Carboxylic acid and phosphate ester derivatives of fluconazole: synthesis and antifungal activities

Two classes of fluconazole derivatives, (a) carboxylic acid esters and (b) fatty alcohol and carbohydrate phosphate esters, were synthesized and evaluated in vitro against Cryptococcus neoformans, Candida albicans, and Aspergillus niger. All carboxylic acid ester derivatives of fluconazole (1a-l), such as O-2-bromooctanoylfluconazole (1g, MIC=111 microg/mL) and O-11-bromoundecanoylfluconazole (1j, MIC=198 microg/mL), exhibited higher antifungal activity than fluconazole (MIC > or = 4444 microg/mL) against C. albicans ATCC 14053 in SDB medium. Several fatty alcohol phosphate triester derivatives of fluconazole, such as 2a, 2b, 2f, 2g, and 2h, exhibited enhanced antifungal activities against C. albicans and/or A. niger compared to fluconazole in SDB medium. For example, 2-cyanoethyl-omega-undecylenyl fluconazole phosphate (2b) with MIC value of 122 microg/mL had at least 36 times greater antifungal activity than fluconazole against C. albicans in SDB medium. Methyl-undecanyl fluconazole phosphate (2f) with a MIC value of 190 microg/mL was at least 3-fold more potent than fluconazole against A. niger ATCC 16404. All compounds had higher estimated lipophilicity and dermal permeability than those for fluconazole. These results demonstrate the potential of these antifungal agents for further development as sustained-release topical antifungal chemotherapeutic agents.     

Effect of iron on fluconazole activity against Candida albicans in presence of human serum or monocyte-derived macrophages

Human serum, transferrin, and apotransferrin are known to profoundly inhibit the growth of Candida albicans by iron deprivation. On the other hand, iron overload (iron saturated transferrin) is a serious risk factor for candidiasis in newborn and in leukemic patients. We tested the efficacy of fluconazole and the previously demonstrated synergy of fluconazole and effector cells against C. albicans under iron overload conditions where efficacy might be diminished. We confirm that exogenous iron completely reversed the inhibitory effect of human serum and report that the efficacy of fluconazole against C. albicans was not significantly compromised in a 24 h assay system. Although exogenous iron inhibited fungistatic activity of monocyte-derived macrophages, it did not interfere with the synergistic candidacidal activity of fluconazole and monocyte-derived macrophages. In 72 h assays, where fluconazole had candidacidal activity, exogenous iron did not compromise efficacy of fluconazole, and fluconazole activity was often increased. These in vitro results suggest that effectiveness of fluconazole therapy would not be compromised in iron overload situations in vivo. This revised version was published online in August 2006 with corrections to the Cover Date.     

氯喹与氟康唑联合对新生隐球菌的体外药敏实验

目的研究氯喹与氟康唑体外联合应用对隐球菌生长作用的影响。方法参考M27-A方案,检测10μmol/L,100μmol/L与1 000μmol/L 3种浓度氯喹与氟康唑联合后对20株菌株的氟康唑对隐球菌最小抑菌浓度与最小杀菌浓度的变化。结果与10μmol/L氯喹组相比,100μmol/L以上浓度的氯喹组可以显著降低氟康唑对隐球菌的M IC与MFC。结论氯喹在体外可以提高氟康唑抗隐球菌的作用,与氟康唑具有协同抗隐球菌的作用。     

白念珠菌SSK1突变株对氟康唑敏感性的初步研究

目的探讨氟康唑作用于白念珠菌双组分信号传导途径SSK1突变株SSK21后药物敏感性的变化。方法采用微量液体稀释法和固醇测定法测定野生株(CAF2-1)和突变株(SSK21)的最小抑菌浓度(MIC);并应用RT-PCR观察氟康唑作用前后,SSK1的表达变化。结果氟康唑对CAF2-1的MIC为16μg/mL,对SSK21为0.032μg/mL。加入氟康唑后,CAF2-1的SSK1表达明显增加,60min时达到最多。结论 SSK21对氟康唑高度敏感,SSK1基因及其相关的重要基因与药物敏感性的关系值得进一步研究,从而为新的抗真菌药物和治疗途径的研发提供参考。     

Diminution of antifungal activity of fluconazole associated with ibuprofen and piroxicam in experimental histoplasmosis of hamsters (Mesocricetus auratus)

The aim of this study was to determine if tha association of non-steroid antiinflammatory drugs (piroxicam and ibuprofen) with fluconazole, affects the antifungal activity of the azole compound, in an experimental model histoplasmosis in hamsters (Mesocricetus auratus). Sixty hamsters were intracardially inoculated with 4x10(6) yeasts of Histoplasma capsulatum var. capsulatum. Treatments began one week after the challenge and continued for three weeks. The hamsters were divided in six groups of ten animals each and received the following treatment: 1- fluconazole 8 mg/kg/day; 2- ibuprofen 20 mg/kg/day; 3- piroxicam 20 mg/kg/day; 4- fluconazole+ibuprofen; 5- fluconazole+piroxicam and 6- only received the solvent of these drugs. One week after ending the treatment, all the animals were sacrified and the evaluation of the treatments was based on the results of blood cultures, on the determination of colony forming units per gram of spleen, and the histopathologic studies of the same organ. The animals treated with fluconazole plus ibuprofen or piroxicam showed more colony colony forming units per gram (3.9x10(7) and 3.3x10(7)) when compared with the animals treated with fluconazole alone (0.9x10(7)). The histopathologic results of the hamsters that received fluconazole showed well-organized granulomas with few yeast-like elements inside the macrophages. In contrast, those which received fluconazole associated with antiinflammatory drugs presented lax granulomas containing numerous yeast-like elements. These findings let us to conclude that non-steroids antiinflammatory drugs diminish the antifungal efficacy of fluconazole in this animal model.     

Silver nanoparticles,a promising treatment against clinically important fluconazole-resistant Candida glabrata

Resistance to azole antifungal agents is a challenging limitation in Candida glabrata treatment. It is associated with decreased intracellular concentrations of antifungal agents as a result of overexpression of efflux pumps on the cellular plasma membranes. This work evaluates the potential of silver nanoparticles (AgNPs) to reverse the resistance of fungal cells to fluconazole. Silver nanoparticles were prepared using wet chemical method and characterised by UV-Vis spectrophotometry, dynamic light scattering, and zeta potential. Broth microdilution and pour plates methods were used to study the anticandidal activity using two C. glabrata fluconazole-resistant strains (DSY565 and CBS138) known to overexpress active efflux pumps, and a standard fluconazole sensitive strain ATCC 22553. Silver nanoparticles–fluconazole combinations decreased concentrations of fluconazole substantially without compromising the activity. These findings suggest that AgNPs enhance the efficacy of fluconazole and offer a promising application in therapy of C. glabrata infections.     

Evolution of drug resistance in experimental populations of Candida albicans

Adaptation to inhibitory concentrations of the antifungal agent fluconazole was monitored in replicated experimental populations founded from a single, drug-sensitive cell of the yeast Candida albicans and reared over 330 generations. The concentration of fluconazole was maintained at twice the MIC in six populations; no fluconazole was added to another six populations. All six replicate populations grown with fluconazole adapted to the presence of drug as indicated by an increase in MIC; none of the six populations grown without fluconazole showed any change in MIC. In all populations evolved with drug, increased fluconazole resistance was accompanied by increased resistance to ketoconazole and itraconazole; these populations contained ergosterol in their cell membranes and were amphotericin sensitive. The increase in fluconazole MIC in the six populations evolved with drug followed different trajectories, and these populations achieved different levels of resistance, with distinct overexpression patterns of four genes involved in azole resistance: the ATP-binding cassette transporter genes, CDR1 and CDR2; the gene encoding the target enzyme of the azoles in the ergosterol biosynthetic pathway, ERG11; and the major facilitator gene, MDR1. Selective sweeps in these populations were accompanied by additional genomic changes with no known relationship to drug resistance: loss of heterozygosity in two of the five marker genes assayed and alterations in DNA fingerprints and electrophoretic karyotypes. These results show that chance, in the form of mutations that confer an adaptive advantage, is a determinant in the evolution of azole drug resistance in experimental populations of C. albicans.     

Characterization of mechanisms of fluconazole resistance in a Candida albicans isolate from a Japanese patient with chronic mucocutaneous candidiasis

We examined the mechanisms of fluconazole resistance in a fluconazole-resistant Candida albicans isolate from a Japanese patient with chronic mucocutaneous candidiasis. It was demonstrated that the highly resistant phenotype of this strain was associated with combined mechanisms of the energy-dependent reduced intracellular accumulation of fluconazole, presumably due to the increased expression of the ATP-binding cassette efflux pump CDR gene(s), and the reduced affinity of the target enzyme, Erg11p, to fluconazole. In particular, the reduced affinity of Erg11p was considered to contribute largely to the fluconazole resistance in the TIMM3209 strain. Biochemical studies indicated that the Erg11p from the TIMM3209 strain showed reduced susceptibility both to fluconazole and itraconazole of cell-free ergosterol biosynthesis, and cytochrome P-450 also showed reduced affinity to fluconazole in the carbon monoxidecytochrome P-450 complex formation assay. We identified two amino acid substitutions, Y132H and G448V, in Erg11p from the TIMM3209 strain. We found that the cytochrome P-450 from the TIMM3209 strain decayed during incubation at 37 C without fluconazole although it is unknown whether or not the phenomenon is linked to the resistant phenotype. These mutations are thought to confer the above-mentioned characteristics to Erg11p.     

In vitro activity of fluconazole and amphotericin B against Candida inconspicua clinical isolates as determined by the time-kill method

Candida inconspicua is an emerging pathogen in immunocompromised patients possessing inherently decreased susceptibility to fluconazole. We determined the MICs and killing activity of fluconazole and amphotericin B against C. inconspicua clinical isolates as well as reference strain C. inconspicua ATCC 16783 for comparison. MICs were determined using the standard broth microdilution method. Killing rates were determined using time-kill methodology at 0.5-16 x MIC fluconazole and amphotericin B concentrations. Fluconazole and amphotericin B MIC values varied between 16-128 mg/l and 0.5-1 mg/l, respectively. In time kill-assays fluconazole showed fungistatic effect at 1-16 x MIC concentrations against all tested strains after 24 h-incubation, but became fungicidal after 48 h at 4-16 x MIC concentrations. The time necessary to achieve fungicidal endpoint at 1 mg/l amphotericin B concentration ranged from 2 to 24 h. Our in vitro results confirm the data that fluconazole is ineffective against C. inconspicua at the fluconazole serum concentration attainable in humans. Amphotericin B due to its rapid killing activity seems to be a good alternative for the treatment of infections caused by C. inconspicua.     

Proliferation of intracellular structure corresponding to reduced affinity of fluconazole for cytochrome P-450 in two low-susceptibility strains of Candida albicans isolated from a Japanese AIDS patient

Three Candida albicans isolates, TIMM 3164, 3165 and 3166 with reduced fluconazole susceptibility, were isolated from two Japanese AIDS patients. We earlier reported that a reduced intracellular accumulation of fluconazole in these isolates played an important role in the resistance mechanism of fluconazole, but we did not exclude the involvement of other factors. We here examined characteristics related to cytochrome P-450 (CYP), especially sterol 14alpha-demethylase encoded by the ERG11 gene which is the target molecule for fluconazole. In TIMM 3164 and 3165, the ergosterol synthesis by cell-free extracts was somewhat less susceptible to fluconazole, due to a decrease in fluconazole affinity for CYP. The nucleotide substitutions in the ERG11 gene were identified to result in three amino acid changes of K143R, E266D and V488I in TIMM 3164, and of E266D, V404L and V488I in TIMM 3165. These amino acid substitutions might contribute to the decreased affinity for CYP in both isolates. However, a single amino acid change, E266D, observed in TIMM 3166 was unrelated to the decreased affinity for CYP. The most prominent finding on the ultrastructure of TIMM 3164 and 3165 was the development of mesh membrane structures of the endoplasmic reticula, which is a location related to sterol synthesis. This phenomenon was not observed in the cells of TIMM 3166 or the susceptible control strains of ATCC 90028 and 10231. In addition to the reduced intracellular accumulation, the decreased affinity of fluconazole for CYP in TIMM 3164 and 3165 is assumed to be associated with the fluconazole-resistance phenotype.     

A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient

A series of 10 strains of Candida albicans, from TIMM 3309 to TIMM 3318, were repeatedly isolated in one myelofibrosis-complicated patient with recurrent candidemia. The latter five isolates, from TIMM 3314 to TIMM 3318, became suddenly resistant to fluconazole during the 10 to 16 weeks after antimycotic therapy. We investigated the resistant mechanism of fluconazole using one susceptible isolate and two of the five resistant isolates in the series. The ergosterol synthesis by cell-free extracts from the two resistant isolates was less susceptible to fluconazole partly as a result of a decreased affinity of cytochrome P-450. Unexpectedly, these two resistant isolates showed higher levels of an intracellular accumulation of [H]fluconazole than the susceptible isolate and the control strain of C. albicans ATCC 10231. In the resistant isolate, TIMM 3318, most intracellular incorporated fluconazole was distributed in the 12,000 X g pellet (P-120) fraction by centrifugation unlike the two susceptible strains. An observation of the ultrastructure of TIMM 3318 showed the most notable alteration to be the characteristic appearance of numerous vesicular vacuoles (diameter, 150 to 400 nm); these vacuoles were not observed, however, in either of the susceptible strains. A direct observation of the subcellular fraction prepared from TIMM 3318 by the electron microscopy negative-staining method suggests that most of the vesicular vacuoles were recovered in the P-120 fraction. These results suggest that fluconazole sequestration caused by vesicular vacuoles of the resistant isolate might act as a novel mechanism of fluconazole resistance besides the decreased affinity of cytochrome P-450.     

两种抗真菌药治疗甲癣成本效果分析

目的：对特比萘芬、氟康唑2种不同抗真菌药物治疗60例甲癣病患者的成本效果分析。方法：选择60例病例,随机分为2组。分别给予特比萘芬、氟康唑治疗,观察各组疗效并运用成本效果法进行分析。结果：2种药物均有较好的抗真菌疗效,特比萘芬疗效优于氟康唑,但氟康唑最具有成本效果。结论：药物经济学分析结果为氟康唑治疗甲癣病较优。     

氟康唑对热带念珠菌活性氧和线粒体膜电位的影响

为了探讨氟康唑作用机制,观察它对热带念珠菌作用后存活率、活性氧(Reactive oxygen species,ROS)、线粒体膜电位(Mitochondrial membrane potential,△Ψm)和细胞周期的变化。参照NCCLS M27-A方案的微量稀释法测定氟康唑对热带念珠菌的最低抑菌浓度(MIC);热带念珠菌与不同浓度氟康唑共同培养后用流式细胞术(Flow cytometry,FCM)分析热带念珠菌存活率、ROS、线粒体膜电位△Ψm和细胞周期的变化。结果表明,氟康唑作用后,热带念珠菌氟康唑耐药株的存活率、ROS、线粒体膜电位△Ψm和细胞周期各期比例均没有明显变化;而热带念珠菌氟康唑敏感株的存活率和线粒体膜电位△Ψm明显下降,ROS明显升高,而且大部分热带念珠菌阻滞于G2/M期,并出现明显凋亡峰,呈一定的时间剂量依赖关系。自由基清除剂谷胱甘肽抑制热带念珠菌ROS的产生,阻止细胞周期G2/M期阻滞和降低凋亡。由此可见,氟康唑可能通过刺激热带念珠菌产生过多ROS,并使线粒体膜电位△Ψm下降,从而诱导热带念珠菌凋亡。     

Calcineurin A of Candida albicans: involvement in antifungal tolerance,cell morphogenesis and virulence

The azole antifungal fluconazole possesses only fungistatic activity in Candida albicans and, therefore, this human pathogen is tolerant to this agent. However, tolerance to fluconazole can be inhibited when C. albicans is exposed to fluconazole combined with the immunosuppressive drug cyclosporin A, which is known to inhibit calcineurin activity in yeast. A mutant lacking both alleles of a gene encoding the calcineurin A subunit (CNA) lost viability in the presence of fluconazole, thus making calcineurin essential for fluconazole tolerance. Consistent with this observation, tolerance to fluconazole was modulated by calcium ions or by the expression of a calcineurin A derivative autoactivated by the removal of its C-terminal inhibitory domain. Interestingly, CNA was also essential for tolerance to other antifungal agents (voriconazole, itraconazole, terbinafine, amorolfine) and to several other metabolic inhibitors (caffeine, brefeldin A, mycophenolic acid, fluphenazine) or cell wall-perturbing agents (SDS, calcofluor white, Congo red), thus indicating that the calcineurin pathway plays an important role in the survival of C. albicans in the presence of external growth inhibitors. Several genes, including PMC1, a vacuolar calcium P-type ATPase, were regulated in a calcineurin- and fluconazole-dependent manner. However, PMC1 did not play a direct role in the survival of C. albicans when exposed to fluconazole. In addition to these different properties, calcineurin was found to affect colony morphology in several media known to modulate the C. albicans dimorphic switch. In particular, calcineurin was found to be essential for C. albicans viability in serum-containing media. Finally, calcineurin was found to be necessary for the virulence of C. albicans in a mice model of infection, thus making calcineurin an important element for adequate adaptation to the conditions of the host environment.     

Comparison between efficacy of allicin and fluconazole against Candida albicans in vitro and in a systemic candidiasis mouse model

The efficacy of allicin compared with fluconazole in alleviating systemic Candida albicans infections was evaluated both in vitro and in vivo through a systemic candidiasis mouse model. Determination of in vitro minimum inhibitory concentrations (MICs) for different C. albicans isolates revealed that both allicin and fluconazole showed different MICs that ranged from 0.05 to 12.5 μg mL(-1) and 0.25 to 16 μg mL(-1) , respectively. A time-kill study showed a significant effect of allicin (P<0.01) against C. albicans, comparable to that of fluconazole. Scanning electron microscopy observation revealed that, similar to fluconazole, allicin produced structural destruction of C. albicans cell surface at low MIC and lysis or puncture at high MIC concentrations. Treatment of BALB/c mice systemically infected with C. albicans showed that although the allicin treatment (at 5 mg kg(-1) day(-1) ) was slightly less efficacious than fluconazole treatment in terms of the fungal load reduction and host survival time, it was still effective against C. albicans in terms of mean survival time, which increased from 8.4 to 15.8 days. These results demonstrate the efficacy of anticandidal effects of allicin both in vitro and in an animal model of candidiasis and affirm the potential of allicin as an adjuvant therapy to fluconazole.     

Search for novel antifungal agents by monitoring fungal metabolites in presence of synthetically designed fluconazole derivatives using NMR spectroscopy

Resistance to currently available antifungal drugs necessitates development of new drugs using rapid, robust and automated methods to test a large number of newly synthesized drugs in less time. We have compared the effect of ketoconazole, fluconazole and its synthesized analogues on Candida albicans ATCC 10231. A metabolic profile of C.albicans ATCC 10231 in presence of drugs has been compared using ¹H NMR. Signals from metabolites have been monitored with time. MIC determined using conventional methods has been compared with Metabolic End Point (MEP) obtained from NMR spectroscopy. Results indicate that the activity of the fluconazole derivatives is in the order fluconazole p-methoxybenzoate > fluconazole = fluconazole benzoate > fluconazole toluate > fluconazole p-nitrobenzoate.     

The First Isolation of Cryptococcus neoformans from Eucalyptus Trees in South Aegean and Mediterranean Regions of Anatolia in Turkey despite Taurus Mountains alkalinity

Between April 2001 and April 2002 were studied 106 women with a clinical diagnosis of vaginal candidiasis seen at the Gynecology and Obstetrics Ambulatory of the Hospital das Clínicas da Universidade Federal de Goiás. The patients were assessed on two occasions, before starting treatment with itraconazole or fluconazole (initial visit) and 14 days after treatment (return). At two visits the signs and symptoms were recorded and vaginal secretion was collected. According to the clinical evaluation, itraconazole was effective in 64.3%, while fluconazole was effective in 71.0% of the patients. The mycological cure rates (negative culture) in the return were 64.3% for the patients treated with itraconazole and 78.9% for the patients treated with fluconazole. The MICs of itraconazole and fluconazole for 80 Candida isolates were determined by Etest method. We investigated the correlation between in vitro susceptibility (Susceptible, Susceptibility Depending Dose and Resistant) to itraconazole and fluconazole with clinical outcome of the patients. The success rates were 63.9% for itraconazole and 90.6% for fluconazole in the susceptible category, 100.0% for both drugs in the susceptible dose dependent category, and 0.0% for both drugs in the resistant category. Our results showed there were a positive correlation between in vitro susceptibility test results with clinical outcome in vaginal Candida infections and that both drugs might be one choice in the treatment of vaginal candidiasis.