Mixed micelle proliposomes for preparation of liposomes containing amphotericin B, in-vitro and ex-vivo studies

The aim of this work was to produce a form of injectable liposomes containing amphotericin B derived from mixed micelle proliposomes. Mixed micelles were derived from a mixture of lecithin/sodium cholate in aqueous media. The solubility of amphotericin B in proliposomes was studied as a function of lipid composition (total lipid concentration, molar ratio of lecithin/sodium cholate), and the dispersion media (pH, ionic strength, presence or absence of human serum albumin), and the temperature. The data show that micelle-->liposome transformation occurs during the dilution of proliposomes containing amphotericin B. These transformations could be followed via transmission electron microscopy (TEM). Data related to dilution of proliposomes as well, show that under no circumstance there occurs any precipitation that might be assigned to the decreased solubility of amphotericin B. These indicate that the incorporated drug also participates during the transformation of the proliposomes into liposomes. It is thus concluded that mixed micelle proliposomes are prime candidates for the production of a form of injectable amphotericin B in liposomes.     

Chitosan‐coated antifungal formulations for nebulisation

Objectives The aim of this study was to produce and characterise amphotericin B (AmB) containing chitosan‐coated liposomes, and to determine their delivery from an air‐jet nebuliser. Methods Soya phosphatidylcholine : AmB (100 : 1) multilamellar vesicles were generated by dispersing ethanol‐based proliposomes with 0.9% sodium chloride or different concentrations of chitosan chloride. These liposomes were compared with vesicles produced by the film hydration method and micelles. AmB loading, particle size, zeta potential and antifungal activity were determined for formulations, which were delivered into a two‐stage impinger using a jet nebuliser. Key findings AmB incorporation was highest for liposomes produced from proliposomes and was greatest (approximately 80% loading) in chitosan‐coated formulations. Following nebulisation, approximately 60% of the AmB was deposited in the lower stage of the two‐stage impinger for liposomal formulations, for which the mean liposome size was reduced. Although AmB loading in deoxycholate micellar formulations was high (99%), a smaller dose of AmB was delivered to the lower stage of the two‐stage impinger compared to chitosan‐coated liposomes generated from proliposomes. Chitosan‐coated and uncoated liposomes loaded with AmB had antifungal activities against Candida albicans and C. tropicalis similar to AmB deoxycholate micelles, with a minimum inhibitory concentration of 0.5 µg/ml. Conclusions This study has demonstrated that chitosan‐coated liposomes, prepared by an ethanol‐based proliposome method, are a promising carrier system for the delivery of AmB using an air‐jet nebuliser, having a high drug‐loading that is likely to be effectively delivered to the peripheral airways for the treatment of pulmonary fungal infections.     

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。     

洛伐他汀自组装前体脂质体的制备及其理化性质的研究

目的:为研究洛伐他汀新剂型,制备洛伐他汀新型前体脂质体,并对其质量进行考察。方法:采用一种新型前体脂质体制备方法将洛伐他汀制成自组装前体脂质体,对水合后脂质体的形态、粒径、Zeta电位、包封率、自组装速度、稳定性等进行考察,验证这种新型前体脂质体制备方法用于制备洛伐他汀脂质体的可行性。结果:所形成的洛伐他汀脂质体包封率为95.4%±6.7%,平均粒径为(327.4±29.6)nm,Zeta电位值为-(22.4±1.5)mV。洛伐他汀自组装前体脂质体可在60 s内自发形成脂质体并达到分散平衡;以人工胃液为稀释介质,洛伐他汀脂质体在12 h内稳定。结论:采用新型前体脂质体制备方法可将洛伐他汀制成洛伐他汀脂质体,形成的脂质体包封率较高且具有良好的稳定性。     

卡铂前体脂质体的制备及安全性的初步评价

目的：制备卡铂前体脂质体,并对用药安全性进行初步评价.方法：采用薄膜挤压法制备卡铂脂质体,加入冻干支持剂冷冻干燥后得到卡铂前体脂质体.对豚鼠全身用药的过敏性、家兔全身用药的血管刺激性以及溶血性进行考察.结果：制备所得的卡铂前体脂质体水合后的包封率为72.0%,载药量为24.0%,平均粒径为125.1 nm.卡铂前体脂质体不引起豚鼠过敏反应,不引起家兔溶血和红细胞凝集反应,静脉注射对家兔血管无刺激性.结论：制备所得的卡铂前体脂质体有较高的包封率和载药量,水合后粒径均匀,形态圆整,且具有较好的用药安全性.     

载基因纳米阳离子脂质体前体制剂的制备及性质研究

目的:制备稳定的载反义寡核苷酸的阳离子脂质体前体制剂。方法:以磷脂-二油酰磷脂酰乙醇胺(dioleoylphophatidylethanolamine,DOPE)-十八胺-胆固醇为类脂成分,采用薄膜超声-挤压制备空白阳离子脂质体,吸附-冷冻干燥法制备载反义寡核苷酸阳离子脂质体前体。激光粒度仪测定冷冻干燥前后脂质体Zeta电位及粒径,透射电镜观察其形态,葡聚糖凝胶柱分离未包封的反义寡核苷酸,紫外法测定冻干前后的载药率。结果:海藻糖与甘露醇及甘氨酸为较好的冻干保护剂,制得的阳离子脂质体前体带正电荷,规则球形,大小较均匀,海藻糖作为保护剂复溶前后平均粒径为175和320 nm左右,复融前后Zeta电位值在+32和+40 mV左右,脂质体的载药率复溶前后分别为87.6%与83.21%。结论:海藻糖作为冻干保护剂,薄膜超声挤压法与冷冻干燥法结合,可成功制备反义寡核苷酸阳离子脂质体前体制剂,稳定性大大改善。     

Solid dispersion and effervescent techniques used to prepare docetaxel liposomes for lung-targeted delivery system: in vitro and in vivo evaluation

A lung-targeting liposomal docetaxel was developed to improve therapeutic index and to reduce side effects. Docetaxel proliposomes composed of docetaxel/Tween-80/Phospholipon 90H/cholesterol/citric acid at molar ratio of 0.18:0.09:3.78:3.78:91.17 were prepared by solid dispersion technique, and then were hydrated with NaHCO₃ solution to obtain docetaxel liposomes by effervescent technique. The stability of proliposomes containing docetaxel, characterization and evaluation of lung-targeting effect of docetaxel liposomes in rabbit were studied. Docetaxel proliposomes were stable at 6?±?2°C for at least 12 months. The particle size, zeta-potential, and entrapment efficiency of the resulted liposomes were 1011?±?22?nm, ?23.7?±?0.26?mv, and 90.12?±?0.36%, respectively. As far as the targeting parameters are concerned, the relative intake rate (R_e) and the ratio of peak concentration (C_e) of lung were 28.91 and 74.28, respectively. Compared with liver, spleen, and kidney, the ratios of targeting efficacy (T_e)_liposomes to (T_e)_injection of lung were increased by a factor of 3.16, 23.00, and 27.83, respectively. In conclusion, the negatively charged docetaxel liposomes with diameter of about 1 µm described in this study have favorable lung-targeting effect and are a promising lung-targeting carrier.     

Changes in the Solid State of Nicergoline,a Poorly Soluble Drug,Under Different Grinding and Environmental Conditions: Effect on Polymorphism and Dissolution

Nicergoline native crystals (Form I) were subjected to different grinding methods for 15, 30, 45, and 60 min: Method A, grinding at 20°C under air atmosphere; Method B, grinding in presence of liquid nitrogen under air atmosphere; Method C, grinding at 20°C under nitrogen atmosphere; and Method D, grinding in presence of liquid nitrogen under nitrogen atmosphere. Scanning electron microscopy, differential scanning calorimetry, X-ray powder diffractometry, thermogravimetry, and infrared spectroscopy were used to follow changes in the particle size and in crystalline structures. Batches from Methods A and C underwent partial amorphization immediately after grinding; Form II was obtained by heating these partially amorphous forms or after spontaneous crystallization after 1 and 5 months storage. Method B promoted the hydration of nicergoline to a monohydrate form. Batch D was stable under grinding and neither amorphization nor hydration were observed. The best intrinsic dissolution rate was that of metastable Form II, followed by Form I, while the worst was that of the Method B monohydrate form. The slowest particle dissolution was observed for hydrated particles, because of the lowest IDR, while the most rapid was exhibited by batch D, because of the very small particle size.     

Proliposomes for oral delivery of dehydrosilymarin: preparation and evaluation in vitro and in vivo

Aim:

To formulate proliposomes with a polyphase dispersed system composed of soybean phospholipids, cholesterol, isopropyl myristate and sodium cholate to improve the oral bioavailability of dehydrosilymarin, an oxidized form of herbal drug silymarin.

Methods:

Dehydrosilymarin was synthesized from air oxidation of silymarin in the presence of pyridine, and proliposomes were prepared by a film dispersion-freeze drying method. Morphological characterization of proliposomes was observed using a transmission electron microscope. Particle size and encapsulation efficiency of proliposomes were measured. The in vitro release of dehydrosilymarin from suspension and proliposomes was evaluated. The oral bioavailability of dehydrosilymarin suspension and proliposomes was investigated in rabbits.

Results:

The proliposomes prepared under the optimum conditions were spherical and smooth with a mean particle size in the range of 7 to 50 nm. Encapsulation efficiency was 81.59%±0.24%. The in vitro accumulative release percent of dehydrosilymarinloaded proliposomes was stable, which was slow in pH 1.2, and increased continuously in pH 6.8, and finally reached 86.41% at 12 h. After oral administration in rabbits, the relative bioavailability of proliposomes versus suspension in rabbits was 228.85%.

Conclusion:

Proliposomes may be a useful vehicle for oral delivery of dehydrosilymarin, a drug poorly soluble in water.     

Exposure of the yeast Candida albicans to the anti-neoplastic agent adriamycin increases the tolerance to amphotericin B

Cancer patients experience a high incidence of fungal infections due to their immuno-suppressed condition. This work has investigated the interaction of an anti-neoplastic agent, adriamycin (doxorubicin), with the yeast Candida albicans and examined whether this drug altered the susceptibility of the yeast to amphotericin B - an anti-fungal agent used for the treatment of systemic fungal infections in cancer patients. Exposure to adriamycin for 24h increased the growth of C. albicans and increased the tolerance to amphotericin B by a small, but statistically significant, extent. Growth in adriamycin-supplemented medium suppressed the respiration rate of C. albicans, which resulted in a decrease in the ergosterol content of the fungal cell membrane. The tolerance to amphotericin B was lost after exposure to adriamycin for 48 h, which coincided with a restoration in the respiration rate and the ergosterol content of the fungal cell membrane. This work demonstrated that short-term exposure (24 h) to adriamycin increased the tolerance of C. albicans for amphotericin B, which may be mediated by a decrease in the ergosterol content as a result of an adriamycin-induced disruption of oxidative phosphorylation.     

水飞蓟素前体脂质体的制备和大鼠药代动力学的研究水飞蓟素前体脂质体的制备和大鼠药代动力学的研究

目的为了提高水飞蓟素的口服生物利用度，研制水飞蓟素前体脂质体并对其理化性质进行考察；研究水飞蓟素前体脂质体的大鼠体内生物利用度。方法采用薄膜载体沉积法制备水飞蓟素前体脂质体，通过研究水合后脂质体的包封率、粒径、稳定性来考察其理化性质；将水飞蓟素前体脂质体在体外进行水合，再给予大鼠灌胃，用RP-HPLC法测定不同时间血浆中总的和游离的水飞蓟素的浓度，通过3P97程序计算药代动力学参数。结果用该法制得的前体脂质体包封率可达90%以上，平均粒径为238.8 nm，稳定性较好；药代动力学研究表明水飞蓟素脂质体在体内吸收较快，生物利用度较高。结论采用薄膜载体沉积法制备水飞蓟素前体脂质体，制备工艺简单，易于工业化生产；将水飞蓟素制备成前体脂质体提高了水飞蓟素的生物利用度。     

挤压器联动装置制备两性霉素B脂质体

目的考察挤压器联动装置制备两性霉素B脂质体制剂的稳定性。方法使用联动装置制备两性霉素B脂质体,用透析法检测两性霉素B脂质体的药物包封率,粒径仪器跟踪脂质体制剂的粒径及其分布,测定稳定性、泄漏率及毒性。结果所用联动装置可使脂质体的制备工艺简化,投料成品一步完成,同时可在线监控粒径。采用此装置制备的两性霉素B脂质体的粒径显著小于手动方式所制备,且粒径分布较窄;药物包封率为97．3％。通过与手动方法制备的脂质体制剂的多项技术指标对比,表明采用联动装置制备的两性霉素B脂质体制剂的药物包封率高,制剂稳定性好,药物渗漏率低。结论挤压器联动装置可用于工业化生产两性霉素B脂质体制剂的产品质量稳定、可控。     

槲皮素前体脂质体的质量考察

目的制备液体型槲皮素前体脂质体,并对制剂质量进行考察。方法采用一种新型前体脂质体制备方法制备液体型槲皮素前体脂质体,将脂质体膜材和药物等以一定比例溶于分散介质中,形成一种无水的澄明溶液。考察其水合后粒子形态、粒径、电位、包封率及自组装速度等理化性质,并评价其体外释药性质。结果槲皮素前体脂质体遇水即可快速自组装成纳米级含药脂质体混悬液,水合后形态多为类球形,平均粒径为228.7nm,Zeta电位为21.2 mV,包封率可达90%以上,体外释药符合Higuchi方程。结论槲皮素口服前体脂质体制备工艺简单可行,包封率高,具有一定的缓释效果。     

Membrane and cell wall targets in Aspergillus fumigatus.

Antifungal drugs directed against the human opportunistic fungal pathogen Aspergillus fumigatus are limited in number and ergosterol-targeted: the polyenes bind to the membrane ergosterol and the azoles block the ergosterol biosynthesis pathway. The efficacy of the drugs currently available for clinical use (amphotericin B and itraconazole) is limited and the frequent occurrence of therapeutic failures in the treatment of invasive aspergillosis emphasizes the need for the development of new agents. Cell wall biosynthetic pathways have been recognized for a long time as essential and unique specific drug targets. Recent studies of the chemical organization of the cell wall of A. fumigatus together with comparative analysis of yeast cell wall data have shown that beta 1-3 glucan branching and chitin-beta 1-3 glucan binding are essential exocellular enzymatic steps in cell wall biosynthesis. The enzymes involved in the biosynthesis and remodeling of cell wall polysaccharides especially in A. fumigatus are reviewed.     

Erythromycin,an inhibitor of mitoribosomal protein biosynthesis,alters the amphotericin B susceptibility of Candida albicans

Exposure of the yeast Candida albicans to the macrolide antibiotic erythromycin (C(37)H(67)NO(13)) results in elevated tolerance to the polyene antifungal amphotericin B. Erythromycin displays no fungistatic activity against C. albicans but inhibits the synthesis of cytochromes, particularly cytochrome aa(3). Consequently there is a reduction in aerobic respiration by up to 90% when cells are exposed to 10 mg mL(-1) erythromycin. Cellular ergosterol levels are also severely reduced. Erythromycin inhibits protein biosynthesis in ribosomes (mitoribosomes) located within the mitochondrion of the yeast cell, which results in a disruption of cytochrome biosynthesis with an adverse effect on respiration. The synthesis of ergosterol is oxygen dependent and consequently ergosterol levels are depleted in erythromycin-treated C. albicans. Ergosterol is the target for amphotericin B and since there is less of this sterol in erythromycin-treated cells, there is an increase in tolerance of the antifungal agent. Our work indicates that co-administration of erythromycin and amphotericin B to control bacterial and fungal infections, respectively, may inadvertently lead to an elevation in the tolerance of C. albicans for this antifungal agent.     

盐酸小檗碱前体脂质体的制备及理化性质研究

目的制备盐酸小檗碱前体脂质体,并对其理化性质进行考察。方法采用薄膜载体沉积法制备盐酸小檗碱前体脂质体,正交实验优选处方;透射电镜观察形态;马尔文激光粒度仪测定粒径;高效液相色谱法测定包封率。结果电镜下观察脂质体形态多为圆形或椭圆形,平均粒径为741nm,包封率为(29.93±1.32)%。结论该方法制备工艺简单,易于工业化生产。     

Particle size analysis of gelatin-acacia coacervate and ethylcellulose walled microcapsules

The Coulter counter has become one of the methods of choice for the measurement of small particle sizes. However, many colloidal walled microcapsules are prone to hydration when dispersed in the electrolyte solution used in Coulter measurements. This hydration causes the walls to swell, producing different size analyses dependent upon the time of measurement. In the present work the change in size with time was studied for microcapsules with gelatin-acacia coacervate, or ethylcellulose, walls. The former, although rendered insoluble with formalin, still hydrated; the latter were almost unaffected by water. A sample size of 30 mg was required to prevent blocking of the orifice with larger samples, or too few particles to permit measurement in smaller samples. The coacervate coated microcapsules showed a bimodal distribution, in part due to aggregate of smaller microcapsules, and this distribution changed significantly over the time of measurement. Although the peak due to larger microcapsules did not disappear, the number of large microcapsules fell significantly, most probably because of the disintegration of aggregated microcapsules. Microcapsules with ethyl cellulose walls did not show a bimodal distribution and the particle size analysis did not alter significantly with time.     

In line final filters for removing particles from amphotericin B infusions.

The feasibility of using membrane filters to remove particles from intravenous infusions of amphotericin B in dextrose 5% (a colloidal solution) was studied. Six types of commercial membrane filters, ranging in pore size from 0.45-1.0 mum, were used. Because of the effect of pH on the particle size of colloidal solutions, each filter was tested at solution pH 4.7, 5.6 and 6.5. Samples of filtrate were analyzed spectrophotometrically for amphotericin content. All filters of pore size 0.22 mum removed amphotericin B from solution and were inappropriate for use with this product. Solutions at pH 4.7 were turbid, filtered slowly and were generally unacceptable for clinical use. At pH 5.6, only filters with pore sizes of 1.0 mum or greater showed acceptable results. At pH 6.5, filters with pore sizes of 0.45 mum or greater gave acceptable results; the use of a filter with a pore size of not less than 1.0 mum would provide a margin for error to compensate for variations in the colloidal particle size of amphotericin B.     

Quantitative structure-activity relationships in amphotericin B derivatives

The quantitative structure-activity relationships studies of amphotericin B and its 16 semisynthetic derivatives obtained by modification at carboxyl and amino groups have been done. The results of five biological tests were subjected to principal component analysis, a numerical method useful in the investigation of large sets of data. For some compounds, also, interaction with lipidic vesicles was investigated by spectroscopic methods. The results obtained indicate that: (i) The presence of positively charged nitrogen atom (protonable or bearing fixed charge) is indispensable for biological activity and antibiotic-sterol interaction; (ii) The lack of free carboxyl group in the molecule favours the differentiation between cholesterol and ergosterol containing cells.     

药物载体空白脂质体前体的制备及性质的研究

以蛋黄卵磷脂、胆固醇为膜材,加入适量的高分子表面活性剂,选择合适的支持剂,采用冷冻干燥法制备了空白脂质体前体,并用它作为药物载体,将药物溶液分散在载体中,制成药物脂质体．研究了空白脂质体前体的再分散性及物理稳定性．空白脂质体前体再分散性良好,平均粒径为０．７５μｍ,粒径分布比较集中,在室温下贮存７个月、９个月后再分散其平均粒径分别为０８１μｍ、０８４μｍ,对５ Ｆｕ的包裹率也没有发生显著改变。用５ Ｆｕ,阿霉素（ＡＤＭ）等为模型药物,考察了影响药物脂质体包裹率的因素,并将最优条件固定．研究结果表明：空白脂质体水相的ｐＨ值、离子强度、再分散药物溶液的浓度（即药物与磷脂的重量比）对药物包裹率有显著影响．