Physicochemical properties and antifungal activity of amphotericin B incorporated in cholesteryl carbonate esters

The antifungal activity of amphotericin B (AmB) incorporated in three cholesteryl carbonate esters (CCEs), sodium cholesteryl carbonate, cholesteryl palmityl carbonate, and dicholesteryl carbonate, was examined to assess their potential for use in a dry powder aerosol. Formulations containing dissolved AmB were stable for 6 months. The particle size varied inversely with liquid crystalline content with observed mass median aerodynamic diameters ranging from 4 to 8 μ m. This was consistent with the visual appearance of the liquid crystals as being low density and free flowing at room temperature. When dispersed in water, the presence of the CCE reduced the rate and extent of AmB release, consistent with the estimated liquid crystal/water partition coefficient. Nevertheless, the rate of AmB release was always sufficient to kill the fungus as established with bioactivity studies. AmB formulated with CCE as a dry powder appears to be promising for use in treating lung fungal infections.     

Polymerized phospholipid vesicles containing amphotericin B: evaluation of toxic and antifungal activities in vitro

We have prepared lipid vesicles (liposomes) composed of polymerized bis[12-(methacryloyloxy)dodecanoyl]-L-alpha-phosphatidylcholine (DPL) which contain the antifungal polyene antibiotic amphotericin B (AMB). It was necessary to devise a novel method for incorporating AMB into the liposomes subsequent to polymerization. The polymer liposome AMB was as effective as AMB in "conventional" liposomes in terms of inhibiting fungal growth in vitro. However, in contrast to "conventional" liposomes, the polymerized DPL vesicles did not protect mammalian cells against AMB induced toxicity.     

Physico-chemical and microbiological comparison of nystatin, amphotericin A and amphotericin B, and structure of amphotericin A

Two polyene antibiotics, nystatin and amphotericin A, were compared by physico-chemical and microbiological methods. The two antibiotics were found to have the same molecular weight, 926, by plasma desorption and electron-impact MS. However, 13C NMR spectrometry and HPLC studies indicated that the two molecules are different. The 200 MHz NMR studies indicated a chemical environment of 24 carbons of amphotericin A identical with that of the carbons of amphotericin B and nystatin. The structure of amphotericin A is identical with that of amphotericin B, except that there is a single bond between carbons 28 and 29 instead of a double bond, as shown by two-dimensional NMR studies.     

Stability of nystatin in mouthrinses; effect of pH, temperature, concentration and colloidal silver addition, studied using an in vitro antifungal activity.

Alkaline low concentration nystatin mouthrinses extemporanely prepared can be used to treat oropharyngeal candidiasis in immunodeficient patients. However, their expiration dates are not distinctly determined.The stability of nystatin, added (as Mycostatine) at a concentration of 14 400 U/ml in 10^–4 N hydrochloric acid, purified water and 1.4% injectable sodium hydrogen carbonate with or without 0.002% colloidal silver (an antiseptic agent added because of its known antifungal potency) was studied after storage in tinted glass bottles at 5 °C and 22 °C over 11 days, and compared with reconstituted 100 000 U/ml aqueous Mycostatine oral suspension. At 2, 4, 7, 9, and 11 days after preparation. Samples were tested for pH, microbial contamination, and assayed by an in vitro microbiological test.Neither significant variation of pH nor microbial contamination were in evidence. Nystatin 14 400 U/ml maintained at least 90% of its initial concentration for 4 days in acid at both temperatures, for 7 days (5 °C) and 4 days (22 °C) in aqueous and alkaline environments, for 9 days (5 °C) and 7 days (22 °C) in 1.4% injectable sodium hydrogen carbonate containing colloidal silver which showed an antifungal potency. The 100 000 U/ml aqueous Mycostatine oral suspension was stable for 9 days and 4 days at 5 °C and 22 °C respectively.An ambulant patient can keep a low concentration alkaline antifungal mouthrinse at home for a week at 5 °C.     

Stability studies with amphotericin B and amphotericin B methyl ester.

In solid form, amphotericin B and amphotericin B methyl ester free base exhibit similar stability. Acid salts of the methyl ester derivative stored under identical conditions are less stable. In solution, amphotericin B is generally more stable than its methy ester salts. However, when pH is adjusted to 6.0 and storage temperature held at 5 degrees C the methyl ester salts reflect the stability exhibited by the parent compound, amphotericin B.     

Imbuing aqueous solubility to amphotericin B and nystatin with a vitamin

Aqueous solubilities of many drugs in current clinical use are very low, necessitating formulations that often present problems for parenteral administration, including toxicities due to the excipients used. Recognizing that pharmacologically active compounds frequently possess amines, we asked whether pyridoxal phosphate (PLP), an inoccuous, water-soluble vitamin, could be utilized to form prodrug-like complexes via the formation of imine or iminium adducts and whether the vitamin would impart solubilizing properties to such complexes. Direct spectroscopic and crystallographic data obtained using model primary and secondary amines showed that PLP forms stable imine adducts with primary amines under entirely aqueous conditions and at physiologic pH, while no reaction was observed for secondary amines; the basis of the exceptional stability appears to be a consequence of favorable H-bond interactions of the imine nitrogen with the 5-OH group of PLP. Amphotericin B and nystatin in their native forms display marked aqueous insolubility and possess lone primary amines. We were able to utilize PLP in achieving excellent solubilization of both of these antifungal agents, surpassing aqueous solubilities of 100 mg/mL. In in vitro bioassays, both polyenes in their PLP-adducted form display attenuated antifungal potencies which are attributable to "prodrug-like" complexes. These results point to the utility of excipient-free, entirely aqueous formulations of amphotericin B for parenteral use, and may also be extended to other primary amine-bearing compounds exhibiting poor aqueous solubility.     

Synthesis and antifungal activity of N-benzyl derivatives of amphotericin B

N-Benzyl derivatives of the polyene macrolide antibiotic amphotericin B were formed by reacting it with benzaldehydes and cyanoborohydride under reductive amination conditions. The physicochemical and biological properties of the resulting amphotericin B derivatives were studied. The N-benzyl derivatives of amphotericin B had high antifungal activity against a broad range of test cultures. Biological studies found that the acute toxicity of the amphotericin B derivatives was three times less than for the starting antibiotic. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 41, No. 7, pp. 20–24, July, 2007.     

抗真菌药物及两性霉素B脂质体制剂

真菌是一种条件致病菌,当机体抵抗力降低、外部因素不良时,就有可能造成全身或局部感染。按国际标准,依据感染部位可分为浅表性真菌病、皮下真菌感染和深部系统性真菌感染。     

Design of amphotericin B oral formulation for antifungal therapy

Amphotericin B (AmB) remains the “gold standard” for systemic antifungal therapy, even though new drugs are emerging as the attractive antifungal agents. Since AmB has negligible oral absorption as a consequence of its unfavorable physicochemical characterizations, its use is restricted to parenteral administration which is accompanied by severe side effects. As greater understanding of the gastrointestinal tract has developed, the advanced drug delivery systems are emerging with the potential to overcome the barriers of AmB oral delivery. Much research has demonstrated that oral AmB formulations such as lipid formulations may have beneficial therapeutic efficacy with reduced adverse effects and suitable for clinical application. Here we reviewed the different formulation strategies to enhance oral drug efficacy, and discussed the current trends and future perspectives for AmB oral administration in the treatment of antifungal infections.     

抗真菌剂两性霉素B的结构修饰

自两性霉素B作为抗真菌剂应用于临床以来,便受到了人们的广泛关注,目前仍为治疗全身或深部真菌病的首选药物.但其毒副作用,水溶件差等缺点存在,使得该抗生素的临床应用受到了一定的限制,因此开发新犁两性霉素B衍生物具有重要的研究和应用价值,近年人们对两性霉素B进行了大量的结构修饰研究,并从中获得了一系列有价值的衍生物.本文对两性霉素B结构修饰的最新研究进展作了一个简要的综述,并重点对几个毒副作用明显降低的两性霉素B衍生物作了较为详细的介绍.     

两性霉素B脂质体体内外毒性及抗真菌活性的研究

对自制的两性霉索B脂质体的体内外毒性以及抗菌活性进行了研究。结果表明,将两性霉索B制成脂质体后,其埘红细胞的溶血作用较市售注射剂有明显的降低,小鼠急性毒性试验的结果表明,两性霉索B脂质体的LD50为(14．89士2．18)mg／kg,约为市售注射剂LD50的6倍,其急性毒性显著下降;体外抗菌活性研究表明．两性霉素B脂质体保持了与市售注射剂相同的体外抑菌、杀菌活性。     

The effect of pH and of temperature on the stability and bioactivity of nystatin and amphotericin B

In the presence of phosphate-citrate buffers, nystatin and amphotericin B are optimally stable between pH 5 and 7. Loss of biological activity followed first-order kinetics, except under acid conditions. Apparent energies, enthalpies, free energies and entropies of activation of the two antibiotics have been calculated at pH 7, and the two sets of figures are similar. Variation in pH between 5 and 8 appeared to have little effect of the activity of amphotericin B against Candida albicans, while nystatin was more active at pH values between 6 and 8. Nystatin was more active at lower incubation temperatures (30-25°), while amphotericin B appeared to be more active at 41°.     

Synthesis and antifungal activity of N-trialkylsilyl derivatives of nystatin

Reactions of the polyene macrolide antibiotic nystatin with trialkylchlorosilanes led to the formation of N-alkylsilyl derivatives. The physicochemical and biological properties of the resulting nystatin derivatives were studied. Organic silicon derivatives of nystatin had high levels of antifungal activity against a wide set of test strains. Biological studies showed that the acute toxicity (LD₅₀) of the resulting nystatin derivatives were 2–3 times lower than that of the initial antibiotic. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 6, pp. 15–18, June, 2008.     

Stability and transdermal absorption of topical amphotericin B liposome formulations

The aim of this study was to characterize the stability and transdermal absorption of amphotericin B (AmB: 0.05 mg/mg lipid) in hydrogenated soya phosphatidylcholine/cholesterol/charged lipid {dicetyl phosphate (−) or stearylamine (+)} liposomes at molar ratios of 1:1:0, 7:2:0, 7:2:1(−) and 7:2:1(+). The AmB contents in liposomes were determined by HPLC with UV detection at 382 nm. Stabilities of AmB in liposome formulations were compared with those in solution and powder forms, during storage at 4, 30 and 45 °C for 90 days. Absorption studies of AmB across the rat skin were conducted, using vertical Franz diffusion cells at 37 °C for 24 h. The slowest degradation was observed in the positive liposome (7:2:1(+)AmB), with shelf life of 1 year (30 °C). In comparison, the shelf lives of AmB in solution and powder were 4 and 14 days, respectively. AmB in positive liposomes seemed to demonstrate the highest flux in stratum corneum (58 ng/cm²/h), while the highest flux in viable epidermis (23 ng/cm²/h) was observed in negative liposomes. AmB entrapped in charged liposomes showed sustained skin absorption. The positively charged liposome might be the best formulation for AmB, due to its higher stability than other formulations.     

Oral administration of amphotericin B nanoparticles: antifungal activity,bioavailability and toxicity in rats

Amphotericin B (AMB) is used most commonly in severe systemic life-threatening fungal infections. There is currently an unmet need for an efficacious (AMB) formulation amenable to oral administration with better bioavailability and lower nephrotoxicity. Novel PEGylated polylactic-polyglycolic acid copolymer (PLGA-PEG) nanoparticles (NPs) formulations of AMB were therefore studied for their ability to kill Candida albicans (C. albicans). The antifungal activity of AMB formulations was assessed in C. albicans. Its bioavalability was investigated in nine groups of rats (n?=?6). Toxicity was examined by an in vitro blood hemolysis assay, and in vivo nephrotoxicity after single and multiple dosing for a week by blood urea nitrogen (BUN) and plasma creatinine (PCr) measurements. The MIC of AMB loaded to PLGA-PEG NPs against C. albicans was reduced two to threefold compared with free AMB. Novel oral AMB delivery loaded to PLGA-PEG NPs was markedly systemically available compared to Fungizone® in rats. The addition of 2% of GA to the AMB formulation significantly (p??790% that of Fungizone®. The novel AMB formulations showed minimal toxicity and better efficacy compared to Fungizone®. No nephrotoxicity in rats was detected after a week of multiple dosing of AMB NPs based on BUN and PCr, which remained at normal levels. An oral delivery system of AMB-loaded to PLGA-PEG NPs with better efficacy and minimal toxicity was formulated. The addition of glycyrrhizic acid (GA) to AMB NPs formulation resulted in a significant oral absorption and improved bioavailability in rats.     

Stability of amphotericin B in four concentrations of dextrose injection.

The stability of amphotericin B in 5%, 10%, 15%, and 20% dextrose injection was investigated. The dextrose solutions were prepared in triplicate from sterile water for injection and 70% dextrose injection and placed in empty 50-mL polyvinyl chloride bags. The pH of each solution was determined before amphotericin B was added to a concentration of approximately 100 micrograms/mL. The bags were stored at 15-25 degrees C and protected from light. Three 1-mL samples were taken from each bag at various times up to 24 hours. One sample was analyzed for precipitation and color and pH changes. Two samples were analyzed in duplicate by stability-indicating high-performance liquid chromatography. No visual changes were observed, and pH did not change substantially. The mean amphotericin B concentration was greater than 90% of the initial concentration at each sampling time. However, the drug concentration in 3 of the 27 samples from the admixtures with 10% dextrose injection and 5 of the 27 samples from the admixtures with 20% dextrose injection fell below 90% of the initial concentration. Amphotericin B 100 micrograms/mL was stable in 5%, 10%, 15%, and 20% dextrose injection when stored for up to 24 hours at 15-25 degrees C and protected from light.     

卡泊芬净与脂质体两性霉素B在血液病患者经验性抗真菌治疗中的比较

目的 评价和比较卡泊芬净与脂质体两性霉素B在血液病患者经验性抗真菌治疗的有效性和安全性。方法 采取随机、对照、开放的临床试验研究，选取出现不明原因发热、广谱抗生素治疗3～7d无效且被真菌感染的高危因素血液病患者。将60例入选患者随机分为两组，卡泊芬净组（32例）静脉滴注卡泊芬净（d170mg，d2起50mg／d）；脂质体两性霉素B组（28例）静脉滴注脂质体两性霉素BE3mg／（kg·d）]。两组治疗时间持续至中性粒细胞恢复和症状消失后5d，或者疗程大于10d。观察两组患者的疗效和不良反应。结果 共有60例患者接受评估，卡泊芬净组32例，总有效率65．6％，不良反应率25．0％；脂质体两性霉素B组28例，总有效率67．9％，不良反应率71．4％。两组总有效率无显著性差异，但卡泊芬净组不良反应率显著低于脂质体两性霉素B组。结论 对血液病患者的经验性抗真菌治疗，卡泊芬净的疗效与脂质体两性霉素B相当，但耐受性较脂质体两性霉素B好。