In Vitro additive effect on griseofulvin and terbinafine combinations against multidrug-resistant dermatophytes

Griseofulvin (GF) and terbinafine (TF) are commonly used drugs to treat dermatophytosis, a fungal infection of the skin. Today there is an increase in drug resistance to these antifungals which highlight the need for alternative synergistic therapies. Minimum Inhibitory Concentration (MIC) of GF and TF were determined against fungi clinical isolates from local hospitals with values ranging 0.03−2.0 μg mL-1 and 0.24−4.0 μg mL-1, respectively. A checkboard test was used to determine the combination of GF:TF which could induce an additive effect against the fungi isolates Multidrug-resistant isolates showed susceptibility after treatment with 16:2 μg mL-1 GF:TF. An MTT assay further verified that GF and TF combinations have greater additive effect against pathological and multidrug-resistant isolates than antifungals alone. Herein we disclose GF:TF combinations that could constitute as a possible new anti-dermatophyte therapy.


INTRODUCTION
Skin infections caused by dermatophytes fungus are common and widespread worldwide.The disease causing fungus result primarily from three genus Epidermophyton, Microsporum, and Trichophyton.These infections are typically limited to the stratum corneum of skin, nails, and scalp, causing tinea-like mycoses and onychomycosis characterized by the symptoms of local irritation, scaling, redness, swelling, and inflammation (Patel, Schwartz, 2011).Although rarely fatal, these fungal diseases are considered difficult to treat, since their therapy is long and frequently recur (Molina de Diego, 2011).
Fungal resistance to conventional antifungals is on the rise which further complicates the treatment (Grover, Arora, Manchanda, 2012).In addition, it is especially difficult to treat immunocompromised patients because of high toxicity associated with many antifungal agents (Vandeputte, Ferrari, Coste, 2012).Griseofulvin (GF) and terbinafine (TF) are among the most commonly used drugs for the treatment of dermatophytosis but often not effective alone (Badali et al., 2015).
The current approach to treat dermatophytosis utilizes a combination of antifungals to overcome fungal resistance, especially in chronic cases (Tamura et al., 2014).This strategy has been successful against other diseases and is particularly attractive to the pharmaceutical industry since approved drugs gain extra lifetime.
GF and TF act on different targets in the fungal cell.GF alters DNA synthesis inhibiting mitosis by interfering with microtubules function (Kathiravan et al., 2012).Whereas, TF inhibits squalene epoxidase leading to ergosterol depletion and squalene accumulation (Campoy et al., 2017;Scorzoni et al., 2017).
Co-administration of drugs of different mechanisms of action can often be synergist through inhibition of complementary targets inside fungal cells (Scorzoni et al., 2017).This strategy has been shown to achieve a wider spectrum of antifungal activity (Mukherjee et al., 2005;Campitielli et al., 2017;Scorzoni et al., 2017).
Herein, we present a study of the in vitro effects of GF and TF combinations against multidrug-resistant fungi using fungi clinical isolates, which are simultaneously or individually resistant to GF and TF.

Microorganisms
C l i n i c a l f u n g i i s o l a t e s ( t e n s t r a i n s o f T. mentagrophytes, eleven of T. rubrum, eight of M. canis, and twelve of M. gypseum) were obtained from the culture collections deposited on the Laboratory of Applied Mycological Research, Universidade Federal do Rio Grande do Sul, Brazil.PCR and direct sequencing, targeting the internal transcribed spacer (ITS) region of rDNA, were used in the identification of T. mentagrophytes isolates employed in this study.

Antifungal solutions
Stock solutions of GF (Wallace Pharmaceuticals, Mumbai, India) and TF (terbinafine hydrochloride -Cristália, São Paulo, Brazil) were prepared by dilution with dimethyl sulfoxide (DMSO; Synth, São Paulo) at 1600 µg mL -1 and stored at −20 °C.For the experiments, antifungal drugs were diluted with RPMI 1640 (Roswell Park Memorial Institute; Gibco) medium supplemented with L-glutamine, without sodium bicarbonate, and buffered at pH 7.0 with MOPS (morpholinepropansulfonic acid; Sigma-Aldrich) buffer 0.165 M.After dilution, the maximum final concentration of DMSO was 2%.

Antifungal susceptibility testing
The minimum inhibitory concentration (MIC) of each antifungal drug was determined by the broth microdilution method using RPMI 1640 medium, according to CLSI protocol M38-A2 (CLSI, 2008).GF and TF resistance was defined as MIC ≥ 3 µg mL -1 (Galuppi et al., 2010) and MIC ≥ 4 µg mL -1 (Mukherjee et al., 2003), respectively, since no breakpoints are available in the CLSI and EUCAST for these drugs.Sterility (without drugs and fungi) and cell viability controls were used respectively and performed in triplicate.

Phenotypic Study of ATP-binding cassette (ABC) efflux pumps
MICs of GF and TF were determined to the multiresistant isolates in the presence of verapamil (100 µM, RPMI) into the culture medium and compared with the MIC values without the addition of this efflux pump inhibitor (Pinto e Silva et al., 2009).Incubation conditions were the same as those used on antifungal susceptibility tests.

Susceptibility assay of the combined antifungal drugs by the checkerboard method
Two-dimensional characterization of the GF:TF interaction was performed in quadruplicate by checkerboard technique as previously described by Lewis et al. (2002).Antifungal interactions were evaluated by comparing the fractional inhibitory concentration index (FICI) expressed as the sum of the fractional inhibitory concentrations (FIC), as defined by the following equation: where MIC GF and MIC TF are the MICs of GF and TF, respectively (Mukherjee et al, 2005).Interactions were defined as synergistic (FICI ≤ 0.5), additive (0.5< FICI < 1), indifferent (1 ≤ FICI < 4), or antagonistic (FICI ≥ 4) (Lewis et al., 2002).

Statistical analysis
The statistical analysis was performed by ANOVA followed by Tukey's test (Minitab 14.0 software, USA).Data expressed as mean ± SEM (Standard Error of Mean).Differences were considered statistically significant when p<0.05.

Antifungal activity of the GF:TF combination
Seven isolates characterized as resistant to either GF or TF, and sixteen isolates susceptible to both antifungals were selected for this study.The FICI values calculated revealed an additive effect in about 70% of these selected strains (Table II).Multidrug-resistant isolates TME 16 and TME 34 showed to be more susceptible to GF:TF mixtures (Table II -bold) which was unexpected but an encouraging result.

Cell damage evaluation by the MTT test
Sixteen isolates, which presented additive effect in the qualitative checkerboard test (Table II) were then selected to have their its interaction substantiated by the quantitative MTT test The MTT test corroborates to the results obtained by the checkerboard assay because the GF and TF mixtures in all cases could inhibit greater amount of cell growth compared to both drugs alone.(Table III).The statistical analysis of cell damage verified that the combinations were significantly (p<0.05)more effective in all isolates tested.
Cell viability was evaluated in the multidrugresistant isolate TME 16 (Figure 1 -A) and the drug sensitive isolate TME 32 (Figure 1 -B) by the MTT assay.It was found that cell damage to TME 16 was decreased by 40% after treatment with GF (32 and 16 µg mL -1 ) and TF (2 µg mL -1 ).In contrast, treatment with the GF:TF mixtures (16:2 and 64:2 µg mL -1 , respectively) resulted in much greater cell damage (>90%) indicating possible hypha death due to synergy (Figure 1 -A).Similar trend in potency improvement was noted with to isolate TME 32 as cell damage could be enhanced to 80% or greater after treatment with GF:TF mixtures (Figure 1 -B).
Statistical analysis of the cell damage further validate that the GF:TF combinations were significantly more effective in all isolates tested (p<0.05).

Phenotypic study of ABC efflux pumps
Effect of ABC efflux-pumps on potency was checked by using Verapamil (Sigma), a known efflux pump inhibitor.Drug efflux is one of the main mechanisms responsible for decrease the MIC within drug resistant organism.It was found that the MICs of GF and TF were not changed with addition of verapamil suggesting that drug efflux is not responsible for drug resistance.

DISCUSSION
According to the critical points chosen from Table I, seven out forty-one isolates assayed were classified as resistant to GF (MIC values ≥ 3 µg mL -1 ) (Galuppi et al., 2010).This finding is consistent with previously reported results (Galuppi et al., 2010;Nardoni et al., 2013) as was the prevalence of GF-resistant isolates compared to TF (Andes et al., 2006).
Two T. mentagrophytes isolates (TME 16 and TME 32) showed resistance to GF and TF.This resistance to distinct antifungal drug classes is alarming, since the probability of therapeutic-treatment failure could be extremely high when these agents are given alone.The multi-drug resistance of dermatophytes to GF and TF is unusual, so further tests was performed focusing on these two multidrug-resistant isolates.
Literature has reported that one particular fungi strain (having resistance to GF, and tioconazole) which was related to the efflux pumps (Fachin, Maffei, Martinez-Rossi, 1996).Therefore, we hypothesized that TME 16 and TME 32 might be drug resistant due to the activity of efflux pumps.This was checked using verapamil to inhibit the ABC efflux pumps and MICs determined.Since the MICs were not improved upon addition of verapamil it is suggestive that the mechanism of resistance is not drug efflux.Additional studies will need to be conducted to further verify this result and uncover the mechanism of drug resistance.
Dermatophytosis is often a chronic, recurrent disease which can require long-term treatment with antifungals.Therefore, it is important to choose the most appropriate and effective drugs in the early stages of infection.Combination therapy of antifungals with different pharmacological effects has shown to have Page 4 / 9     merit for treatment of dermatophytosis (Scorzoni et al., 2017).
TF combined with amorolfine was checked in a randomized study of severe dermatophyte onychomycosis.Patients outcome were good and this combination treatment demonstrated an improvement cost per cure ratio (Baran et al., 2000).Onychomycosis caused by dermatophytes were also successfully treated by combination of oral terbinafine with ciclopyroxolamine, imidazole and other such topical antifungals.(Romano et al., 2005).Fusarium oxysporum infections respond positively to TF and topical imidazole treatment and this combination led to clinical and mycological healing, which is often refractory to antimycotics (Romano et al., 2005).Patient recovery to dermatophytosis was also achieved when GF plus cyclopyroxolamine lotion and GF with topical imidazole were chosen and used in combination therapy (Romano et al., 2005).Several other studies of combination therapy were reported, however most of these studies were lacking quantitative data regarding drug combinations in vitro (Spader et al., 2013;Semis et al., 2015).
The qualitative checkerboard assay enabled us to identify combinations of GF and TF which resulted in additive drug effects against sixteen pathogenic fungi (Table II).Subsequent evaluation by the quantitative MTT test using the same sixteen isolates identified by the checkerboard assay was performed and resulted in the determination of GF:TF concentrations that caused over 80% of cellular damage (Figure 1).
Of great importance was discovery of GF:TF drug combination that could cause 90% and 80% cellular damage to multidrug-resistant isolates TME 16 and TME 32, respectively.
It is possible that additive effect was achieved based upon the mechanism of action of GF (nucleic acid inhibitor) and TF (ergosterol synthesis inhibitor).Alteration of the membrane integrity can be linked to squalene epoxidase inhibition by TF, which would promote cellular internalization of GF; while intracellular accumulation would decreases DNA synthesis (Favre, Ryder, 1997;Polak, 1993).
While this is only one biochemical hypothesis, it is also possible that intermolecular bonds in between GF and TB molecules could be responsible for additive effect observed.Our group will be pursuing additional studies to identify these possible interactions and the results will be reported in the due course.
As reported previously we found that GF has low solubility in water (Kahsav et al., 2013).In fact, it was noted that GF partially precipitates in aqueous solution at 35 °C.Consequently, it was necessary to mediate drug precipitation during incubation to avoid misleading results.This was first attempted by using MOPS as a buffer to impart enhanced compound solubility.However, it is not possible to make the desired stock solutions of GF in concentrations higher than 10 µg mL -1 in MOPS buffer at 35 °C described at the CLSI for MIC determinations (CLSI, 2008).As an alternative, we found that we could make stock solution 1.6 mg mL -1 of GF in DMSO and dilution were made with RPMI 1640 media thereby allowing for proper MICs determinations.
In conclusion, herein we report the first investigation of GF and TF combination drug assessments against a panel of fungi clinical isolates.While, these two drugs have been evaluated with other antifungals (Baran et al., 2000;Romano et al., 2005) our findings are new and noteworthy particularly when pathological dermatophytes have become drug resistant to many antifungal drugs (including GF and TB alone).Through a checkerboard assay, combining GF and TF results in additive effect and has impressive efficacy to several clinical dermatophytes including two hard to kill multidrug-resistant fungi isolates.This enhanced activity might be due to having selected drugs of different mechanisms of actions: inhibition of mitosis (GF) and ergosterol depletion (TF), as it is believed that TF might be altering the membrane by blocking the squalene epoxidase which ultimately facilitates cellular internalization of GF allowing for resensitizing via DNA damage of the multidrug-resistant isolates.

FIGURE 1 -
FIGURE 1 -Hyphal damage expressed in percentage of the griseofulvin (GF) () and terbinafine (TF) () alone and in combination ().A) Hyphal damage of the TME 16 drug resistant isolate to the combination of TF and GF antifungals at concentrations below MIC; B) Hyphal damage of TME 32 drug sensitive isolate to combination of TF and GF antifungals at concentrations below MIC.Different letters above the hyphal damage represent a statistically significant difference (p <0.05).

TABLE I -
MICs to GF and TF against dermatophyte isolates Isolates having MIC ≥ 4 µg/ml are classified as resistant to the drugs.Bold: multi-resistant isolates.

TABLE II -
Fractional inhibitory concentration index (FICI) and type of interaction obtained for hyphae by testing GF:TF combinations according to the checkerboard microdilution method (qualitative methodology) Synergistic (Syn); additive (Add); indifferent (Ind).*multi-resistant isolates.

TABLE III -
Cell damage after treatment with GF, TF, and GF:TF combinations (quantitative methodology)

TABLE III -
Cell damage after treatment with GF, TF, and GF:TF combinations (quantitative methodology) (cont.)