两性霉素B脂质体的制备及稳定性研究

采用薄膜－超声法制备了两性霉素B脂质体,稳定性考察的结果表明,制得的两性霉素B脂质在较低温度（4℃）条件下贮存时能保持良好的物理和化学稳定性;在近似生理条件下能较长时间地保持比较完整的脂质体结构,稳定性良好。     

Role of Phospholipid Transfer Protein on the Plasma Distribution of Amphotericin B Following the Incubation of Different Amphotericin B Formulations

Purpose The purpose of this study was to investigate the role of phospholipid transfer protein (PLTP) on the plasma distribution of amphotericin B (AmpB) following incubation with different AmpB formulations in human plasmas with varying lipid profiles. Methods In a first set of experiments, plasma distribution profiles of AmpB were determined following the incubation of Fungizone? and lipid-based formulations (Abelcet? and AmBisome?) at a concentration of 20 μg AmpB/mL for 5–120 min at 37°C in the plasma obtained from six different individuals (total cholesterol concentrations range between 62 and 332 mg/dL). In a second set of experiments, Abelcet?, and AmBisome? at a concentration of 20 μg AmpB/mL were incubated for 5 min at 37°C in human plasma (total cholesterol = 163 mg/dL) that had been pretreated with an antibody raised up against PLTP (1:400 v/v dilution from stock solution) for 20 min at 37°C. Following incubation, the human plasma was separated into its lipoprotein and lipoprotein-deficient fractions by density gradient ultracentrifugation and analyzed for AmpB content by high-performance liquid chromatography. Results The majority of AmpB was covered in the lipoprotein-deficient plasma and high-density lipoprotein (HDL) fractions following incubation of Fungizone? in human plasma. The majority of AmpB (48.7–87.2%) was recovered in the HDL fraction following incubation of Abelcet? and AmBisome? in human plasma. The presence of the PLTP antibody resulted in a 20% decrease in the percentage AmpB recovered in the HDL fraction following the incubation of Abelcet?. However, the plasma distribution of AmpB remained unchanged following the incubation of AmBisome? in plasma containing the PLTP antibody. Conclusions Taken together, these findings suggest indirect evidence that PLTP may play an important role in the plasma distribution profile of AmpB following the incubation of Abelcet? and may be one of the factors responsible for the preferential association of AmpB with HDL when administered as Abelcet?.     

HPLC法测定复方两性霉素B眼用凝胶的含量

目的建立同时测定复方两性霉素B眼用凝胶中两性霉素B和利福平含量的高效液相色谱法。方法采用DiamonsilC18柱(250mm×4.6mm,5μm),以0.005mol/L乙二胺四乙酸二钠-乙腈(65∶35)为流动相,流速为1.0ml/min,检测波长为334nm,柱温为20℃。结果两性霉素B的线性范围为4.816～48.16μg/ml,r=0.9999,平均回收率为99.3%,RSD=1.0%(n=9);利福平的线性范围为1.739～17.39μg/ml,r=0.9999,平均回收率为99.4%,RSD=1.1%(n=9)。结论该方法操作简便,快速,结果准确,可适用于产品的质量控制。     

Lyophilized Lecithin Based Oil-Water Microemulsions as a New and Low Toxic Delivery System for Amphotericin B

Purpose. To develop and investigate lecithin based oil-water microemulsions as potential amphotericin B (AmB) delivery systems and to evaluate their in vivo acute toxicity. Methods. AmB was added to the microemulsion and its location was evaluated by partitioning studies and UV-visible spectrophotometric analysis of the drug. Both, non-lyophilized and reconstituted microemulsions were characterised and assessed for their stability. Single-dose acute toxicity of the AmB microemulsion was studied on male albino Webster-derived CD-1 mice and compared with Fungizone®. Results. The studies performed showed that AmB was intercalated on the oil-water interface of the microemulsion as a complex formed with lecithin molecules. AmB addition did not seem to modify the rheological properties of the original system, but had an effect on its particle size distribution. Lyophilization of the microemulsion led to an oily cake, easily reconstituted and stable at the conditions studied. Single-dose acute toxicity studies proved that the LD₅₀ of AmB microemulsions was of 4 mg kg^–1 of animal weight, compared with 1 mg kg^–1 found for Fungizone®. Conclusions. Lyophilized lecithin based oil-water microemulsions appear to be valuable systems for the delivery of AmB in terms of easy and low-cost manufacturing, stability and safety compared with the formulations already in market.     

两性霉素B脂质体鞘内注射治疗新型隐球菌性脑膜炎疗效观察

目的：探讨两性霉素B鞘内注射治疗新型隐球菌性脑膜炎的可行性。方法：将常规静脉注射两性霉素B后、病情仍难以控制的（脑脊液中新型隐球菌性仍难以转阴,颅内高压持续不降）24例新型隐球菌性脑膜炎患者,随机分为两性霉素B鞘内注射组、两性霉素B脂质体鞘内注射组,鞘内注射剂量从0.1 mg开始,逐渐增加剂量,直到出现难以耐受的不良反应或每次注射剂量达到1 mg。结果：患者可以较好地耐受两性霉素B脂质体鞘内注射,鞘内注射两性霉素B脂质体发生不良反应的几率及严重程度明显低于普通两性霉素B。结论：两性霉素B脂质体鞘内注射治疗新型隐球菌性脑膜炎具有可行性。     

Block Copolymer Micelles for the Encapsulation and Delivery of Amphotericin B

Purpose. To assess the effect of fatty acid substitution of a micelle-forming poly(ethylene oxide)-block-poly(N-hexyl stearate-L-aspartamide) (PEO-b-PHSA) on the encapsulation, hemolytic properties and antifungal activity of amphotericin B (AmB). Methods. PEO-b-PHSA with three levels of stearic acid substitution were synthesized and used to encapsulate AmB by a solvent evaporation method. Size exclusion chromatography and UV spectroscopy were used to confirm and measure levels of encapsulated AmB. The hemolytic activity of encapsulated AmB toward human red blood cells and its minimum inhibitory concentration against Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans were obtained and compared to AmB alone. Results. An increase in the level of stearic acid substitution on PEO-b-PHSA improved the encapsulation of AmB while reducing its hemolytic activity. PEO-b-PHSA micelles having 50% and 70% stearic acid substitution (mol fatty acid: mol reacted and unreacted hydroxyls) were completely non-hemolytic at 22 g/ml. At 11% stearic acid substitution, AmB caused 50% hemolysis at 1 g/ml. AmB in PEO-b-PHSA micelles was as effective as AmB alone against pathogenic fungi. Conclusions. PEO-b-PHSA micelles with a high level of stearic acid side chain substitution can effectively solubilize AmB, reduce its hemolytic activity yet retain its potent antifungal effects.     

Enhanced Encapsulation of Amphotericin B into Liposomes by Complex Formation with Polyethylene Glycol Derivatives

Purpose. A highly efficient method was developed for the encapsulation of amphotericin B (AmB) in liposomes, and the mechanism involved was characterized. Methods. AmB was encapsulated in dipalmitoylphosphatidylcholine/cholesterol (DPPC/CH, 2:1) liposomes after complex formation with distearoyl-N-(monomethoxy poly(ethylene glycol) succinyl) phosphatidylethanolamine (DSPE-PEG). Hydration of lipids was done with 9% sucrose solution. Results. The encapsulated amount of AmB was 111 g/mg lipid, which was much higher than that obtained by the same method without DSPE-PEG (14 g/mg lipid). The amount encapsulated increased with amount of DSPE-PEG used and with PEG molecular weight. Encapsulation efficacy was also influenced by the type of PEG derivatives used and by the modification of AmB, suggesting the involvement of complex formation between AmB and DSPE-PEG. Absorption and ³¹P-NMR spectral analyses indicated that interactions between the amino and phosphate groups and between the polyene and PEG moieties in AmB and DSPE-PEG, respectively, play an important role in the complex formation. Conclusions. Complex formation of AmB with DSPE-PEG allows the highly efficient encapsulation of the drug in liposomes. This simple technique should be applicable to other hydrophobic drugs.     

The reformulation of Amphotericin B for oral administration to treat systemic fungal infections and visceral leishmaniasis

Amphotericin B (AmB) is a parenterally administered broad-spectrum antifungal and leishmanicidal drug that has been on the market for over sixty years. Unfortunately, significant infusion-related side effects and renal toxicity often accompany treatment, limiting its clinical applications. Lipid-based formulations have somewhat ameliorated the associated toxicity, but the increased cost of formulations restricts widespread use. AmB is amphipathic and exhibits low solubility and permeability, resulting in negligible absorption when administered orally. Advances in drug delivery systems have overcome some of the solubility issues that prevent oral bioavailability and new formulations are currently in development. The existence of an effective, safe and inexpensive oral formulation of amphotericin B would have significant applications for the treatment of disseminated fungal infections and would dramatically expand access to treatment of visceral leishmaniasis by introducing a readily available highly tolerated oral formulation of a drug with known efficacy.     

两性霉素B脂质体粒度测定方法研究

目的：建立两性霉素B脂质体粒度检测方法，通过测定一组性质不同的样品，找出最佳测定方法。方法：用计算机的图像一数字处理技术结合扫描电镜、透射电镜和激光光散射粒度测定仪分别测定两性霉素B脂质体的粒度。结果：电镜法测定两性霉素B脂质体的粒度为20—100nln，平均粒径为55—75nm；激光光散射法测定两性霉素B脂质体的粒度为30—200nm，平均粒径为50—180nm。结论：激光光散射法能较好反映两性霉素B脂质体在使用时的真实粒度，且方法快速、简便，是一种较好的两性霉素B脂质体粒度测定方法。     

Amphotericin B lipid complex for the treatment of invasive fungal infections

Amphotericin B lipid complex (ABLC; Abelcet^®, Enzon Pharmaceuticals) has become the dominant marketed lipid amphotericin B compound to emerge since the approval of these agents from the mid-1990s onwards. This agent is a 1:1 combination of amphotericin B and a lipid moiety consisting of dimyristoyl phosphatidylcholine and dimyrisoyl phosphatidylcholine, which exists in a ribbon-like molecular structure. ABLC undergoes rapid reticuloendothelial uptake from the circulation and achieves significantly higher tissue concentrations in the liver, spleen and lung compared to comparably dosed conventional amphotericin B. ABLC is approved by the FDA for all mycoses in amphotericin B-intolerant or -refractory infection. Randomised, controlled trials of amphotericin B have shown comparable efficacy in candidiasis and an improved outcome in invasive aspergillosis versus historical controls. ABLC has demonstrated a reduced incidence of nephrotoxicity and infusion reactions versus amphotericin B. Comparative studies against other lipid formulations are quite limited and have shown variable differences in infusion toxicity, nephrotoxicity, hepatotoxicity and clinical efficacy. Postapproval experience has shown substantial efficacy for less common mycotic pathogens including zygomycosis. The precise position of ABLC versus both other lipid formulations and expanding formulary of new antifungal agents is in flux. Future studies which examine its clinical efficacy, role in combination therapy, toxicity and cost-effectiveness in these complex patients are needed.     

Poor and Unusually Prolonged Oral Absorption of Amphotericin B in Rats

Pharmaceutical Research -     

两性霉素B复合磷脂纳米脂质体Beagle犬体内药动学研究

目的 采用LC-MS/MS测定Beagle犬血浆中两性霉素B的含量,研究受试物两性霉素B复合磷脂纳米脂质体(liposomal amphotericin B,L-AmB)和参比药物AmBisome在Beagle犬体内的药动学行为。方法 选取成年健康Beagle犬12只,♀♂各半。采用双交叉试验设计,周期间洗脱期为2周。经前肢静脉注射给药(6 mg·kg^-1,1.0 mg·mL^-1),采集给药前以及给药后各时间点血浆样品,应用经验证的LC-MS/MS方法测定血浆中两性霉素B浓度,DAS 2.1.1中求算药动学参数。结果 Beagle犬静脉注射给予6 mg·kg^-1的受试物和参比药物后,动物体内两性霉素B的AUC_(0-t)分别为(705 520±178 252),(677 310±166 326)μg·h·L^-1;C_max分别为(142 683±29 823),(121 992±37 983)ng·mL^-1;t_1/2分别为(8.9±1.5),(9.7±0.6)h。结论 L-AmB及参比制剂AmBisome在Beagle犬体内的药动学参数差异无统计学意义。     

Study the effects of PLGA-PEG encapsulated Amphotericin B nanoparticle drug delivery system against Leishmania donovani

Abstract

Drug delivery systems are a promising technology to increase poor solubility and bioavailability of compounds. Therefore we have developed PLGA-PEG encapsulated amphotericin B nanoparticles (NPs) drug delivery technology to increase the solubility of amphotericin B and target the macrophage of infected tissues during visceral leishmaniasis. The structural characterization by transmission electron microscopy and dynamic light scattering revealed the nano-size of the particle (30–35 nanometers). Fourier transform infrared spectroscopy confirmed the PLGA-PEG encapsulation. The mean cytotoxic assay (0.0803?+?0.0253) of extracellular promastigote of PLGA-PEG encapsulated amphotericin B is significantly lower than that of amphotericin B (0.1134?+?0.0153) and inhibition of amastigotes in the splenic tissue was significantly more than with conventional amphotericin B (93.02?+?6.63 versus 74.42?+14.78). Amphotericin B encapsulated PLGA-PEG nanoparticles were found to be more effective than free amphotericin B in terms of therapeutic efficacy during in vitro and in vivo study.     

Unidirectional Inhibition of Lipid Transfer Protein I-Mediated Transfer of Cholesteryl Esters Between High-Density and Low-Density Lipoproteins by Amphotericin B Lipid Complex

No HeadingPurpose. The purpose of this study was to determine whether Fungizone or amphotericin B lipid complex (ABLC; ABELCET^®) affects the transfer of cholesteryl ester (CE) by lipid transfer protein I (LTP I; also known as cholesteryl ester transfer protein) between HDL and LDL (bidirectional transfer HDL to LDL and LDL to HDL).Methods. Increasing concentrations of either Fungizone or ABELCET^® (1.25–12.5 g AmpB/ml) were incubated with HDL and [3H]CE-LDL or [3H]CE-HDL and LDL (the amount of each fraction added was equivalent to 10 g of cholesterol) and LTP I in delipidated human plasma at 37C for 90 min. As a positive control, TP2, a monoclonal antibody directed against LTP-1, was added instead of drug. After incubation, manganese and phosphate reagents were then added to precipitate out all of the LDL. The supernatant, consisted of only HDL, was counted for radioactivity to determine the amount of CE transferred from LDL. Similarly, the precipitate consisted of only LDL, was counted for radioactivity to determine the amount of CE transferred from HDL.Results. For Fungizone, the transfer of cholesteryl ester (CE) between HDL and LDL were not significantly different compared to nontreated controls. For ABELCET^®, CE transfer from HDL to LDL was significantly decreased at 12.5 g AmpB/ml compared to control. However, transfer from LDL to HDL was not significantly different compared to non-treated controls. Similar results were observed with the major lipid component of ABELCET^®, dimyristoylphosphatidylcholine. CE transfer from HDL to LDL and LDL to HDL was significantly decreased when using the positive control (TP2).Conclusions. Fungizone does not affect LTP I–mediated transfer of CE between HDL and LDL. ABELCET^® inhibits transfer from HDL to LDL, but has no effect on CE transfer from LDL to HDL. This uni-directional inhibition may contribute to the high recovery of AmpB in HDL but the very low presence of drug in the LDL fraction following ABELCET^® incubation.     

Biodistribution of Amphotericin B When Delivered Through Cholesterol Hemisuccinate Vesicles in Normal and A. Fumigatus Infected Mice

Purpose. This study compared the biodistribution of two amphotericin B formulations in normal and Aspergillus infected mice. Amphotericin B cholesterol hemisuccinate vesicles (ABCV) which reduces the toxicity of amphotericin B and thereby enhances its therapeutic efficacy in a murine model of aspergillosis was compared with conventional amphotericin B deoxycholate suspension (AmB_DOC). Methods. ABCV (12 mg/kg wt) and AmB_DOC (2 mg/kg wt) were intravenously administered to normal and A.fumigatus infected mice. The concentration of amphotericin B in plasma and other organs was determined at different time points. Results. It was observed that ABCV had a significantly different pharmacokinetic profile compared to conventional amphotericin B. In comparison to AmB_DOC significantly lower levels of amphotericin B were observed in kidneys and plasma, the major target organs of toxicity. Animals receiving ABCV demonstrated high levels of amphotericin B in liver (38% retention till 48 h) and spleen (2.6% retention till 48 h) in comparison to AmB_DOC (7.3% and 0.21% retention in liver and spleen respectively till 48 h). Biodistribution studies of ABCV in infected mice demonstrated that there was a moderate enhancement in levels of amphotericin B in liver, spleen, lungs and kidneys as compared to normal mice and the plasma levels were reduced. However, such observations were not made after AmB_DOC administration to infected mice except for kidneys in which there was a marked increase in uptake as compared to normal mice. Conclusions. Our results suggest that prolonged retention of high concentrations of ABCV in reticuloendothelial system organs is the reason for its reduced toxicity. Enhanced localization of the drug at the infected site may lead to improvement in therapeutic efficacy.     

Elucidation of Human Amphotericin B Pharmacokinetics: Identification of a New Potential Factor Affecting Interspecies Pharmacokinetic Scaling

Purpose. To elucidate the pharmacokinetics of amphotericin B in rats, mice and humans, and to perform interspecies scaling to humans using allometry. Methods. Plasma concentrations following intravenous bolus administration in rats, and mice were determined by HPLC. Human pharmacokinetic parameters elucidated from literature data were validated in a preliminary study involving a patient receiving daily infusion dose for 27 days. A critical literature review was conducted to identify appropriate pharmacokinetic parameter values in other species for interspecies scale-up. Interspecies allometric scale-up was performed across mice, rats, rabbits and dogs and the resulting predictions in humans were compared to observed values. Results. A triexponential decline in rat, mouse and human plasma concentrations were observed. No gender differences in rat pharmacokinetics were observed. In contrast to allometry, mouse CL was smaller (82 vs 116 ml/h/kg) and T_0.5 (33 vs 20 h) was longer compared to rat. In the preliminary human study, Cpeak and Cmin values remained relatively constant over the duration of therapy, and a CL, MRT, T_0.5, Vss and Vdarea of 26 ml/h/kg, 10 and 23 days, 6.2 and 20 L/kg, respectively, were estimated. The relative contributions of the terminal phase area in rat, mouse and human were 75%, 92% and 31%, respectively. Interspecies allometric scale-up predictions of human CL (41 ml/h/kg), CLu (467 ml/h/kg) and Vss (3.3 L/kg) were similar to reported values, whereas poor predictions of human Vuss (33 L/kg), Vdarea (4.1 L/kg) and T_0.5 (3 days) were obtained. Conclusions. Insignificant accumulation in humans inspite of the long terminal T_0.5 was rationalized to be due to the small terminal-phase area contribution. While human CL and Vss were sucessfully predicted in the interspecies scaling, poor predictions of human Vdarea and T_0.5 were obtained, which was attributed to disposition pattern differences between humans and other species, a potential new critical factor affecting interspecies scale-up.     

Amphotericin B-Loaded Poly(lactic-co-glycolic acid) Nanofibers: An Alternative Therapy Scheme for Local Treatment of Vulvovaginal Candidiasis

Vulvovaginal candidiasis is an inflammation localized in the vulvovaginal area. It is mostly caused by Candida albicans. Its treatment is based on the systemic and local administration of antifungal drugs. However, this conventional therapy can fail owing to the resistance of the Candida species and noncompliance of patients. Amphotericin B-loaded poly(lactic-co-glycolic acid) nanofibers are single-use, antifungal, controlled drug delivery systems, and represent an alternative therapeutic scheme for the local treatment of vulvovaginal candidiasis. Nanofibers were characterized by analytical techniques and with an in vitro drug delivery study. In vitro and in vivo fungicidal activity of amphotericin B released from nanofibers was evaluated using the agar diffusion method and an experimental murine model of vulvovaginal candidiasis, respectively. Analytical techniques showed that amphotericin B was physically mixed in the polymeric nanofibers. Nanofibers controlled the delivery of therapeutic doses of amphotericin B for 8 consecutive days, providing effective in vitro antifungal activity and eliminated the in vivo vaginal fungal burden after 3 days of treatment and with only one local application. Amphotericin B-loaded poly(lactic-co-glycolic acid) nanofibers could be potentially applied as an alternative strategy for the local treatment of vulvovaginal candidiasis without inducing fungal resistance, yet ensuring patient compliance.     

Heat-Treated Fungizone Retains Amphotericin B Antifungal Activity Without Renal Toxicity in Rats Infected with Aspergillus Fumigatus

PURPOSE: The purpose of this study was to assess the antifungal activity and renal and hepatic toxicity of amphotericin B (AmpB) following administration of Fungizone (FZ) and a heat-treated form of FZ (HFZ) to rats infected with Aspergillus fumigatus. METHODS: Infected rats were administered FZ and HFZ at a dosing regimen of 1 mg/kg i.v. once daily for 4 consecutive days. Following administration the number of colony forming units (CFUs) of Aspergillus fumigatus in different organs and serum creatinine concentrations were determined. RESULTS: FZ and HFZ had similar overall effectiveness in decreasing the total number of Aspergillus fumigatus CFUs found in all organs analyzed compared to controls. Except for the serum creatinine concentrations reported in the nontreated infected control rats, none of the treatment groups tested displayed a greater than 50% increase in serum creatinine. CONCLUSIONS: Taken together, these findings suggest that HFZ at 1 mg/kg once daily x 4 days appears to be as effective as FZ as an antifungal agent without renal toxicity.     

两性霉素B联合用药的研究进展

两性霉素B （amphotericin B,AmB）属于多烯类抗真菌药物,因其耐药率低、抗菌谱广,被称为治疗深部真菌感染的“金标准”,但是严重的毒副作用（尤其是肾毒性）大大限制了AmB在临床上的应用.在目前高效、低毒的抗真菌药物较少的情况下,联合使用不同作用机制的抗真菌药物,使其发挥协同作用,缩短疗程、减少药物毒副作用、扩大药物抗菌谱、避免耐药性的出现,是当前深部真菌感染治疗研究领域的一个重要研究方向.本文对AmB与不同种类抗真菌药物联合使用的研究进展作一综述.     

Assessment safety and efficacy of a combination therapy of topical amphotericin B and subconjunctival fluconazole for the treatment of fungal keratitis

Purpose: The study was aimed at evaluating the treatment of fungal keratitis using a combination therapy of a low concentration of topical amphotericin B (0.2?mg/mL) eye drops together with subconjunctival injections of 2?mg/mL of fluconazole.

Methods: The study was carried out in the Ophthalmology Department of Zagazig University Hospital and included 12 patients with 12 pairs of eyes with resistant corneal ulcers that had the following clinical features suggesting fungal keratitis: thick elevated ulcer, feathery edge, and gutter formation. Cultures of corneal specimens were grown on Sabouraud agar medium. Topical amphotericin B eye drops in a concentration of 0.2?mg/mL in dextrose 5% were used every 2 hours for 21 days together with subconjunctival injections of fluconazole 2?mg/mL.

Results: Among the eyes subjected to culture of corneal specimens, 8 eyes had positive results: Candida was detected in 5 eyes (66%) and filamentous fungi grew from the cultures of 3 eyes. Nine eyes improved as a result of treatment, showing complete healing of the ulcers (75%). Three cases showed no improvement (25%); 2 cases developed endophthalmitis; and 1 case was negative. A single case with corneal perforation had negative results on culture.

Conclusion: The use of a combination of topical amphotericin B eye drops at a concentration of 0.2?mg/mL in dextrose 5% with subconjunctival injection of fluconazole 2?mg/mL had the advantage of a lower incidence of the complications of local use of amphotericin B and a broader spectrum of antifungal coverage. This study reports a relatively high success rate of healing of fungal keratitis, with a significant reduction of the potential side effects of the local use of antifungal agents.