In Vitro Sensitivity of Torulopsis glabrata to Amphotericin B, 5-Fluorocytosine, and Clotrimazole (Bay 5097)

Thirty-five strains of Torulopsis glabrata were tested by a tube dilution method for their susceptibility to amphotericin B, 5-fluorocytosine, and clotrimazole (Bay 5097). Amphotericin B was the most active in vitro, inhibiting all strains at a concentration of 1 μg/ml and killing all strains at 2 μg/ml. 5-Fluorocytosine inhibited over 80% of strains at 0.24 μg/ml, but three strains required ≥7.8 μg/ml for killing. A concentration of 2 μg of clotrimazole per ml inhibited less than 50% of strains, and 8 μg/ml killed only 10% of strains. Most strains of T. glabrata were killed by therapeutically achievable concentrations of amphotericin B and 5-fluorocytosine, but not clotrimazole.     

Effect of synthetic antifungal drugs on the adherence of Candida albicans to the mouth mucosa epithelium in vitro

Ten Candida albicans strains were tested for their adherence to buccal epithelial cells in vitro in the presence of synthetic antimycotic drugs as 5-fluorocytosine, clotrimazole and ketoconazole. The drugs when applied in therapeutic concentrations inhibited adherence stronger than when applied in subinhibitory concentrations although in both situations the inhibition was statistically significant in comparison to control experiments without these drugs (p less than 0.01). The highest inhibition of adherence was seen with 5-fluorocytosine and the weakest with clotrimazole. Out of imidazole derived drugs strong inhibition of adherence was achieved with ketoconazole.     

Sensitivity of fungi of the genus Candida to antifungal drugs

Seventy two Candida strains isolated from patients with candidiosis of the oral mucosa were studied with respect to their sensitivity to nystatin, levorin, decamine, ethonium, sanguiritrin and clotrimazole. At concentrations of 0.5 to 5 micrograms/ml all the Candida species i.e. C. albicans, C. tropicalis, C. krusei and C. quilliermondii were highly sensitive to clotrimazole. Fungistatic action of levorin, nystatin and sanguiritrin was observed in 91, 67 and 38 per cent of the strains respectively. The Candida strains were resistant to decamine and ethonium used in the above concentrations.     

Influence of incubation time in the in vitro antifungal activity of terbinafine against Trichophyton rubrum.

Antifungal susceptibility tests are influenced by a number of technical variables, including inoculum size, temperature, medium formulation and duration of incubation. In this study, we have compared the in vitro susceptibility of 20 strains de Trichophyton rubrum against clotrimazole and terbinafine, and studied the influence of incubation time on MICs of both drugs. The assay was performed by agar dilution, the medium used was Saboraud glucose agar without an antibiotic. The MIC was evaluated at 15, 30 and 45 days' incubation. The MICs ranges of terbinafine were 0.002 to 0.0975 microg/ml, 0.0975 to 0.39 microg/ml and 0.195 to 0.39 microg/ml at 15, 30 and 45 days' incubation, respectively. The MICs ranges of clotrimazole at 15, 30 and 45 days' incubation were 3.125 to 50 microg/ml. T. rubrum was markedly more susceptible to terbinafine than to clotrimazole (p<0.001). In addition, we observed that an increase of incubation time causing an increase in the MIC value of terbinafine (p<0.001), but MIC values for clotrimazole remained constant with time (p=0.464). In conclusion, the MIC is dependent on reading time and the antifungal compound.     

Determination of biological activity of hydrogel preparations based on ketoconazole and clotrimazole]

Biological activity of original hydrogel preparations based on ketoconazole and clotrimazole was estimated biologically with the 3-dose variant of the agar-diffusion method. The optimal concentrations of the active substances in the hydrogels were the following: 2% of ketoconazole and 1% for clotrimazole.     

Antifungal susceptibility testing of Trichosporon beigelii to imidazole compounds

Twenty-two strains of Trichosporon beigelii have been tested for susceptibility to imidazole compounds. Ten strains were isolated from untreated genital white piedra lesions and 12 were from the same patients following treatment failure with imidazole compounds. Agar dilution and disk elution methods were compared using two media: yeast nitrogen base and antibiotic assay medium 3 (Difco). Antifungal agents tested were econazole, miconazole, ketoconazole, clotrimazole, and amphotericin B in concentrations of 0.0625-32 micrograms/mL. The most consistent results occurred with antibiotic assay medium 3 and the agar dilution method giving minimal inhibitory concentrations between 0.0625 and 0.25 micrograms/mL. Using yeast nitrogen base agar, minimal inhibitory concentrations were higher ranging from 0.0625 to 2.0 micrograms/mL. End points of growth in the disk elution method were not clearly delineated and ranged from 0.0625 to 8.0 micrograms/mL. The distribution of minimal inhibitory concentrations obtained using different media and methods were compared by chi 2 analysis, and the medium was found to significantly change the minimal inhibitory concentrations. There was no difference in the susceptibility of strains of T. beigelii to imidazole compounds whether isolated before or after treatment. It was concluded that in vitro susceptibility of T. beigelii to imidazole compounds did not necessarily predict efficacy in vivo.     

Preparation and in vitro evaluation of liposomal/niosomal delivery systems for antifungal drug clotrimazole

Clotrimazole, an imidazole derivative antifungal agent is widely used for the treatment of mycotic infections of the genitourinary tract. In order to develop alternative formulation for the vaginal administration of clotrimazole to provide sustained and controlled release of appropriate drug for local vaginal therapy, liposomes/niosomes were evaluated as delivery vehicles. To optimize the preparation of liposomes/niosomes with regards to size and entrapment efficiency, multilamellar liposomes/niosomes containing drug were prepared by lipid hydration method. The ability of the systems to deliver clotrimazole into and through the mucosa was evaluated in vitro using rabbit vaginal mucosa with vertical Franz diffusion cells. The in vitro permeation data showed that the liposomes/niosomes system increased the clotrimazole total penetration through the vaginal mucosa by 1.6, 1.5-fold, the accumulation of clotrimazole into the mucosa was increased by 3.1, 2.3-fold, respectively, as compared with control during 24 hr. These results suggest that the studied liposomes/niosomes systems may be appropriate vesicles for the vaginal mucosa delivery of clotrimazole for local vaginal therapy.     

Clotrimazole preferentially inhibits human breast cancer cell proliferation, viability and glycolysis

Background

Clotrimazole is an azole derivative with promising anti-cancer effects. This drug interferes with the activity of glycolytic enzymes altering their cellular distribution and inhibiting their activities. The aim of the present study was to analyze the effects of clotrimazole on the growth pattern of breast cancer cells correlating with their metabolic profiles.

Methodology/Principal Findings

Three cell lines derived from human breast tissue (MCF10A, MCF-7 and MDA-MB-231) that present increasingly aggressive profiles were used. Clotrimazole induces a dose-dependent decrease in glucose uptake in all three cell lines, with K_i values of 114.3±11.7, 77.1±7.8 and 37.8±4.2 µM for MCF10A, MCF-7 and MDA-MB-231, respectively. Furthermore, the drug also decreases intracellular ATP content and inhibits the major glycolytic enzymes, hexokinase, phosphofructokinase-1 and pyruvate kinase, especially in the highly metastatic cell line, MDA-MB-231. In this last cell lineage, clotrimazole attenuates the robust migratory response, an effect that is progressively attenuated in MCF-7 and MCF10A, respectively. Moreover, clotrimazole reduces the viability of breast cancer cells, which is more pronounced on MDA-MB-231.

Conclusions/Significance

Clotrimazole presents deleterious effects on two human breast cancer cell lines metabolism, growth and migration, where the most aggressive cell line is more affected by the drug. Moreover, clotrimazole presents little or no effect on a non-tumor human breast cell line. These results suggest, at least for these three cell lines studied, that the more aggressive the cell is the more effective clotrimazole is.     

Clinical and experimental keratitis caused by the Colletotrichum state of Glomerella cingulata and Acrophialophora fusispora

Two cases of mycotic keratitis caused by the Colletotrichum state of Glomerella cingulata and Acrophialophora fusispora are reported for the first time. Both the isolates produced experimental corneal lesions in rabbit eyes but A. fusispora was more pathogenic. The experimental infection was more severe, with both the fungi, in rabbits pretreated with cortisone as compared with untreated animals. In vitro A. fusispora was most sensitive to miconazole and tolciclate followed by clotrimazole, amphotericin B and lactones while clotrimazole exerted maximum inhibitory effect on Colletotrichum followed by miconazole, lactones, amphotericin B and arnebins. Arnebins and tolciclate were inactive respectively against A. fusispora and Colletotrichum. Of the 3 drugs tested in vivo, against A. fusispora keratitis in rabbit, amphotericin B showed better results than tolciclate and miconazole.     

Sensitivity of scopulariopsis brevicaulis to some antimicrobial agents

Sensitivity tests were done against two isolates of Scopulariopsis brevicaulis, using amphotericin B in combination with chloramphenicol, Chloramphenicol alone, amphotericin B in combination with 5-fluorocytosine, and 5-fluorocytosine, myxin and clotrimazole alone. Results indicated that the effectiveness of amphotericin B was improved in the presence of chloramphenicol or 5-fluorocytosine. Growth inhibitory values recorded for chloramphenicol alone and combined with amphotericin B did not show much variation. Resistance of the fungus has been noticed to 5-fluorocytosine; but the organism's response was much better when tested against 5-fluorocytosine in the presence of amphotericin B. Both myxin and clotrimazole proved very effective and their ED50 was 50 and 2.5 ug/ml of the medium, respectively. Thus, clotrimazole may be the drug of choice in the cases of deep scopulariopsis.     

Chemotherapeutic Evaluation of Clotrimazole [Bay b 5097, 1 (o-Chloro-α-α-Diphenylbenzyl) Imidazole]

Clotrimazole has a broad spectrum of activity against yeast and filamentous fungi in vitro and also in vivo when given orally or parenterally to experimentally infected mice and when administered orally or topically to infected guinea pigs. In vitro a distinct inoculum effect has been observed with a number of strains of Candida and Torulopsis; minimal inhibitory concentrations have tended to increase with increased incubation time. With prolonged incubation times, resistance can be developed to clotrimazole in vitro, but this resistance is readily reversible upon passage in drug-free broth. The degree of in vivo activity of clotrimazole against Candida depends on the severity of infection used. Orally it appears to be more effective when administered by gavage than when given mixed in the diet. Pretreatment with the agent may decrease its efficacy because of drug inactivation. Against dermatophytes, clotrimazole has a degree of activity similar to griseofulvin when given orally, but it is less active than tolnaftate topically in cutaneous infection of Trichophyton mentagrophytes in guinea pigs. In vitro, but not in vivo, some gram-positive and gram-negative bacteria are inhibited by low concentrations of clotrimazole.     

Clotrimazole as an amplifier of hemin-induced damage to human erythrocytes. Antioxidant-independent protective effects of flavonoids

This study is focused on the effect of the antifungal drug clotrimazole (CLT), also possessing anti-malarial and anticancer activities, on hemin-induced hemolysis and changes in ion permeability and filter-ability of human erythrocytes. In the presence of 10 μM clotrimazole, the hemolytic response of erythrocytes to exogenous hemin at concentrations as low as 2–8 μM was considerably potentiated and their filterability, as measured by passing them through a 5-μm nuclepore filter, dropped sharply. Flavonoids quercetin (Q) and taxifolin (DHQ), unlike the standard antioxidant Trolox, abolished the effects of clotrimazole, suggesting that protection of hemin-treated erythrocytes by flavonoids is not related to their antioxidant properties.     

短期联合应用灰黄霉素、克霉唑和地塞米松对红色毛癣菌的影响

目的研究灰黄霉素、克霉唑及灰黄霉素、克霉唑与地塞米松组合成的联合药物对红色毛癣菌在体内、外活性影响。方法在体外分别应用灰黄霉素、地塞米松、克霉唑、灰黄霉素：克霉唑：地塞米松=5：5：1联合药物的不同药物浓度作用于红色毛癣菌,确立药物最小抑菌浓度（MIC）和联合药物的分数抑菌浓度（FIC）;制作由红色毛癣菌引起皮肤癣的豚鼠动物模型,根据豚鼠用药部位分成四组,对豚鼠皮疹进行疗效的评估。结果体外实验联合药物的MIC小于单药的MIC,且联合药物起协同和次协同作用;体内实验中联合用药组对动物受损局部皮肤给药后,对红色毛癣菌所致皮损积分为（1．89±0．68）,与对照组（3．33±0．69）比较,可明显减轻局部皮肤损伤病变程度,该药优于克霉唑组（2．33±0．69）、灰黄霉素组（2．11±0．58）。结论短期内灰黄霉素、克霉唑与地塞米松组合成的联合药物有较强的抗红色毛癣菌和抗炎作用,其抗菌活性强于单独药物的灰黄霉素和克霉唑。     

Effects of imidazole derivatives on cytochromes P450 from human hepatocytes in primary culture.

The expression of several forms of cytochrome P450 including P450 1A2, 2D6, 2E1, and 3A was investigated in human hepatocytes maintained in primary culture for 96 h in the absence or presence of 50 microM of various imidazole derivatives. These included ketoconazole, clotrimazole, miconazole, fluconazole, secnidazole and metronidazole. In addition, the typical inducers rifampicin and beta-naphthoflavone were used for comparison. Western and Northern blot analysis of microsomes and RNA prepared from these cultures as well as de novo synthesis experiments revealed that, among the imidazole derivatives tested, only clotrimazole was a strong rifampicin-like inducer of P450 3A. The expression of the other forms of P450 tested was not affected by the treatments. Analysis of the inhibition of 13 monoxygenase activities, including ethoxyresorufin and phenacetin O-deethylases, coumarin 7 alpha-, lauric acid 11- and 12-, mephenytoin 4-, debrisoquin 4-, and aniline hydroxylases, benzphetamine, aminopyrine, mephenytoin and erythromycin demethylases, and cyclosporin oxidase (representative of 10 different forms of P450 in human liver microsomes) revealed that ketoconazole was a strong and selective in vitro inhibitor of P450 3A (cyclosporin oxidase) with a Ki less than 1 microM. Clotrimazole and miconazole were also strong inhibitors of P450 3A-mediated activities in contrast to the other imidazole derivatives.     

Complete inhibition of the Pdr5p multidrug efflux pump ATPase activity by its transport substrate clotrimazole suggests that GTP as well as ATP may be used as an energy source

The yeast Pdr5p transporter is a 160 kDa protein that effluxes a large variety of xenobiotic compounds. In this study, we characterize its ATPase activity and demonstrate that it has biochemical features reminiscent of those of other ATP-binding cassette multidrug transporters: a relatively high Km for ATP (1.9 mM), inhibition by orthovanadate, and the ability to specifically bind an azidoATP analogue at the nucleotide-binding domains. Pdr5p-specific ATPase activity shows complete, concentration-dependent inhibition by clotrimazole, which is also known to be a potent transport substrate. Our results indicate, however, that this inhibition is noncompetitive and caused by the interaction of clotrimazole with the transporter at a site that is distinct from the ATP-binding domains. Curiously, Pdr5p-mediated transport of clotrimazole continues at intracellular concentrations of substrate that should eliminate all ATPase activity. Significantly, however, we observed that the Pdr5p has GTPase and UTPase activities that are relatively resistant to clotrimazole. Furthermore, the Km(GTPase) roughly matches the intracellular concentrations of the nucleotide reported for yeast. Using purified plasma membrane vesicles, we demonstrate that Pdr5p can use GTP to fuel substrate transport. We propose that Pdr5p increases its multidrug transport substrate specificity by using more than one nucleotide as an energy source.     

用聚类法分析受抗真菌物质处理后的酵母细胞全基因表达谱

微阵列DNA芯片技术可以并行分析成千上万个基因的表达情况,它为研究药物的作用机制提供了一个新的高效技术平台.用9种已知和未知作用机理的抗真菌化合物处理酵母细胞,并得到酵母细胞的全基因表达谱,然后对其进行聚类分析.结果表明作用机制类似的化合物具有相近的聚类关系.两性霉素B和制霉菌素、酮康唑和克霉唑都是已知的作用机制类似的抗真菌药物.通过对基因表达谱进行聚类分析,发现前一组和后一组分别被聚类在一起.另外已知澳洲茄胺抑制的是细胞膜上麦角固醇的合成,聚类分析表明它与酮康唑,克霉唑的聚类很靠近.对微阵列DNA芯片产生的基因表达谱进行聚类分析,由于作用机制相似的药物会被聚类在一起,因此根据未知药物和已知药物的聚类关系,可以了解未知药物的作用机制,这对于加速新药开发的步伐具有十分重要的意义.     

Comparison of terbinafine and clotrimazole in treating tinea pedis.

OBJECTIVE--To compare the efficacy and safety of terbinafine 1% cream and clotrimazole 1% cream in the treatment of tinea pedis. DESIGN--Multicentre, double blind parallel group study. SETTING--32 general practices and one hospital. PATIENTS--256 patients with mycologically confirmed tinea pedis. Of the 211 patients evaluable, 107 were randomised to terbinafine (75 male, 32 female; mean (range) age 40 (12-81) years) and 104 to clotrimazole (79 male, 25 female; mean (range) age 36 (12-71) years). INTERVENTIONS--Terbinafine 1% cream applied twice daily for one week and inert cream applied twice daily for the next three weeks. Clotrimazole 1% cream applied twice daily for four weeks. MAIN OUTCOME MEASURES--Mycological cure (negative results on microscopy and culture) and effective treatment (mycological cure plus no or minimal signs and symptoms) measured at weeks 1, 2, 3, 4, and 6. RESULTS--At week four rates of mycological cure were 93.5% for terbinafine and 73.1% for clotrimazole (p = 0.0001); and at week six 97.2% for terbinafine and 83.7% for clotrimazole (p = 0.001). Rates of effective treatment at week 4 were 89.7% for terbinafine and 58.7% for clotrimazole (p = 0.0001); and 89.7% for terbinafine and 73.1% for clotrimazole (p = 0.002) at week 6. CONCLUSION--These results indicate that a one week course of terbinafine 1% cream is more effective in the treatment of tinea pedis than a four week course of clotrimazole 1% cream, both in terms of mycological cure and effective treatment.     

Synthesis and in vitro activity of 2-thiazolylhydrazone derivatives compared with the activity of clotrimazole against clinical isolates of Candida spp

In this paper, we report on the synthesis of a novel series of 2-thiazolylhydrazone derivatives and the influence of the substituents on the thiazole ring on antifungal activity. All synthesized compounds were screened for their in vitro activities against 22 clinical isolates of Candida spp., representing six different species, compared to clotrimazole as a reference compound. Some of the tested compounds were found to possess significant antifungal activity when compared to clotrimazole, in particular compound 14 which exhibited higher potency against most of the Candida spp. considered. The compounds that were most active as anti-Candida agents were also submitted to cytotoxic screening by the Trypan Blue dye exclusion assay and in general they were shown to induce low cytotoxic effects.     

Liquid chromatographic determination of ketoconazole, a potent inhibitor of CYP3A4-mediated metabolism

A high-performance liquid chromatographic assay with UV detection has been developed for the determination of ketoconazole in human plasma. Quantitative extraction was achieved by a single solvent extraction involving a mixture of acetonitrile–n-butyl chloride (1:4, v/v). Ketoconazole and the internal standard (clotrimazole) were separated on a column packed with Inertsil ODS-80A material and a mobile phase composed of water–acetonitrile–tetrahydrofuran–ammonium hydroxide–triethylamine (45:50.2:2.5:0.1:0.1, v/v). The column effluent was monitored at a wavelength of 206 nm with a detector range set at 0.5. The calibration graph was linear in the range of 20–2000 ng/ml, with a lower limit of quantitation of 20.0 ng/ml. The extraction recoveries for ketoconazole and clotrimazole in human plasma were 93±9.7% and 83±10.0%, respectively. The developed method has been successfully applied to a clinical study to examine the pharmacokinetics of ketoconazole in a cancer patient.     

Broth dilution and disc diffusion methods in the susceptibility testing of pathogenic Candida albicans against four antimycotics

Susceptibility of Candida albicans isolated from mouthrinse specimens during episodes of acute pseudomembranous candidiasis in patients with haematological malignancies was tested by the broth dilution and disc diffusion methods using 24 and 48 h of incubation. The time factor did not significantly affect the results with 5-fluorocytosine. With amphotericin B, prolonged incubation doubled the geometric mean MIC of C. albicans as well as the number of isolates with intermediate sensitivity. With the shorter incubation in the disc diffusion assay, few isolates showed lowered sensitivity to clotrimazole; at 48 h, however, this figure was as high as 54%. The yeasts were highly sensitive to ketoconazole at 24 h, whereas at 48 h the results were bizarre. At 24 h, correlations between disc diffusion and broth dilution methods were satisfactory, with clotrimazole and ketoconazole accounting for most of the discordancies. Accordingly, the disc diffusion results should be recorded at 24 h.