiments for amorolfine plus terbinafine and in 13 of 18 experiments for ciclopirox plus terbinafine (Table 1, Figure 1).

TA B L E 1
Results of susceptibility testing of T. rubrum, T. interdigitale and S. brevicaulis clinical isolates (triplicate testing of three strains (#) for each fungal species).Geometric means (GMs) were calculated for each triplicate and for all isolates of each species.A combined fractional inhibition concentration (CFIC) of <1 indicates synergistic activity (SYN), and CFIC of 1 to 2 shows an additive effect of the two antifungals (ADD) and an CFIC >2 suggests antagonism (ANT). 15The sum of the FIC values were used to create the CFIC score.Trichophyton spp.for terbinafine combinations with amorolfine and ciclopirox, respectively.However, none of the isolates showed uniform antagonism in triplicate measurements in any of the combinations (Table 1, Figure 1).
We repeated this experiment and more strikingly, the terbinafineresistant strain MYA-4438 was inhibited by terbinafine in concentrations as low as 0.0625 mg/L if it was used in combination with amorolfine at 0.25 mg/L, indicating synergistic activity (Figure 3).No inhibition of a terbinafine-resistant dermatophyte strain was observed with terbinafine and ciclopirox in combination (data not shown).

| Moulds
The MIC (99%) values of the S. brevicaulis clinical isolates (n = 3) varied between 2 and 8 mg/L for terbinafine, and between 0.5 and 1 mg/L for amorolfine (Table 1A).Antagonism was observed in 1 experiment with the latter combination (Table 1B, Figure 1).

| Sublimation of antifungal drugs
In the initial experiments, we noticed that several wells of the growth control row (no antifungal drug added) showed reduced growth of T.
rubrum.The affected wells were located adjacent to those containing amorolfine at 2 mg/L and terbinafine at descending concentrations from 2.0 to 0.25 mg/L.
Growth of dermatophytes was also inhibited by adjacent singlesubstance terbinafine-containing wells for Trichophyton rubrum strains.These effects were absent after the plating pattern was changed to a layout with alternating empty rows between drugloaded and growth control wells.
A highly similar result was seen with amorolfine and terbinafine used alone, while ciclopirox did not show this effect.
This effect is plausibly explained by the sublimation of the antifungal agents leading to substance transfer via air-filled distances to adjacent wells.Results of single studies have been confirmed by a respective meta-analysis, proving additional evidence that amorolfine 5% nail lacquer in combination with systemic terbinafine (and itraconazole) leads to significant higher complete clearance rates than oral monotherapy. 17mbination of ciclopirox 8% nail lacquer with systemic terbinafine resulted in higher mycological cure but could not show significantly higher complete cure rates in randomised, prospective clinical trials. 4,18,19The in vitro results of our study provide additional evidence of additive effects and synergy between topical amorolfine and systemically administered terbinafine in vivo.
Antagonism was rarely observed and consistent antagonism was not found in any of the isolates.A terbinafine-resistant clinical T. rubrum strain was rendered susceptible to terbinafine if administered in combination with subinhibitory amorolfine.Limitations of this study include the limited number of tested clinical isolates and the non-testing Epidermophyton floccosum from our sample dermatophyte strains.
Given the increasing trend of antifungal-resistance of fungal strains involved in onychomycosis 19 -particularly against terbinafine 20-22 -combination therapy with a topically applied drug such as amorolfine is expected to add significant and clinically relevant advantages, improving the effective treatment of onychomycosis.This notion is further supported by ex vivo mass spectroscopic imaging data indicating that amorolfine penetrates effectively from the lacquer layer into the infected nail plate. 23Diffusion of ciclopirox was less pronounced in this ex vivo model.

Synergistic and additive effects of combined antifungals should
further augment the benefits of using topical antifungals that penetrate the nail plate from the surface together with systemically applied drugs that diffuse bottom-up from the nail bed and via matrix.Synergistic effects may be of particular value in patients with nail matrix involvement, severe onychomycosis, nail destruction, or impaired perfusion of nailbed capillaries, for example, in diabetic patients with microangiopathy-given the elevated prevalence of onychomycosis in this population. 24,25e sublimation effect of amorolfine and terbinafine in these experiments are consistent with the results of Polak et al. (2003). 21is phenomenon may support the penetration of these antifungals in air-filled cavities in and under the nail plate (in dermatophytoma), aiding effective fungal eradication from these spaces that presumably harbour persistent reservoirs of viable fungal cells and spores.
Generally, amorolfine appears to exhibit a synergistic effect in combination with terbinafine against S. brevicaulis and an additive effect against Trichophyton spp.whereas for ciclopirox and The fractional inhibitory concentration (FIC) was calculated via dividing the MIC of an antifungal agent used with another drug by the MIC observed for the same antifungal agent when used as a single substance.The combined fractional inhibitory concentration (CFIC) value was calculated by adding the FICs of two different antifungals used in a single experiment.Results were interpreted as follows: a CFIC value <1 indicated synergistic activity, a CFIC value in the range of 1 through 2 indicated additive activity, and a CFIC >2 indicated antagonistic activity of the two drugs used in the individual experiment.15 Synergistic activity was observed for amorolfine plus terbinafine in 5 of 9 experiments with 3 strains (in one isolate, the effect was uniformly synergistic in the triplicate tests), versus none for ciclopirox plus terbinafine.Additive activity was seen for amorolfine plus terbinafine in 4 of 9 experiments with 2 strains; and for ciclopirox plus terbinafine in 8 of 9 experiments with 3 strains.

TA B L E 1
(Continued) F I G U R E 1 Interaction of antifungal effects of terbinafine used in combination with amorolfine and ciclopirox.(A) Terbinafine and amorolfine and (B) Terbinafine and ciclopirox.Our experiments revealed that combining amorolfine or ciclopirox with terbinafine results in at least additive efficacy against a substantial fraction of isolates from fungal pathogens commonly involved in onychomycosis.These results are in accordance with data from clinical studies that demonstrated benefits of combining oral (i.e.systemic) and topical treatments with different antifungal drugs.

F I G U R E 2
MICs for terbinafine and amorolfine as single substances and in combination for a terbinafine-resistant reference strain of T. rubrum.F I G U R E 3Experimental setup and interpretation.Two different antifungal drugs were present at decreasing dilutions in the wells 1 through 12 and A through H, respectively.As expected, the ATCC strain T. rubrum MYA-4438 (terbinafine-resistant, MIC >2 mg/L) showed no sensitivity (i.e.full growth, dark blue) to single-substance terbinafine (not shown).The MIC for amorolfine alone was 0.5 mg/L.However, when both antifungal agents were combined, terbinafine at 0.625-2 mg/L completely inhibited fungal growth (clear wells) at subinhibitory concentrations of amorolfine (0.25 mg/L), as did terbinafine 1.0 and 2.0 mg/L combined with 0.125 mg/L amorolfine.The checkerboard diagram represents the experiment shown in the photograph below.