Tolnaftate inhibits ergosterol production and impacts cell viability of Leishmania sp.

Leishmaniasis is an infectious disease caused by protozoan parasites of the genus Leishmania. The treatment of all forms of leishmaniasis relies on first-line drug, pentavalent antimonial, and in cases of drug failure, the second-line drug amphotericin B has been used. Besides the high toxicity of drugs, parasites can be resistant to antimonial in some areas of the World, making it necessary to perform further studies for the characterization of new antileishmanial agents. Thus, the aim of the present work was to evaluate the leishmanicidal activity of tolnaftate, a selective reversible and non-competitive inhibitor of the fungal enzyme squalene epoxidase, which is involved in the biosynthesis of ergosterol, essential to maintain membrane physiology in fungi as well as trypanosomatids. Tolnaftate eliminated promastigote forms of L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) infantum (EC50 ~ 10 µg/mL and SI ~ 20 for all leishmanial species), and intracellular amastigote forms of all studied species (EC50 ~ 23 µg/mL in infections caused by dermatotropic species; and 11.7 µg/mL in infection caused by viscerotropic species) with high selectivity toward parasites [SI ~ 8 in infections caused by dermatotropic species and 17.4 for viscerotropic specie]. Promastigote forms of L. (L.) amazonensis treated with the EC50 of tolnaftate displayed morphological and physiological changes in the mitochondria and cell membrane. Additionally, promastigote forms treated with tolnaftate EC50 reduced the level of ergosterol by 5.6 times in comparison to the control parasites. Altogether, these results suggest that tolnaftate has leishmanicidal activity towards Leishmania sp., is selective, affects the cell membrane and mitochondria of parasites and, moreover, inhibits ergosterol production in L. (L.) amazonensis.

Enhancement of the topical tolnaftate delivery for the treatment of tinea pedis via provesicular gel systems.

Tolnaftate is a thiocarbamate antifungal drug which is therapeutically active against dermatophytes that cause various forms of tinea. Due to the small amount of tolnaftate released from ordinary ointment bases and insufficient penetration through the infected skin layers the need to incorporate the drug in a more suitable pharmaceutical form has evolved. A provesicular system is one such form that can solve these problems. Once in contact with the skin, dilution with moisture occurs and the provesicular system rapidly transforms into a vesicular one. Provesicular systems were prepared according to full-factorial experimental design. Plain provesicular systems were compared with systems containing Phospholipon 80 H and Lipoid S45 as penetration enhancers. Design expert software was used to analyze the effect of formulation variables (type of Span used as well as the presence or the absence of the penetration enhancer and its type) on the dependent variables: percent encapsulation efficiency (EE%), vesicle size and percent in vitro drug released). Three formulations were chosen; a plain provesicular system (PV-2), one containing Phospholipon 80H (PV-6) and another containing Lipoid S45 (PV-10) with the goal to reveal the effect of penetration enhancer on morphology, rheological properties and ex vivo permeation using confocal laser scanning microscopy (CLSM). Analysis of CLSM results showed that the penetration enhancing effect for the tested formulations followed the order PV-10 > PV-6 > PV-2. Promising clinically active treatment for tinea patients could be expected as shown by the in vivo permeation results for the provesicular systems as suggested by the CLSM results.

Characterization of iprodione resistance in Botrytis cinerea from strawberry and blackberry.

Gray mold, caused by the fungal pathogen Botrytis cinerea, is one of the most destructive diseases of strawberry. Control of the disease in commercial fields is largely dependent on the application of fungicides, including the dicarboximide iprodione. Single-spore isolates were collected from strawberry fields in Florida, North Carolina, and South Carolina and subjected to an assay using conidial germination that distinguished sensitive (S) isolates from isolates with various levels of resistance to iprodione. Of the 245 isolates, 1 was highly resistant (HR), 5 were moderately resistant (MR), and 43 had low resistance (LR) to iprodione. LR and MR strains were found in the Florida population and in 9 of 11 locations from North Carolina and South Carolina, indicating that resistance was widespread but accounted for only a relatively small percentage of the B. cinerea population. Sequence analysis of the target gene bos1, which codes for a class III histidine kinase, revealed that the MR phenotype was associated with Q369P and N373S mutations and that the LR phenotype was associated with either a I365S or a I365N mutation. The I365S and I365N mutations were also present in five additionally included HR isolates from North Carolina and South Carolina blackberry fields and one HR isolate from a Virginia strawberry field but no mutation or mutation combinations in bos1 were uniquely associated with the HR phenotype. Expression analysis of bos1 in S and HR isolates did not reveal convincing evidence of the gene's involvement in HR resistance either. The six HR isolates had three different phenotypes with respect to their sensitivity to fludioxonil; two were S, two were LR, and two were MR. The fludioxonil LR and MR isolates were also resistant to tolnaftate, an indication of multidrug efflux pump activity. These data suggest that, in addition to point mutations in bos1, drug efflux pump activity and potentially a third mechanism of resistance may be contributing to the iprodione HR phenotype. Detached fruit studies showed that field rates of Rovral 4 Flowable (iprodione) did not control iprodione MR and HR isolates.

Nonplanar property study of antifungal agent tolnaftate-spectroscopic approach.

Vibrational analysis of the thionocarbamate fungicide tolnaftate which is antidermatophytic, antitrichophytic and antimycotic agent, primarily inhibits the ergosterol biosynthesis in the fungus, was carried out using NIR FT-Raman and FTIR spectroscopic techniques. The equilibrium geometry, various bonding features, harmonic vibrational wavenumbers and torsional potential energy surface (PES) scan studies have been computed using density functional theory method. The detailed interpretation of the vibrational spectra has been carried out with the aid of VEDA.4 program. Vibrational spectra, natural bonding orbital (NBO) analysis and optimized molecular structure show the clear evidence for electronic interaction of thionocarbamate group with aromatic ring. Predicted electronic absorption spectrum from TD-DFT calculation has been compared with the UV-vis spectrum. The Mulliken population analysis on atomic charges and the HOMO-LUMO energy were also calculated. Vibrational analysis reveals that the simultaneous IR and Raman activation of the C-C stretching mode in the phenyl and naphthalene ring provide evidence for the charge transfer interaction between the donor and acceptor groups and is responsible for its bioactivity as a fungicide.

Biochemical characterization of terbinafine-resistant Trichophyton rubrum isolates.

We investigated the biochemical basis for resistance in six sequential clinical isolates of Trichophyton rubrum, from the same patient, which exhibited high-level primary resistance to terbinafine. Cellular ergosterol biosynthesis was measured by incorporation of [14C]acetate, and microsomal squalene epoxidase was assayed by conversion of [3H]squalene to squalene epoxide and lanosterol. Direct comparison was made with a terbinafine-susceptible reference strain of T. rubrum in which squalene epoxidase was previously studied. Resistant isolates displayed normal cellular ergosterol biosynthesis, although slight accumulation of radiolabeled squalene suggested reduced squalene epoxidase activity. Ergosterol biosynthesis in the resistant isolates was only inhibited by terbinafine concentrations above 1 microg/ml (IC50 5 microg/ml). In the reference strain, ergosterol biosynthesis was eliminated by terbinafine at 0.03 microg/ml in accordance with historical data. There was no significant difference in sensitivity between the six resistant isolates. Squalene epoxidase from resistant strains was three orders of magnitude less sensitive than normal enzyme to terbinafine (IC50 of 30 micromol/l and 19 n mol/l respectively). The epoxidase in the resistant strains was also unresponsive to tolnaftate. Resistance to terbinafine in these T. rubrum isolates appears to be due to alterations in the squalene epoxidase gene or a factor essential for its activity.

Current therapy of dermatophytosis.

In the past dermatophytes were treated with topical agents or, in the case of more recalcitrant or extensive disease, with oral antifungals (griseofulvin or ketoconazole). Topical therapies may be effective in many cases, but they have limitations. They may be viewed as inconvenient by the patient, thereby affecting compliance. Therapy with early oral antifungals entails long treatment periods until complete cure is obtained. For ketoconazole rare but serious side effects can occur, particularly with prolonged use. Griseofulvin is still the drug of choice for the treatment of tinea capitis of the Microsporum type. In recent years a few new antimycotic agents have been developed for systemic therapy of superficial fungal infections. Itraconazole is a broad-spectrum triazole. Fluconazole belongs to the same chemical class and was used mainly in systemic yeast infections and mucosal candidosis. Terbinafine is an allylamine and has been found to be effective and safe in brief therapy of dermatophyte infections. Short-duration therapy of most dermatophyte infections is also possible with itraconazole. The high and specific activity against the causative agents, together with their pharmacokinetic properties, explains the good results obtained with these new drugs and their improved safety profile. Their mode of action, pharmacokinetics, and treatment schedules will be discussed.

Two mechanisms of butenafine action in Candida albicans.

The mechanism of action of a new benzylamine antimycotic, butenafine hydrochloride, was studied in Candida albicans by using the thiocarbamate antimycotic tolnaftate as a reference drug. Butenafine completely inhibited the growth of a test strain of C. albicans at 25 micrograms/ml and was cidal at 50 micrograms/ml. Tolnaftate did not show any growth-inhibitory activity up to 100 micrograms/ml. Both butenafine and tolnaftate inhibited squalene epoxidation in C. albicans, with 50% inhibitory concentrations being 0.57 and 0.17 microgram/ml, respectively. Butenafine, but not tolnaftate, induced the release of appreciable amounts of Pi from C. albicans cells at 12.5 micrograms/ml. This effect of butenafine was augmented when the cells were pretreated with tolnaftate. The results suggest that the direct membrane-damaging effect of butenafine may play a major role in its anticandidal activity and that the drug-induced alteration in the cellular sterol composition renders the cell membrane more susceptible to the membrane-damaging effect of this drug.

Studies on the mode of action of tolnaftate in Microsporum gypseum.

Studies were performed on the mode of action of tolnaftate and resistance to this drug in Microsporum gypseum. Cells grown in the presence of tolnaftate (at the IC 50) showed a reduced content of total phospholipids and sterols whereas there was an increase in total RNA content. Incubation of cells with tolnaftate (at 10 x MIC), followed by addition of different macromolecule precursors revealed inhibition of the biosynthesis of all macromolecules except for RNA. The activity of membrane-bound enzymes did not change on treatment with tolnaftate (10 x MIC) whereas an increase in the leakage of intracellular 32P was observed. The content of total phospholipids was higher in tolnaftate-resistant cells, whereas the content of total sterols, DNA, RNA and protein was comparable to that of susceptible cultures. Activity of phosphodiesterase decreased and 5'-nucleotidase increased in tolnaftate-resistant cells. Our results suggest that the antifungal activity of tolnaftate is due to differential action on various targets site(s) which are modified in strains resistant to the drug.

In vitro and in vivo activities of piritetrate (M-732), a new antidermatophytic thiocarbamate.

Piritetrate (M-732), a new topical antifungal agent belonging chemically to the thiocarbamates, was demonstrated to possess a potent selective antidermatophytic activity. In terms of its MICs in susceptibility testing, mainly done by using Sabouraud dextrose agar plates, piritetrate exhibited several- to 10-fold-stronger antidermatophytic activity than tolnaftate, a reference thiocarbamate. Furthermore, piritetrate was found to show a broader antifungal spectrum than tolnaftate; relatively many species and strains of dematiaceous fungi, dimorphic fungi, and some other filamentous fungi as well as a few strains of Cryptococcus neoformans were fairly susceptible to piritetrate, while almost all the tested species and strains were resistant to tolnaftate. All the tested species of the genus Candida were, however, resistant to both compounds. Variables which can influence antimicrobial activity caused few changes in the MICs of either compound against Trichophyton mentagrophytes; however, an increase in the inoculum size resulted in a significant increase in the MICs. The antidermatophytic activities of piritetrate and tolnaftate were fungistatic but not fungicidal. Piritetrate also exhibited a more potent in vitro anti-T. mentagrophytes activity than clotrimazole or tolciclate. Piritetrate and tolnaftate had no antibacterial activity. The in vivo activity of topically administered piritetrate against experimental dermal infection of guinea pigs with T. mentagrophytes was more effective than that of tolnaftate both mycologically and clinically. Piritetrate manifested no acute toxicity in laboratory animals when administered even in large quantities by the oral, intraperitoneal, and topical routes.

Inhibitory effect of a new mycotic agent, piritetrate on ergosterol biosynthesis in pathogenic fungi.

A new antimycotic agent piritetrate, a thiocarbamate derivative, was found to interfere with fungal sterol biosynthesis. Sterol biosynthesis was measured by means of incorporation of radioactivity from [14C]acetate into individual sterol fractions and their precursor compounds extracted from cells of Candida albicans, Cryptococcus neoformans, and Trichophyton mentagrophytes. Piritetrate was a much more potent inhibitor of fungal squalene epoxidation than a related drug, naphthiomate. The greater antifungal efficacy of the former was reflected in its greater inhibitory action on sterol biosynthesis than the latter. Such inhibitory effects were also demonstrated in in vitro labelling experiments with [14C]mevalonate on the S10 fraction (10,000 g supernatant of homogenate) of C. albicans cells. At 5 x 10(-7) M concentration, piritetrate gave almost 90% inhibition of ergosterol biosynthesis in the S10 fraction of C. albicans cells. Furthermore, this agent was highly specific for fungal enzymes involved in ergosterol biosynthesis, with no detectable effects on mammalian cholesterol biosynthesis in rat liver at the therapeutic concentration.

Effect of antifungal agents on lipid biosynthesis and membrane integrity in Candida albicans.

Eight antifungal agents were examined for effects on lipid biosynthesis and membrane integrity in Candida albicans. Lipids were labeled in vivo or in vitro with [14C]acetate and analyzed by thin-layer and gas chromatography. Membrane integrity was measured by a recently developed [14C]aminoisobutyric acid radiolabel release assay. The imidazole antifungal agents miconazole, econazole, clotrimazole, and ketoconazole, at concentrations inhibiting ergosterol biosynthesis (0.1 microM), decreased the ratio of unsaturated to saturated fatty acids in vivo but not in vitro. Similarly, naftifine, tolnaftate, and the azasterol A25822B, at concentrations inhibiting ergosterol biosynthesis (10, 100, and 1 microM, respectively), decreased the ratio of unsaturated to saturated fatty acids in vivo only. This suggests that the effect on fatty acids observed with ergosterol biosynthesis inhibitors may be secondary to the effect on ergosterol. With imidazoles, oleic acid antagonized inhibition of cell growth but not inhibition of ergosterol. This suggests that, with the C-14 demethylase inhibitors, decreased unsaturated fatty acids, rather than decreased ergosterol, are responsible for growth inhibition. Cerulenin, previously reported to be a potent inhibitor of both fatty acid and ergosterol biosynthesis, was found in the present study to inhibit the former (at 5 microM) but not the latter (up to 100 microM). Of the antifungal agents tested, econazole and miconazole (at 100 microM) produced complete release of [14C]aminoisobutyric acid, which is consistent with membrane damage.

Ergosterol biosynthesis inhibition by the thiocarbamate antifungal agents tolnaftate and tolciclate.

The thiocarbamate antimycotics tolnaftate and tolciclate blocked sterol biosynthesis in fungal cells and cell extracts, with accumulation of squalene. This point of action was confirmed by the direct inhibition of microsomal squalene epoxidase from Candida albicans. There was no inhibition of other steps in ergosterol biosynthesis. In whole Candida cells, ergosterol biosynthesis inhibition was not complete at drug concentrations up to 100 mg/liter, whereas full inhibition occurred in a cell-free test system. Rat liver cell-free cholesterol biosynthesis was much less sensitive to the drugs. The biochemical action of tolnaftate and tolciclate is thus similar to that of the allylamine antimycotics naftifine and terbinafine.

The mode of antifungal action of tolnaftate.

The anti-dermatophyte agent tolnaftate was compared with the allylamine antifungal compounds naftifine and terbinafine. Tolnaftate was shown to inhibit sterol biosynthesis at the level of squalene epoxidation and squalene was shown to accumulate in dermatophytes grown in its presence. Biochemical studies in whole and broken cells supported this conclusion and showed that the compound was active against squalene epoxidation in broken C. albicans cells, but was much less potent against whole cells. These results suggested there was a barrier to penetration in these yeasts.

Effects of antifungal agents on ergosterol biosynthesis in Candida albicans and Trichophyton mentagrophytes: differential inhibitory sites of naphthiomate and miconazole.

The effects of naphthiomate and miconazole showing ergosterol biosynthesis inhibition on Candida albicans and Trichophyton mentagrophytes were investigated by measuring [14C]acetate incorporation into sterols and its precursors. Naphthiomate, like allylamine compounds, was found to interfere with fungal ergosterol biosynthesis by preventing the conversion of squalene into squalene epoxide which is mediated by squalene epoxidase. This metabolic inhibition resulted in a considerable decrease of ergosterol with a corresponding increase of squalene, which is more distinct in T. mentagrophytes than in C. albicans. This indicates that naphthiomate blocks the utilization of squalene by inhibition of squalene epoxidation; unlike N-substituted imidazole, miconazole has been known to inhibit ergosterol formation by inhibiting C14-demethylation. In addition, it is of interest to note that miconazole causes a great accumulation of lanosterol in C. albicans cells, while in T. mentagrophytes cells there was instead a drastic increase of 24-methylenedihydrolanosterol but not lanosterol. The results obtained from this study indicate that repression of ergosterol synthesis by naphthiomate and miconazole is due to inhibition of squalene epoxidation for the former and C14-demethylation for the latter.

Otomycosis: an experimental evaluation of six antimycotic agents.

One hundred-and eight-six fungi were isolated from one hundred-and-eighty cases clinically diagnosed as otomycosis. They comprise 59 species of 26 genera of moulds, and 2 genera of yeasts. This large variety of mould isolates provided ample material for in vitro experiments, to evaluate the anti-fungal activity of six antimycotic substances found in the preparations currently used for the treatment of otomycosis. The antifungal activity of clotrimazole and tolnaftate in vitro was evident. For over 94 per cent of the 59 fungus species tested, the MIC was less than 0.1 micrograms/ml, and for 6 per cent it was between 0.4 and 1 microgram/ml. Concerning the other 4 antimycotic substances (iodochlorhydroxyquin, fluonilid, natamycine and polymyxin B sulphate) the MIC ranged from greater than 100 micrograms/ml to 1 microgram/ml for the majority of tested fungi. The clinical observations were mostly in accordance with these findings.

In vitro activity of naftifine, a new antifungal agent.

Naftifine exhibits an interesting in vitro spectrum of activity against dermatophytes (38 strains; minimal inhibitory concentration [MIC] range 0.1 to 0.2 microgram/ml), aspergilli (6 strains; MIC range, 0.8 to 12.5 microgram/ml), Sporothrix schenckii (2 strains; MICs, 0.8 and 1.5 microgram/ml), and yeasts of the genus Candida (77 strains; MIC range, 1.5 to greater than 100 microgram/ml). Its degree of efficacy is unaffected by the organism density in the test medium, and it is primarily fungicidal against dermatophytes as well as yeasts. Its in vitro efficacy is pH dependent and rises with increasing pH values.

Mechanism of action of antifungal drugs, with special reference to the imidazole derivatives.

Currently used antifungal drugs are distinct in terms of spectrum of activity, potency, therapeutic index, development of resistance, and mode of use. An important factor in the usefulnesss of a compound is the mechanism by which it attacks the structure and function of the fungal cell. The target organelles have been established for most antifungal drugs. Polyenes bind irreversibly to cell membranes. Alteration of the permeability of these structures precedes metabolic disruption and cell death. Griseofulvin deteriorates spindle and cytoplasmic microtubules, influencing cell division and outgrowth of hyphal tips. Flucytosine is deaminated to 5-fluorouracil, which is then phosphorylated and incorporated into RNA; protein synthesis is consequently impaired. A mechanism of action via inhibition of DNA synthesis is an alternative explanation. The imidazole derivatives inhibit the biosynthesis of ergosterol, the main sterol in membranes of fungi. These agents also affect the synthesis of triglycerides and phospholipids. Changes in oxidative and peroxidative enzyme activities, leading to an intracellular buildup of toxic concentrations of hydrogen peroxide, may contribute to the observed deterioration of subcellular organelles and to cell necrosis. The imidazole derivatives inhibit the transformation of blastospores of Candida albicans into the invasive mycelial form. This inhibition probably facilitates the task of host defense cells and may be the principal factor leading to clearance of infection.