Antifungal activity of azoles, allylamines, and 8-hidroxiquinolines, alone and in combination, against Malassezia pachydermatis in vitro and in vivo.

Malassezia pachydermatis is often reported as the causative agent of dermatitis in dogs. This study aims to evaluate the in vitro and in vivo antifungal activity of azoles and terbinafine (TRB), alone and in combination with the 8-hydroxyquinoline derivatives (8-HQs) clioquinol (CQL), 8-hydroxyquinoline-5-(n-4-chlorophenyl)sulfonamide (PH151), and 8-hydroxyquinoline-5-(n-4-methoxyphenyl)sulfonamide (PH153), against 16 M. pachydermatis isolates. Susceptibility to the drugs was evaluated by in vitro broth microdilution and time-kill assays. The Toll-deficient Drosophila melanogaster fly model was used to assess the efficacy of drugs in vivo. In vitro tests showed that ketoconazole (KTZ) was the most active drug, followed by TRB and CQL. The combinations itraconazole (ITZ)+CQL and ITZ+PH151 resulted in the highest percentages of synergism and none of the combinations resulted in antagonism. TRB showed the highest survival rates after seven days of treatment of the flies, followed by CQL and ITZ, whereas the evaluation of fungal burden of dead flies showed a greater fungicidal effect of azoles when compared to the other drugs. Here we showed for the first time that CQL is effective against M. pachydermatis and potentially interesting for the treatment of malasseziosis.

In vitro determination of the combination of ciclopirox and terbinafine in the treatment of dermatophytosis.

INTRODUCTION: The cases of dermatophytosis are increasing and they are associated with a higher number of therapeutic failures leading the doctor to prescribe combinations of antifungals as therapy. The objective was to evaluate the interaction of terbinafine and ciclopirox, the most commonly antifungals used in the clinic, in dermatophyte isolates. METHODOLOGY: The minimum inhibitory concentrations (MIC) of ciclopirox and terbinafine were determined by the broth microdilution method according CLSI and the checkerboard assay was used to evaluate the interaction between the antifungal agents. RESULTS: For terbinafine the mic50 was 0.125 ug/mL and mic90 was 0.250 ug/mL. For ciclopirox the values were 2.0 ug/mL for mic50 and 4.0 ug/mL for mic90. No synergistic interaction was observed for the dermatophyte isolates tested. CONCLUSION: These results suggest that the use of terbinafine in combination with ciclopirox, which is widely used in the clinic, may not be a good choice for the treatment of onychomycosis.

Drosophila melanogaster as a model of systemic dermatophytosis.

BACKGROUND: Dermatophytosis is one of the most common fungal infections worldwide. The distribution of dermatophytes varies across continents, but the genera Trichophyton and Microsporum have emerged as the main isolated agents in humans and animals. OBJECTIVES: To validate Drosophila melanogaster flies as a fast and feasible model to study dermatophytic infections. METHODS: Wild-type (WT) and Toll-deficient D. melanogaster flies were infected by Trichophyton rubrum, T. mentagrophytes, Microsporum canis and Nannizzia gypsea by pricking with a needle previously dipped in inoculum concentrations ranging from 103 to 108 colony-forming units/mL. Establishment of infection was confirmed by survival curves, histopathological analysis and fungal burden. Thereafter, flies were treated with terbinafine, itraconazole and clioquinol. RESULTS: WT flies were predominantly resistant to the infection, whereas Toll-deficient flies succumbed to the four dermatophyte genera tested. The antifungal drugs protected flies from the infection, except for N. gypsea whose survival curves did not differ from the untreated group. CONCLUSIONS: This pilot study confirms that D. melanogaster is a suitable model to study the virulence and antifungal drug efficacy in dermatophyte species.

Clioquinol and 8-hydroxyquinoline-5-sulfonamide derivatives damage the cell wall of Pythium insidiosum.

AIMS: To evaluate the antimicrobial activity and to determine the pharmacodynamic characteristics of three 8-hydroxyquinoline derivatives (8-HQs) against Pythium insidiosum, the causative agent of pythiosis. METHODS AND RESULTS: Antimicrobial activity was tested by broth microdilution and MTT assays. The antimicrobial mode of action was investigated using sorbitol protection assay, ergosterol binding assay, and scanning electron microscopy. Clioquinol, PH151, and PH153 were active against all isolates, with MIC values ranging from 0.25 to 2 µg ml-1. They also showed a time- and dose-dependent antimicrobial effect, damaging the P. insidiosum cell wall. CONCLUSIONS: Together, these results reinforce the potential of 8-HQs for developing new drugs to treat pythiosis.

8-hydroxyquinoline-5-(N-4-chlorophenyl) sulfonamide and fluconazole combination as a preventive strategy for Candida biofilm in haemodialysis devices.

Introduction. The presence of Candida biofilms in medical devices is a concerning and important clinical issue for haemodialysis patients who require constant use of prosthetic fistulae and catheters.Hypothesis/Gap Statement. This prolonged use increases the risk of candidaemia due to biofilm formation. PH151 and clioquinol are 8-hydroxyquinoline derivatives that have been studied by our group and showed interesting anti-Candida activity.Aim. This study evaluated the biofilm formation capacity of Candida species on polytetrafluoroethylene (PTFE) and polyurethane (PUR) and investigated the synergistic effects between the compounds PH151 and clioquinol and fluconazole, amphotericin B and caspofungin against biofilm cells removed from those materials. Further, the synergistic combination was evaluated in terms of preventing biofilm formation on PTFE and PUR discs.Methodology. Susceptibility testing was performed for planktonic and biofilm cells using the broth microdilution method. The checkerboard method and the time-kill assay were used to evaluate the interactions between antifungal agents. Antibiofilm activity on PTFE and PUR materials was assessed to quantify the prevention of biofilm formation.Results. Candida albicans, Candida glabrata and Candida tropicalis showed ability to form biofilms on both materials. By contrast, Candida parapsilosis did not demonstrate this ability. Synergistic interaction was observed when PH151 was combined with fluconazole in 77.8 % of isolates and this treatment was shown to be concentration- and time-dependent. On the other hand, indifferent interactions were predominantly observed with the other combinations. A reduction in biofilm formation on PUR material of more than 50 % was observed when using PH151 combined with fluconazole.Conclusion. PH151 demonstrated potential as a local treatment for use in a combination therapy approach against Candida biofilm formation on haemodialysis devices.

Evaluation of activity and toxicity of combining clioquinol with ciclopirox and terbinafine in alternative models of dermatophytosis.

Dermatophytosis is a superficial fungal infection that affects humans and is very common in small animals. The treatment using the most commonly used antifungals is failing, and new therapeutic alternatives are required to combat the resistance of these fungal infections. Previous studies by the group have shown that clioquinol is an important therapeutic alternative in the treatment of dermatophytosis. The object was to conduct studies of antidermatophytic activity and the irritant potential from the double and triple combinations of clioquinol, terbinafine and ciclopirox in ex vivo and in vivo alternative models. To evaluate the irritant potential of antifungal combinations, the alternative HET-CAM method (chicken egg test chorioallantoic membrane) was used. Ex vivo models were used to assess the effectiveness of antifungal combinations, using pig hooves and veterinary fur. Any possible tissue damage was to assess through in histopathology of swine ears. HET-CAM results showed that all combinations can be classified as non-irritating, corroborated by the results of the histopathological evaluation of the pig's ear skin. Only the double combinations managed to remove 100% of the colony-forming units (CFU) formed on the pig's hooves. The clioquinol + terbinafine combination and the triple combination were more effective than clioquinol + ciclopirox in eradicating the preformed biofilm in fur of veterinary origin. These results show the potential of formulations of clioquinol in combination with antifungals for use in humans and in the veterinary field to combat dermatophytosis, as an important alternative therapy, for use in the near future.

Ex vivo potential of a quinoline-derivative nail lacquer as a new alternative for dermatophytic onychomycosis treatment.

Introduction. Onychomycosis infections currently show a significant increase, affecting about 10 % of the world population. Trichophyton rubrum is the main agent responsible for about 80 % of the reported infections. The clinical cure for onychomycosis is extremely difficult and effective new antifungal therapy is needed.Hypothesis/Gap Statement. Ex vivo onychomycosis models using porcine hooves can be an excellent alternative for evaluating the efficacy of new anti-dermatophytic agents in a nail lacquer.Aim. Evaluation of the effectiveness of a nail lacquer containing a quinoline derivative on an ex vivo onychomycosis model using porcine hooves, as well as the proposal of a plausible antifungal mechanism of this derivative against dermatophytic strains.Methodology. The action mechanism of a quinoline derivative was evaluated through the sorbitol protection assay, exogenous ergosterol binding, and the determination of the dose-response curves by time-kill assay. Scanning electron microscopy evaluated the effect of the derivative in the fungal cells. The efficacy of a quinoline-derivative nail lacquer on an ex vivo onychomycosis model using porcine hooves was evaluated as well.Results. The quinoline derivative showed a time-dependent fungicidal effect, demonstrating reduction and damage in the morphology of dermatophytic hyphae. In addition, the ex vivo onychomycosis model was effective in the establishment of infection by T. rubrum.Conclusion. Treatment with the quinoline-derivative lacquer showed a significant inhibitory effect on T. rubrum strain in this infection model. Finally, the compound presents high potential for application in a formulation such as nail lacquer as a possible treatment for dermatophytic onychomycosis.

In vitro antidermatophytic synergism of double and triple combination of clioquinol with ciclopirox and terbinafine.

BACKGROUND: Dermatophytoses are the most frequent fungal infections worldwide and there have been described clinical resistance to the commonly used antifungals. Clioquinol is an antimicrobial that had the oral formulations withdrawn from the market in the 70s due to the report of neurotoxicity and recently has been considered as an effective alternative for the treatment of dermatophytosis. OBJECTIVES: To evaluate the effect of the double and triple association between clioquinol with terbinafine and ciclopirox on clinical isolates of dermatophytes. The cytotoxicity of these associations on human leukocytes was also verified. METHODS: Checkerboard method was used to evaluate the interaction between antifungal agents. Time-kill assay was used to verify fungicidal action and evaluate the combination with greater effect for TRU47 isolate. Cell viability was assessed by loss of integrity of the leukocyte membrane in order to verify the toxicity. RESULTS: Synergistic interaction was observed in 42% of isolates when terbinafine was associated with clioquinol and in 50% of isolates when ciclopirox was associated with clioquinol. The triple association resulted in synergistic interaction for 75% of the isolates. Clioquinol + terbinafine and triple combination were more effective for TRU47 isolate, and the combinations exhibited a time-dependent fungicidal effect. Furthermore, the results of cell viability demonstrated that clioquinol and terbinafine combination is not cytotoxic to human leukocytes. CONCLUSIONS: Clioquinol in combination with antifungals in the treatment of dermatophytosis can be a therapeutic strategy to overcome problems related to resistance, action spectrum and toxicity of the antifungal drugs used in the clinic.

Synergistic effect of ibuprofen with itraconazole and fluconazole against Cryptococcus neoformans

The present study investigated the association of the non-steroidal anti-inflammatory drug ibuprofen with itraconazole, fluconazole and amphotericin B against Cryptococcus neoformans isolates. The minimal inhibitory concentration (MIC) was found according to M27-A3 protocol and in vitro interactions were evaluated using checkerboard microdilution method. Synergism was demonstrated between azoles and ibuprofen for most isolates. However, no synergistic effects were seen when amphotericin B was combined with ibuprofen. Therefore, our results suggest that ibuprofen presents clinical potential when combined with azole drugs in the treatment of cryptococcosis.

Chloroacetamide derivatives as a promising topical treatment for fungal skin infections.

The aim of this study was to evaluate the antifungal potential of 11 chloroacetamide derivatives and derivative incorporated into a film-forming system (FFS) as a potential alternative for the topical treatment of superficial and skin mycoses. The minimum inhibitory concentration (MIC) evaluation followed Clinical and Laboratory Standards Institute protocols M27-A3 (Candida) and M28-A2 (dermatophytes). Compounds 2, 3, and 4 were the most effective against Candida species (MIC range: 25-50 µg/mL) and dermatophytes (MIC range: 3.12-50 µg/mL). Compound 2 maintained its antifungal activity when incorporated in a FFS, with MIC values equivalent to the free compound. In addition, the compound does not act through complexation with ergosterol, suggesting that it may act on other targets of the fungal cell membrane. Chloroacetamide derivatives presented anti-Candida and anti-dermatophytic effectiveness. The FFS containing compound 2 has shown to be superior to traditional topical treatment of superficial and cutaneous fungal infections. It was found that these new chemical entities, with their applicability, are an excellent alternative to the topical treatment of fungal skin infections.

Oral clioquinol is effective in the treatment of a fly model of Candida systemic infection.

BACKGROUND: Clioquinol was used in the 1950s-1970s as antimicrobial but its oral formulations were withdrawn from the market due to suspected neurotoxicity. Currently, there is possibility of repositioning of oral clioquinol formulations. OBJECTIVES: To evaluate the antifungal activity and toxicological parameters of clioquinol and the other two 8-hydroxyquinoline derivatives using alternative animal models and to study the interaction dynamic of clioquinol with Candida albicans. METHODS: We used Toll-deficient Drosophila melanogaster to test the protective effect of 8-hydroxyquinolines against C. albicans infection. Toxicological parameters were investigated in chicken embryo. A mathematical model-based analysis of the time-kill data of clioquinol was performed to obtain pharmacodynamic characteristics. RESULTS: Clioquinol fully protected D. melanogaster from the infection. The 8-hydroxyquinolines did not cause changes in opening of the beak and movement of the chicken embryo; however, clioquinol and compound 2 increased arterial pulsation. Compound 3 was lethal at 1 mg mL-1 . Effective concentration found in modelling indicated that clioquinol was highly effective against C. albicans (0.306 µg mL-1 ) in easily achievable serum levels; clioquinol rapidly achieved kill rate reaching the maximum effect after 13 hours. CONCLUSIONS: These results support the potential of clioquinol to be used as a systemic antifungal agent.

New insights into the mechanism of antifungal action of 8-hydroxyquinolines.

The 8-hydroxyquinoline core is a privileged scaffold for drug design explored to afford novel derivatives endowed with biological activity. Our research aimed at clarifying the antifungal mechanism of action of clioquinol, 8-hydroxy-5-quinolinesulfonic acid, and 8-hydroxy-7-iodo-5-quinolinesulfonic acid (three 8-hydroxyquinoline derivatives). The antifungal mode of action of these derivatives on Candida spp. and dermatophytes was investigated using sorbitol protection assay, cellular leakage effect, ergosterol binding assay, and scanning electron microscopy. Clioquinol damaged the cell wall and inhibited the formation of pseudohyphae by C. albicans. The 8-hydroxy-5-quinolinesulfonic acid derivatives compromised the functional integrity of cytoplasmic membranes. To date no similar report was found about the antifungal mechanism of 8-hydroxyquinolines. These results, combined with the broad antifungal spectrum already demonstrated previously, reinforce the potential of 8-hydroxyquinolines for the development of new drugs.

Lipid core nanoparticles as a broad strategy to reverse fluconazole resistance in multiple Candida species.

Fungal resistance is the major problem related to fluconazole treatments. This study aims to develop innovative lipid core nanocapsules and nanostructured lipid carriers containing fluconazole, to study in vitro antifungal activity and to assess the possibility of resistance reversion in Candida albicans, C. glabrata, C. krusei, and C. tropicalis isolates. The action mechanism of nanoparticles was investigated through efflux pumps and scanning electron microscopy studies. The lipid core nanocapsules and nanostructured lipid carriers were prepared by interfacial deposition of preformed polymer and high-pressure homogenization methods, respectively. Both nanostructures presented sizes below 250 nm, SPAN < 1.6, negative zeta potential, pH slightly acid, high drug content and controlled drug release. The nanostructured lipid carriers were unable to reverse the fungal resistance. Lipid core nanoparticles displayed advantages such as a reduction in the effective dose of fluconazole and resistance reversion in all isolates tested - with multiple mechanisms of resistance. The main role of the supramolecular structure and the composition of the nanoparticles on antifungal mechanisms of action were discussed. The results achieved through this study have an impact on clinical therapy, with a potential application in the treatment of fungal infections caused by resistant isolates of Candida spp.

Rapid tools to gain insights into the interaction dynamics of new 8-hydroxyquinolines with few fungal lines.

The combination of tools such as time-kill assay with subsequent application of mathematical modeling can clarify the potential of new antimicrobial compounds, since minimal inhibitory concentration (MIC) value does not provide a very detailed characterization of antimicrobial activity. Recently, our group has reported that the 8-hydroxy-5-quinolinesulfonic acid presents relevant antifungal activity. However, its intrinsic acidity could lead to an ionization process, decreasing fungal cell permeability. To overcome this potential problem and enhance activity, the purpose of this study was to synthesize and evaluate a novel series of hybrids between the 8-hydroxyquinoline core and sulfonamide and to prove their potential using broth microdilution method, obtaining the pharmacodynamic parameters of the most active derivatives combining time-kill studies and mathematical modeling and evaluating their toxicity. Compound 5a was the most potent, being active against all the fungal species tested, with low toxicity in normal cells. 5a and 5b have presented important antibacterial activity against Staphylococcus aureus strain. The EC50 values obtained by combination of time-kill studies with mathematical model were similar to those of MIC, which confirms the potential of compounds. In addition, these derivatives are non-irritant molecules with the absence of topical toxicity. Finally, 5a and 5b are promising candidates for treatment of dermatomycosis and candidiasis.

Clioquinol is a promising preventive morphological switching compound in the treatment of Candida infections linked to the use of intrauterine devices.

PURPOSE: Candida biofilm infections are frequently linked to the use of biomaterials and are of clinical significance because they are commonly resistant to antifungals. Clioquinol is an antiseptic drug and is effective against multidrug-resistant Candida. We investigated the effect of clioquinol and two other 8-hydroxyquinoline derivatives on Candida biofilm. METHODOLOGY: The ability to inhibit biofilm formation, inhibit preformed biofilm and remove established biofilms was evaluated using in vitro assays on microtitre plates. The action of clioquinol on biofilm in intrauterine devices (IUDs) was also investigated, describing the first protocol to quantify the inhibitory action of compounds on biofilms formed on IUDs. RESULTS: Clioquinol was found to be the most effective 8-hydroxyquinoline derivative among those tested. It prevented more than 90 % of biofilm formation, which can be attributed to blockade of hyphal development. Clioquinol also reduced the metabolic activity of sessile Candida but the susceptibility was lower compared to planktonic cells (0.031-0.5 µg ml-1 required to inhibit 50 % planktonic cells and 4-16 µg ml-1 to inhibit 50 % preformed biofilms). On the other hand, almost complete removal of biofilms was not achieved for the majority of the isolates. Candida spp. also showed the ability to form biofilm on copper IUD; clioquinol eradicated 80-100 % of these biofilms. CONCLUSION: Our results indicate a potential application in terms of biomaterials for 8-hydroxyquinoline derivatives. Clioquinol could be used as a coating to prevent morphological switching and thus prevent biofilm formation. Furthermore, clioquinol may have future applications in the treatment of Candida infections linked to the use of IUDs.

Antifungal mechanism of action of Schinus lentiscifolius Marchand essential oil and its synergistic effect in vitro with terbinafine and ciclopirox against dermatophytes.

OBJECTIVES: The aim of this study was to evaluate the antifungal, antichemotactic and antioxidant activities of Schinus lentiscifolius essential oil, as well as its combined effect with terbinafine and ciclopirox, against dermatophytes. METHODS: Essential oil was analysed by GC-MS. The antifungal activity and the mechanism of action were determined by broth microdilution, sorbitol and ergosterol assays, as well as scanning electron microscopy. The checkerboard method was used for evaluating the interactions with commercial antifungal agents. The antioxidant and antichemotactic activities were measured using the DPPH and the modified Boyden chamber methods, respectively. KEY FINDINGS: Chemical analysis revealed the presence of 33 compounds, the primary ones being γ-eudesmol (12.8%) and elemol (10.5%). The oil exhibited 97.4% of antichemotactic activity and 37.9% of antioxidant activity. Antifungal screening showed effect against dermatophytes with minimum inhibitory concentration values of 125 and 250 µg/ml. Regarding the mechanisms of action, the assays showed that the oil can act on the fungal cell wall and membrane. Synergistic interactions were observed using the combination with antifungals, primarily terbinafine. CONCLUSIONS: Schinus lentiscifolius essential oil acted as a chemosensitizer of the fungal cell to the drug, resulting in an improvement in the antifungal effect. Therefore, this combination can be considered as an alternative for the topical treatment of dermatophytosis.

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

ABSTRACT 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.

Evaluation of 8-Hydroxyquinoline Derivatives as Hits for Antifungal Drug Design.

Clioquinol is an 8-hydroxyquinoline derivative that was widely used from the 1950s to 1970s as an oral antiparasitic agent. In 1970, the oral forms were withdrawn from the market due to reports of toxicity, but topical formulations for antifungal treatment remained available. Thus, the purpose of this study was to evaluate the toxicity, anti-Candida and antidermatophyte activity and to determine pharmacodynamic characteristics of clioquinol and other 8-hydroxyquinoline derivatives (8-hydroxy-5-quinolinesulfonic acid and 8-hydroxy-7-iodo-5-quinolinesulfonic acid). Antifungal activity was tested by broth microdilution and the fungicidal or fungistatic effect was checked by a time-kill assay. Permeation and histopathological evaluation were performed in Franz diffusion cells with ear skin of pigs and examined under light microscopy. An HET-CAM test was used to determine the potential irritancy. The three compounds were active against all isolates showing anti-Candida and antidermatophyte activity, with MIC ranges of 0.031-2 µg/ml, 1-512 µg/ml, and 2-1024 µg/ml for clioquinol, 8-hydroxy-5-quinolinesulfonic acid, and 8-hydroxy-7-iodo-5-quinolinesulfonic acid, respectively. All compounds showed fungistatic effect for Candida, 8-hydroxy-5-quinolinesulfonic acid, and 8-hydroxy-7-iodo-5-quinolinesulfonic acid showed a fungicidal effect for M. canis and T. mentagrophytes, and clioquinol showed a fungicidal effect only for T. mentagrophytes. Furthermore, they presented a fungicidal effect depending on the time and concentration. The absence of lesions was observed in histopathological evaluation and no compound was irritating. Moreover, clioquinol and 8-hydroxy-5-quinolinesulfonic acid accumulated in the epithelial tissue, and 8-hydroxy-7-iodo-5-quinolinesulfonic acid had a high degree of permeation. In conclusion, 8-hydroxyquinoline derivatives showed antifungal activity and 8-hydroxy-5-quinolinesulfonic acid demonstrated the potential for antifungal drug design.

Synergistic antifungal activity of the lipophilic fraction of Hypericum carinatum and fluconazole

ABSTRACT Hypericum species, Hypericaceae, are recognized as a source of therapeutical agents. Purified fractions and isolated compounds have been shown antimicrobial activity. As the indiscriminate use of antifungals and the increase of infections caused by emerging species are leading to the search of new alternative treatments, the aim of this study was to continue the study with Hypericum carinatum Griseb. lipophilic fraction, rich in phloroglucinol derivatives, investigating the effect of its association with fluconazole against emerging yeasts (Candida krusei, C. famata, C. parapsilosis and Cryptococcus neoformans). The synergistic activity between H. carinatum lipophilic fraction and fluconazole was assessed by two methodologies for multiple dose–response analysis: checkerboard and isobologram. Regarding synergistic experiments, the effect of the association was higher than the effect of fluconazole alone against Candida krusei and C. famata isolates (MIC fluconazole decreased about eight and four folds, respectively), suggesting that, somehow, H. carinatum lipophilic fraction compounds are facilitating the action of this drug. On the other hand, when tested against Cryptococcus neoformans and C. parapsilosis, fluconazole showed better results than the association. Thus, against Candida krusei and C. famata, the lipophilic fraction of H. carinatum was able to reduce the MIC values of fluconazole and could be considered as a potential alternative to be used against emerging yeast species.

In vitro synergism of a water insoluble fraction of Uncaria tomentosa combined with fluconazole and terbinafine against resistant non-Candida albicans isolates.

CONTEXT: Uncaria tomentosa D.C. (Rubiaceae) has several biological activities, including activity against resistant Candida strains. The synergistic interaction with terbinafine or fluconazole can be an important alternative to overcome this resistance. OBJECTIVES: The potential synergy between a water insoluble fraction (WIF) from Uncaria tomentosa bark and the antifungals terbinafine (TRB) and fluconazole (FLZ) against non-Candida albicans resistant strains was investigated. MATERIALS AND METHODS: TRB and FLZ, alone and combined with WIF, were tested by the checkerboard procedure using the micro-dilution technique against seven isolates of Candida glabrata and C. krusei. The molecular interactions occurring outside the cell wall were evaluated by scanning electron microscopy, Fourier transform infrared (FT-IR) and differential scanning calorimetry (DSC) analysis. RESULTS: The checkerboard inhibitory assay demonstrated synergy for WIF:TRB and WIF:FLZ combinations, respectively. The best synergistic cell damage was demonstrated unequivocally for the associations of WIF and TRB (1.95:4.0 µg/mL) and WIF and FLZ (1.95:8.0 µg/mL). The comparison of the FT-IR spectra of the antifungal alone, and in combination with WIF, allows recognizing clear differences in 3000, 1600, 1400, and 700-800 cm-1 bands. Additionally, modifications on TRB and FLZ thermograms were clearly noticed after their combination with WIF. CONCLUSIONS: DSC and infrared analysis demonstrated intermolecular interactions between WIF and either TRB or FLZ. Hence, quite likely the synergistic effect is related to interaction events occurring outside the cell wall between antifungal and cat's claw proanthocyanidins. A direct action on the cell wall is suggested, without connection with the ABC efflux pump mechanism.