Reduced toxicity of amphotericin B methyl ester (AME) vs. amphotericin B and fungizone in tissue culture

Summary The comparative toxicities of amphotericin B methyl ester (AME), the parent antibiotic amphotericin B (AB), and the deoxycholate solubilized complex of AB, Fungizone² (FZ), toward five cell lines has been determined as measured by early membrane damage (⁵¹Cr release), 24 hr survival, 72 hr viability, and growth rate. Cells used were of turtle (TH-1), marsupial (PT K2), human MA 160), rabbit (RK-13) and hamster (BHK-21) origin. AME: (a) caused less membrane damage at 1 hr than AB or FZ; (b) was less toxic than AB or FZ as indicated by 24 hr cell survival and 72 hr cell viability; and (c) was required in higher levels than AB or FZ to reduce the growth rate of all five cell lines. Spectrophotometric analysis of residual polyene levels indicated that AME had good stability in tissue culture medium. Previous studies have indicated that AME has the same in vitro antifungal activity as the parent antibiotic AB (1, 2). These findings suggest that AME may prove to be superior to AB and FZ for use as an antifungal agent in tissue culture systems. Fungizone^R. Trade mark. E. R. Squibb and Sons. This investigation was supported in part by Contract NIH 69-2161, NIH Grant No. AI-02095 and NIH Training Grant No. GM 507 from the National Institute of General Medical Sciences.     

两性霉素B联合伊曲康唑或卡泊芬净治疗恶性血液病并发侵袭性真菌感染

目的观察两性霉素B联合伊曲康唑或卡泊芬净治疗恶性血液病患者并发侵袭性真菌感染(IFI)的临床疗效及安全性。方法收集我院联合治疗患者9例,对其疗效及毒副反应进行分析研究。结果9例患者7例有效,1例无效,1例停药。低钾是最常见的副反应,其他副反应包括畏寒、发热及肝、肾功能受损。结论两性霉素B联合伊曲康唑或卡泊芬净治疗IFI,经济、有效,患者依从性较好。     

米卡芬净对分离自中国的念珠菌和曲霉临床株体外抑菌活性的研究

目的了解新型抗真菌药物米卡芬净(micafungin,MFG)对分离自中国的念珠菌和曲霉临床株的体外抑菌活性。方法参照CLSI(Clinical and Laboratory Standards Institute,以前为NCCLS)制定的M27-A2和M38-A方案测定86株念珠菌和35株曲霉的最低抑菌浓度(MIC)或最低有效浓度(MEC)。结果MFG对大多数念珠菌属和曲霉属均有较好的抑菌作用。对念珠菌属的MIC90从高到低依次为:氟康唑(FLC)敏感的白念珠菌、热带念珠菌、光滑念珠菌为0.125μg/ml,FLC耐药和剂量依赖敏感株为0.25μg/ml,克柔念珠菌为0.5μg/ml,近平滑念珠菌8μg/ml,季也蒙念珠菌>16μg/ml。MFG对烟曲霉的MEC90为≤0.03μg/ml,对非烟曲霉的曲霉属MEC90为0.06μg/ml。MFG与唑类药物、两性霉素B(AMB)不存在交叉耐药,对FLC耐药的念珠菌、伊曲康唑耐药的曲霉、AMB不敏感的曲霉均有好的抑菌活性。结论MFG对多数念珠菌属和曲霉属(包括对唑类耐药和AMB不敏感的菌株)有较好的体外抑菌作用。     

In vitro and in vivo antifungal activities of liposomal amphotericin B,and amphotericin B lipid complex

The in vitro and in vivo antifungal activities of liposomal amphotericin B (L-AMPH) and amphotericin B lipid complex (ABLC), which is composed of amphotericin B and the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol, were compared with those of conventional amphotericin B (Fungizone®, AMPH). The acute intravenous toxicity was markedly lower in BALB/c mice; 50% lethal doses (LD_50s) were 2.75 mg/kg in AMPH, 32.9 mg/kg in L-AMPH and >75 mg/kg in ABLC. In vitro antifungal activities againstCandida albicans, C. parapsilosis, C. tropicalis, C. glabrata, andC. krusei were evaluated by the agar plate dilution method. The activities were unchanged againstC. albicans, but MICs increased more than four fold in 18 of the 20 strains other thanC. albicans in L-AMPH and in 9 of the 20 in ABLC. L-AMPH and ABLC were as efficacious as AMPH in the treatment of mice infected withC. albicans, and at a dose of 0.5 and 1.0 mg/kg of body weight, ABLC was more efficacious on survival. A ten-times larger dose (10 mg/kg) of L-AMPH and ABLC was administered to mice with 100% survival, suggesting improved tolerability as compared to amphotericin B.     

Discrepancies between MIC and MLC values of amphotericin B against isolates ofAspergillus species

There is little information addressing the phenomena of discrepancy between minimal inhibitory concentrations (MIC) and minimal lethal concentrations (MLC) values of amphotericin B (AMB) to clinical isolates of fungi. This study assessed in vitro activity of AMB against 70 clinical isolates of aspergilli: 30 strains ofAspergillus fumigatus, 20 strains ofAspergillus flavus and 20 strains ofAspergillus niger. Susceptibility tests were accomplished using a macro broth dilution procedure, with special emphasis on ascertainment of MLCs. AMB exhibited low MIC values against all clinical isolates. While we did not identify any AMB resistant isolates among 70Aspergillus spp. studied as judged by MIC levels, analysis of the data demonstrated a clear discrepancy between the MIC and MLC levels of AMB obtained against clinical isolates ofAspergillus spp. The MLC values of AMB were significantly higher than the MIC values with MIC 50 and MIC 90 of 0.29 and 0.5 µg/ml, respectively, at the second reading time, and MLC 50 and MLC 90 of 2.31 and 9.24 µg/ml, respectively (p<0.001). Additionally, minimal lethal concentrations in 36/70 (51%) of aspergillal isolates studied produced drug concentrations above those which can usually be sustained in patient plasma or tissue.     

Multicenter evaluation of commercial frozen plates for microdilution broth antifungal susceptibility testing of yeasts and comparison of MIC limits recommended in NCCLS M27-A2

A commercial kit, Frozen Plate for Antifungal Susceptibility Testing of Yeasts, Eiken (Eiken Chemical Co., Ltd., Tokyo), was tested in a multi-institute study to evaluate the agreement between interinstitute MICs and National Committee for Clinical Laboratory Standards (NCCLS) M27-A2 recommendation limits of MIC value. The kit was reported as a method equivalent to the standardized guidelines for antifungal susceptibility testing by the Committee for Clinical Laboratory Standards-1994, the Japanese Society for Medical Mycology, and which is widely used in Japan for amphotericin B, flucytosine, fluconazole, miconazole, and itraconazole. The degrees of inter-institute and NCCLS agreements were good to excellent especially with 48-hr incubation for all antifungal agents. However, the percent agreements to NCCLS recommendations against itraconazole were poor. Overall, MIC values obtained using the frozen plate antifungal susceptibility testing kit, with 48-hr incubation, were thought to be reliable and convenient alternatives to the data obtained by the NCCLS M27-A2 reference macrodilution and microdilution method. This kit will allow matching of results between international laboratories. However, the MIC value for itraconazole requires careful interpretation.     

Investigations on feasibility of in situ development of amphotericin B liposomes for industrial applications

Amphotericin B (AmB) liposome formulations are very successful in the treatment of fungal infections and leishmaniasis. But higher cost limits its widespread use among people in developing countries. Therefore, we have developed a modified ethanol-injection method for the preparation of AmB liposomes. Two liposomal formulations were developed with dimyristoyl phosphatidylcholine [F-1a] and soya phosphatidylcholine [F-2a], along with egg phosphatidyl glycerol and cholesterol. AmB was dissolved in acidified dimethyl acetamide and mixed with ethanolic lipid solution and rapidly injected in 5% dextrose to prepare liposomes. Liposomes were characterized on the basis of size (~100?nm), zeta (–43.3?±?2.8 mV) and percent entrapment efficiency (>95%). The in vitro release study showed an insignificant difference (P?≥?0.05) for 24-hour release between marketed AmB liposomes (AmBisome) and F-1a and F-2a. Proliposome concentrate, used for the preparation of in situ liposomes, was physically stable for more than 3 months at experimental conditions. Similarly, AmB showed no sign of degradation in reconstituted liposomes stored at 2–8°C for more than 3 months. IC₅₀ value of Ambisome (0.18 µg/mL) was comparatively similar to F-1a (0.17 µg/mL) and F-2a (0.16 µg/mL) against intramacrophagic amastigotes. Under experimental conditions, a novel modified method for AmB liposomes is a great success and generates interest for development as a platform technology for many therapeutic drug products.     

Comparative efficacy of fluconazole and amphotericin B in the parenteral treatment of experimental paracoccidioidomycosis in the rat

Patients with severe and complicated paracoccidioidomycosis are treated with amphotericin B by the intravenous route. Fluconazole is active in vitro against Paracoccidioides brasiliensis and can also be administered intravenously, but few clinical or experimental data are available about its action against the infection caused by this fungus. In the present study, the efficacy of fluconazole andamphotericin B was assessed comparatively in rats inoculated parenterally with P. brasiliensis. The treatment was performed 3 times a week for 4 weeks starting one week after infection. Fluconazole administered intraperitoneally (14 mg/kg bodyweight/dose) was more effective (P > 0.001)than amphotericin B (2 mg/kg body weight/dose) in reducing the number of colony forming units in the lungs and spleen. When administered intravenously at the dose of 3 mg/kg body weight, fluconazole was as effective as amphotericin B (0.8 mg/kg body weight) in reducing the pulmonary fungal burden. Under these conditions, the rats treated with fluconazole had a smaller number of colony forming units than untreated animals (P > 0.001), but amphotericin B was more effective than fluconazole in reducing spleen infection (P > 0.005). Except for this result obtained with a low dose, fluconazole showed an antifungal action equal to or higher than that of amphotericin B. The activity of fluconazole at doses equivalent to those used for human treatment suggests that this antifungal agent may be an alternative to amphotericin B for the early intravenous treatment of patients with paracoccidioidomycosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

伊曲康唑联合特比萘芬治疗孢子丝菌病疗效及其对病原真菌体外抗菌活性研究

目的 初步探讨伊曲康唑和特比萘芬联合治疗孢子丝菌病的疗效,评价两药体外联合对申克孢子丝菌菌丝相和酵母相的抗菌活性.方法 口服伊曲康唑200mg/d和特比萘芬250mg/d治疗孢子丝菌病;体外联合药敏试验采用棋盘微量稀释法,计算分数抑菌浓度(FIC)指数判定两药相互作用具有协同、拮抗或无关作用.结果 伊曲康唑和特比萘芬联...     

In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: Review of the literature

Voriconazole (Vfend™) is a new triazole that currently is undergoing phase III clinical trials. This review summarizes the published data obtained by NCCLS methods on the in vitro antifungal activity of voriconazole in comparison to itraconazole, amphotericin B, fluconazole, ketoconazole and flucytosine. Voriconazole had fungistatic activity against most yeasts and yeastlike species (minimum inhibitory concentrations [MICs] <2 μg/ml) that was similar or superior to those of fluconazole, amphotericin B, and itraconazole. Against Candida glabrata and C. krusei, voriconazole MIC ranges were 0.03 to 8 and 0.01 to >4 μg/ml, respectively. For four of the six Aspergillus spp. evaluated, voriconazole MICs (< 0.03 to 2 μg/ml) were lower than amphotericin B (0.25 to 4 μg/ml) and similar to itraconazole MICs. Voriconazole fungistatic activity against Fusarium spp. has been variable. Against F. oxysporum and solani, most studies showed MICs ranging from 0.25 to 8 μg/ml. Voriconazole had excellent fungistatic activity against five of the six species of dimorphic fungi evaluated (MIC₉₀s < 1.0 μg/ml). The exception was Sporothrix schenckii (MIC₉₀s and geometric mean MICs ≥ 8 μg/ml). Only amphotericin B had good fungistatic activity against the Zygomycetes species (voriconazole MICs ranged from 2 to >32 μg/ml). Voriconazole showed excellent in vitro activity (MICs < 0.03 to 1.0 μg/ml) against most of the 50 species of dematiaceous fungi tested, but the activity of all the agents was poor against most isolates of Scedosporium prolificans and Phaeoacremonium parasiticum (Phialophora parasitica). Voriconazole had fungicidal activity against most Aspergillus spp., B. dermatitidis, and some dematiaceous fungi. In vitro/in vivo correlations should aid in the interpretation of these results. This revised version was published online in June 2006 with corrections to the Cover Date.     

Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances

The aim of broth and agar dilution methods is to determine the lowest concentration of the assayed antimicrobial agent (minimal inhibitory concentration, MIC) that, under defined test conditions, inhibits the visible growth of the bacterium being investigated. MIC values are used to determine susceptibilities of bacteria to drugs and also to evaluate the activity of new antimicrobial agents. Agar dilution involves the incorporation of different concentrations of the antimicrobial substance into a nutrient agar medium followed by the application of a standardized number of cells to the surface of the agar plate. For broth dilution, often determined in 96-well microtiter plate format, bacteria are inoculated into a liquid growth medium in the presence of different concentrations of an antimicrobial agent. Growth is assessed after incubation for a defined period of time (16-20 h) and the MIC value is read. This protocol applies only to aerobic bacteria and can be completed in 3 d.     

Effects of amphotericin B on the electrical properties ofNecturus gallbladder: Intracellular microelectrode studies

Summary Intracellular microelectrode techniques were employed to study the mechanism by which amphotericin B induces a transient mucosa-negative transepithelial potential (V _ms) in the gallbladder ofNecturus. When the tissue was incubated in standard Na-Ringer's solution, the antibiotic reduced the apical membrane potential by about 40 mV, and the basolateral membrane potential by about 35 mV whereas the transepithelial potential increased by about 5 mV. The electrical resistance of the apical membrane fell by 83%, and that of the basolateral membrane by 40%; the paracellular resistance remained unchanged. Circuit analysis indicated that the equivalent electromotive forces of the apical and basolateral membranes fell by 35 and 11 mV, respectively. Changes in potentials and resistances produced by ionic substitutions in the mucosal bathing medium showed that amphotericin B produces a nonselective increase in apical membrane small monovalent cation conductance (K, Na, Li). In the presence of Na-Ringer's on the mucosal side, this resulted in a reduction of the K permselectivity of the membrane, and thus in a fall of its equivalent emf. During short term exposure to amphotericin B,P _Na/P _Cl across the paracellular pathway did not change significantly, whereasP _K/P _Na doubled. These results indicate that V _ms is due to an increase of g_Na across the luminal membranes of the epithelial cells (Cremaschiet al., 1977,J. Membrane Biol. 34:55); the data do not support the alternative hypothesis (Rose & Nahrwold, 1976.J. Membrane Biol. 29:1) that V _ms results from a reduction in shuntP _Na/P _Cl acting in combination with a rheogenic basolateral Na pump.     

鞘内注射两性霉素 B 治疗隐球菌脑膜炎的 META 分析

目的 评价鞘内注射两性霉素B用于隐球菌脑膜炎患者治疗的有效性和安全性.方法 采用循证医学系统评价方法,检索Cochran图书馆注册临床对照试验数据库2012年第1期,PubMed、Ovid、Springer、外文生物医学期刊全文数据库,中国生物医学文献数据库、中文生物医学期刊文献数据库、中国知网、万方数据库、维普中文期刊数据库、中华医学会数字期刊数据库.文献检索时间从建库至2012年2月,纳入探讨鞘内注射两性霉素B治疗隐球菌脑膜炎的临床对照试验,并逐个进行质量评价和资料提取,运用RevMan 5.1.6软件进行统计分析.结果 共检出89篇文献,最终纳入6篇,涉及临床对照试验6项,纳入患者175例.Meta分析提示:鞘内注射给药组较对照组有效率高[OR =4.59,95％CI (2.19,9.64),P＜0.0001].可使脑膜刺激征持续时间平均缩短8.56 d(95％CI-11.74～-5.38,P＜0.000 01),但相应不良反应较多.结论 鞘内注射两性霉素B治疗隐球菌脑膜炎可以提高疗效,但较多的不良反应限制了其广泛应用,能否作为首选治疗手段有待于更大样本的高质量临床对照研究证实.     

Determination of the minimal inhibitory concentration of Amphotericin B and Miconazole for 21 strains of Aspergillus

The susceptibility of 21 strains ofAspergillus (11 ofA. fumigatus, 8 ofA. niger, and 2 ofA. flavus) isolated from human pathologic specimens to Amphotericin B and Miconazole has been comparatively studied. Determination of the minimal inhibitory concentration of both drugs in a liquid medium showed a noticeably variability for the different strains. The values obtained for Amphotericin B varied between 0.25g/ml (2 strains) and 1.25g/ml (5 strains) after 48 hours, and between 1.25g/ml (1 strain) and 50g/ml (1 strain) after 10 days. For Miconazole the results varied between 0.1g/ml (1 strain) and 25g/ml (1 strain) after 48 hours of incubation, and between 0.5g/ml (5 strains) and > 100g/ml after 10 days. The variability of these results indicates the usefulness of carrying ourin vitro sensitivity studies whenever it is possible.     

AmphotericinB在全细胞穿孔膜片钳技术中的应用研究

目的：为研究海德氏突触的电生理特性,建立用amphotericinB穿孔的膜片钳技术。方法：本文应用两性霉素B（amphotericinB）在小鼠脑干的calyx细胞上进行穿孔膜片钳技术的研究。结果：应用amphotericinB进行穿孔后,通道电流衰减现象显著变慢。AmphotericinB的最适浓度为400tc／ml。结论：本研究摸索出了一种稳定的穿孔膜片钳全细胞记录技术,可以更有效,更真实的反应神经元通道电流的电生理特性。为穿孔膜片钳技术在听觉信息传导和调控研究中的应用提供了基础资料。     

Interactions of the drug amphotericin B with phospholipid membranes containing or not ergosterol: new insight into the role of ergosterol

Amphotericin B (AmB) is an amphipathic polyene antibiotic which permeabilizes ergosterol-containing membranes, supposedly by formation of pores. In water, AmB forms chiral aggregates, modelled as stacks of planar dimers in which the joined polyene chains in each dimer turn round, from one dimer to the following in these stacks, by forming a helical array. Studies of the binding of AmB with L-dipalmitoylphosphatidylcholine (L-DPPC) and L-dilauroylphosphatidylcholine (L-DLPC) bilayers disclose the main following results. (1) An inversion of the helicity of the L-DPPC-bound AmB aggregates, when the L-DPPC bilayers are in the gel phase, is inferred from the evolution of the circular dichroism spectra of AmB+L-DPPC mixtures. (2) An AmB-induced gel-to-subgel transformation of L-DPPC bilayers, in the previous mixtures, is revealed by a differential scanning calorimetry study. (3) The role played by ergosterol in the location of phospholipid-bound AmB aggregates with respect to a phospholipid bilayer is directly demonstrated from atomic force microscopy observations of mica-supported AmB+L-DLPC mixtures, in the presence or absence of ergosterol. While in the absence of ergosterol AmB aggregates remained at the surface of the bilayer, in the presence of ergosterol they appeared embedded within this bilayer and became hollow-centered. As such an embedding in the hydrophobic core of a bilayer requires a rearrangement of the aggregates with respect to their architecture in water, this rearrangement is held responsible for the hollowing of aggregates. The hollow-centered sublayer-embedded AmB aggregates are thought to be the precursors of the formation of AmB pores.     

Nanoethosomal formulation for skin targeting of amphotericin B: an in vitro and in vivo assessment

Abstract

The present study is envisaged to develop nanoethosomal formulation for enhanced topical delivery of amphotericin B (AmB) for the treatment of cutaneous fungal infections. AmB encapsulated nanoethosomes were prepared using mechanical dispersion method in a strength of 0.1% w/w similar to the strength of marketed topical formulation. Vesicle size of nanoethosomal formulations was found to be in the range of 186?±?2 to 298?±?4?nm. The optimized nanoethosomal formulation was further incorporated in gel base to form AmB nanoethogel formulation. Rheological characterization study of nanoethogel demonstrated its viscoelastic nature with high elasticity and resistance to deformation at 37?°C. The yield stress value was found to be 108.05?±?2.4 and 52.15?±?0.9?Pa for nanoethogel and marketed gel formulation, respectively. The nanoethogel formulation exhibited 2.7- and 3.5-fold higher steady state transdermal flux and skin deposition of AmB, respectively, in comparison to marketed formulation. Confocal laser scanning microscopy (CLSM) study also revealed enhanced skin permeation and deposition with nanoethogel formulation. In vivo study showed that topical application of nanoethogel does not exhibit any skin irritation as tested by Draize test. The developed formulation, in comparison to the marketed gel, demonstrated a remarkable increase in the antifungal activity against Candida albicans. It is thus corroborated from the above results that nanoethosomal formulation represents an efficacious carrier for effective topical delivery of AmB.     

Minimum inhibitory concentration (MIC) of crude preparations of Brevibacillus laterosporus SA14 bioactive material compared to vancomycin and oxacillin, against clinical isolates of methicillin-resistant Staphylococcus aureus

Secondary metabolites, particularly bioactive compounds, from probiotic bacteria, are good candidates for replacing antibiotics to which bacteria have become resistant. In order to compare bioactive crude material from strain SA14 of Brevibacillus laterosporus with two antibiotics, the MICs of this bioactive crude and those of antibiotics vancomycin and oxacillin, against methicillin-resistant Staphylococcus aureus (MRSA), were determined. The result indicated that the MIC (3.6–19.2 μg/ml) of bioactive crude was higher than vancomycin (MIC = 1.28–2.56 μg/ml) when tested against MRSA. Interestingly, all tested strains of MRSA were susceptible to bioactive crude and were approximately 5.2-fold more potent than oxacillin (MIC > 100 μg/ml). Its activity against MRSA gives support for further evaluation, and the development of this substance for therapeutic use.