耐药结核病治疗用噬菌体干粉吸入剂的研究
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摘要
结核病是由结核杆菌感染引起的人畜共患传染病,一直以来严重威胁人类健康。多重耐药结核、广泛耐药结核的出现,以及有的超级耐药结核菌对目前已知的抗结核药物几乎全部耐药,使其感染者的病死率高达90%以上,这势必给结核病的治疗带来重大挑战。已有研究证实噬菌体D29可以在体外环境中杀灭巨噬细胞内的结核分枝杆菌,在动物试验中也证明对结核感染的豚鼠具有治疗效果,且对豚鼠无副作用,这些结果预示分枝杆菌噬菌体D29具有治疗结核病的潜在价值。肺结核是结核杆菌感染中对人类危害最大的一种临床疾病,感染的主要病灶器官为肺脏,采用肺部给药可以直接将药物输送至病灶部位,是临床治疗肺结核的较理想途径。而目前报道的噬菌体治疗结核分枝杆菌的研究中,多采用皮下注射、滴鼻给药等方式,噬菌体到达肺部的有效剂量低,因此需要在给药方式上进行改进。本文旨在研究一种可以避免传统给药方式缺陷的耐药结核病治疗用噬菌体干粉吸入剂。首先建立了噬菌体D29的培养及计数方法。噬菌体D29的增殖采用半固体双层琼脂平皿法,效价检测方法采用双层琼脂培养测定法。一般在培养后24-48h内对噬菌体斑计数统计,使用平皿计数值在30-300的结果。建立了干粉吸入剂的质量评价体系。包括振实密度、干粉形态、干粉粒径、干粉粒度分布、流动性、含水量、引湿性、含量均一性、排空率、体外沉积率。以粉体综合特性测试仪、扫描电镜、激光粒度分析仪及双层液体碰撞器等作为分析手段。选择HPLC法以荧光素钠为化学示踪剂测定噬菌体的体外沉积率。采用了不同方法来制备可吸入颗粒,对喷雾干燥法制备的可吸入颗粒的制备工艺、颗粒性质评价和保护剂的筛选进行了深入探讨。采用筛分法、研磨法、重结晶法、冷冻干燥法、喷雾干燥法来制备乳糖颗粒,以所制得颗粒的粒径大小和形态以及含水量为主要评价指标,考察处方和工艺因素对颗粒特性的影响,筛选出可吸入颗粒的制备方法和工艺；为了提高噬菌体在干粉中的生物活性,初步筛选了相应的保护剂。结果表明,采用喷雾干燥法,当入口温度(120℃)、气流速度(150L/min)、喷雾速率(100%)、喷头盖(4.0μm),溶液浓度(10%)时可得到收率达80%以上,含水量为1.17%左右,平均粒径在3.00μm左右的圆整球形颗粒,适合于吸入给药；其他方法所获得颗粒平均粒径大、形状不规则,用于吸入给药需要进一步处理。糖类、蛋白类对噬菌体喷干均有较好的保护作用,而多元醇类、NaCl的保护效果差(喷干后存活率低于1%)。各类保护剂组合配方,效果好于单一保护剂。综合考虑,以糖类和氨基酸组合保护效果最佳,优化工艺参数喷干后,可得到收率80%以上,含水量2%以下,平均粒径在3.00μm左右球形的颗粒,噬菌体活性可保持60%以上。稳定性实验表明,噬菌体以固体形式保存,在常温、4℃条件下放置3个月效价没有明显变化。噬菌体D29干粉吸入剂在加速试验条件(40℃,RH75%)下1个月内效价下降不到一个数量级。
Tuberculosis (TB) is a kind of infectious disease from which both the human being and animals suffer,caused by Mycobacterium tuberculosis. tuberculosis has been seriously threatened to human health. The prevelance of Multi-drug resistant tuberculosis (MDR-TB) and the extensively-drug resistant tuberculosis,and some of super-drug resistant tuberculosis almost resistanting to the currently known anti-TB drugs that the fatality rate of persons who infected tuberculosis is as high as90%or more.Those will pose a tremendous challenge to tuberculosis treatment.Studies have confirmed that the phage D29can kill Mycobacterium tuberculosis within macrophages in vitro environment. And the phage D29also has a therapeutic effect with guinea pigs who infected tuberculosis in animal trials. moreover,it has no side effects in guinea pigs.These results indicate Mycobacterium phage D29has a potential value of the treatment of tuberculosis.Pulmonary tuberculosis is Mycobacterium tuberculosis infection in a clinical disease which has been seriously hazarded to human beings.Lung was the infection viscera of tuberculosis so pulmonary drug delivery directly carried drugs to the lesion is the ideal way to treat tuberculosis.Currently phage D29treatment of mycobacterium tuberculosis research mainly administers by subcutaneous injection and intranasal administration, and the effective dosage of the phage to reach the infectious lung is low, therefore, we need to improve on the administration.This paper aims to research about dry powder inhalation of phage against drug-resistant tuberculosis infection disease which can avoid the defects of the traditional delivery method.Firstly we established the cultivation and counting of phage D29, using semi-solid double-layer agar and double-layer agar method for the replication and titer detection of Phage D29, respectively. And we counted phage plaques after24h to48h, used the results of the count in30to300.Then we established a series of quality evaluation systems for dry powder inhaler, including the tapped density, powder morphology, powder particle size, powder particle size distribution, flowability, moisture content, hygroscopicity, content uniformity, emitted dose, the deposition rate in vitro. We used powder comprehensive characteristic tester, scanning electron microscopy, laser particle size analyzer, twin stage impinger as analytical tools and selected a HPLC method for determination of the phage in vitro deposition with fluorescein as a chemical tracer.We use different methods to prepare respirable particles, discussing deeply in the preparation process of respirable particles by spray drying, the evaluation of the particle characteristics and screening of protective agents in this paper.The lactose particles were prepared by different methods:sieving, grinding, re-crystallization, freeze drying,spray drying. The particle size and morphology and water content were chosen as the main evaluation index to investigate the effect of formulation and process factors on them. In order to improve the biological activity of the phage D29in the dry powders, we initially screened the appropriate protective agents.The results show that, when the inlet temperature is120℃, air flow rate is150L/min, spray rate is100%, the nozzle cap is4.0μm, concentration is10%, we can obtain the particles which are spherical with the average particle size about3.0μm by spray drying process.The particles's yield is more than80%, moisture content is about1.17%, which were suitable for inhalation. All other methods prepared a larger average particle size, irregular particles, which needed further process for inhalation.The sugars, proteins on the phage D29which spray-dried have a better protective effect, polyols and NaCl have a poor protective effect (after spray-dried the survival rate is less than1%). Various types of protective agent with combination formulation, which protective effect is better than a single protective agent. By comprehensive consideration, sugars and amino acids with combination have a best protective effect. Optimizing the process parameters after the spray-dried, we can prepare the spherical particles which income rate is80%, moisture content is below2%, average particle size is about3.00μm, and the survivality of phage D29reach60%.Stability experiments showed that the phage D29stored in solid form at room temperature and4℃is stable in3months.The titer of phage D29dry-powder inhaler in the accelerated test conditions (40℃, RH75%) decrease less than an magnitude within one month.
Tuberculosis (TB) is a kind of infectious disease from which both the human being and animals suffer,caused by Mycobacterium tuberculosis. tuberculosis has been seriously threatened to human health. The prevelance of Multi-drug resistant tuberculosis (MDR-TB) and the extensively-drug resistant tuberculosis,and some of super-drug resistant tuberculosis almost resistanting to the currently known anti-TB drugs that the fatality rate of persons who infected tuberculosis is as high as90%or more.Those will pose a tremendous challenge to tuberculosis treatment.Studies have confirmed that the phage D29can kill Mycobacterium tuberculosis within macrophages in vitro environment. And the phage D29also has a therapeutic effect with guinea pigs who infected tuberculosis in animal trials. moreover,it has no side effects in guinea pigs.These results indicate Mycobacterium phage D29has a potential value of the treatment of tuberculosis.Pulmonary tuberculosis is Mycobacterium tuberculosis infection in a clinical disease which has been seriously hazarded to human beings.Lung was the infection viscera of tuberculosis so pulmonary drug delivery directly carried drugs to the lesion is the ideal way to treat tuberculosis.Currently phage D29treatment of mycobacterium tuberculosis research mainly administers by subcutaneous injection and intranasal administration, and the effective dosage of the phage to reach the infectious lung is low, therefore, we need to improve on the administration.This paper aims to research about dry powder inhalation of phage against drug-resistant tuberculosis infection disease which can avoid the defects of the traditional delivery method.Firstly we established the cultivation and counting of phage D29, using semi-solid double-layer agar and double-layer agar method for the replication and titer detection of Phage D29, respectively. And we counted phage plaques after24h to48h, used the results of the count in30to300.Then we established a series of quality evaluation systems for dry powder inhaler, including the tapped density, powder morphology, powder particle size, powder particle size distribution, flowability, moisture content, hygroscopicity, content uniformity, emitted dose, the deposition rate in vitro. We used powder comprehensive characteristic tester, scanning electron microscopy, laser particle size analyzer, twin stage impinger as analytical tools and selected a HPLC method for determination of the phage in vitro deposition with fluorescein as a chemical tracer.We use different methods to prepare respirable particles, discussing deeply in the preparation process of respirable particles by spray drying, the evaluation of the particle characteristics and screening of protective agents in this paper.The lactose particles were prepared by different methods:sieving, grinding, re-crystallization, freeze drying,spray drying. The particle size and morphology and water content were chosen as the main evaluation index to investigate the effect of formulation and process factors on them. In order to improve the biological activity of the phage D29in the dry powders, we initially screened the appropriate protective agents.The results show that, when the inlet temperature is120℃, air flow rate is150L/min, spray rate is100%, the nozzle cap is4.0μm, concentration is10%, we can obtain the particles which are spherical with the average particle size about3.0μm by spray drying process.The particles's yield is more than80%, moisture content is about1.17%, which were suitable for inhalation. All other methods prepared a larger average particle size, irregular particles, which needed further process for inhalation.The sugars, proteins on the phage D29which spray-dried have a better protective effect, polyols and NaCl have a poor protective effect (after spray-dried the survival rate is less than1%). Various types of protective agent with combination formulation, which protective effect is better than a single protective agent. By comprehensive consideration, sugars and amino acids with combination have a best protective effect. Optimizing the process parameters after the spray-dried, we can prepare the spherical particles which income rate is80%, moisture content is below2%, average particle size is about3.00μm, and the survivality of phage D29reach60%.Stability experiments showed that the phage D29stored in solid form at room temperature and4℃is stable in3months.The titer of phage D29dry-powder inhaler in the accelerated test conditions (40℃, RH75%) decrease less than an magnitude within one month.
引文
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庆医科大学.2006
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[16] Chougule MB, Padhi BK, Misra A. Nano-liposomal dry powder inhaler of Amiloride Hydrochloride Nanosci Nanotechnol,2006,6(9-10): 3001-3009
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[19] Lo YL, Tsai JC, Kuo JH. Liposomes and disaccharides as carriers in
spray-dried powder formulations of superoxide dismutase. Control Release,2004,94(2-3):259-272
[20] Millqvist-Fureby A, Malmsten M, Bergenstahl B. Spray-drying of trypsin-surface characterization and activity preservation. Int J Pharm, 1999,188(2):243-253
[21] Rabbani NR, Seville PC. Influence of storage humidity on the aerosolization performance of spray-dried powders.Aerosol Med, 2005,18(9):251
[22] Broadhead J, Rouan SK, Hau I, et al. The effect of process and formulation variables on the properties of spray-dried beta-galactosidase. Pharm Pharmacol,1994,46(6):458-467
[23] Bosquinon C, Rouxhet PG, Ahimou F, et al. Aerosalization properties, surface composition and physical state of spray-dried protein powders. Control Release,2004,99(3):357-367
[24] Chan HK, Clark AR, Feeley JC, et al. Physical stability of salmon calcitonin spray-dried powders for inhalation. Pharm Sci, 2004,93(3);792-804.
[25] Yang M, Velaga S, Yamamoto H, et al. Characterization of salmon calcitonin in spray-dried powder for inhalation, Effect of chitosan. Int J Pharm,2007,331(2):176-181
[26] Guntur VP, Dhand R. Inhaled insulin:extending the horizons of inhalation therapy. Respir Care,2007,52(7):911-922
[27] Rabbani NR, Seville PC. The influence of formulation components on the aerosolization properties of spray-dried powders.Control Release,2005,110(1)130-140
[28]Najafabadi AR, Gilani K, Barghi M, et al. The effect of vehicle on
physical properties and aerosolization behaviour of disodium cromoglycate microparticles spray dried alone or with L-leucine. Int J Pharm,2004,285(1-2):97-108.
[29] Chew NY, Shekunov BY, Tong HH, et al. Effect of amino acids on the dispersion of disodium cromoglycate powders. Pharm Sci, 2005,94(10):2289-3000
[30] Seville PC, LearoydTP, Li H-Y, etc. Aminoacid-modified spray-dried powders with enhanced aerosolization properties for pulmonary drug delivery. Powder Technol,2007,178(1):40-50.
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[34] Rabbani NR, Seville PC. The use of amino acids as formulation excipients in lactose-based spray-dried powders. Pharm Pharmacol, 2004,56(Suppl.):S?32-33
[35] Ely L, Roa W, Finlay WH, et al. Effervescent dry powder for respiratory drug delivery. Eur J Pharln Biopharm,2007,65(3):346-353
[36] Learoyd TP, Burrows JL, French E, et al. Chitosan-based spray-dried respirable powders for sustained delivery of terbutaline sulfate. Eur J
Pharm Biopharm.2008,68(2),224-234
[37] Li HY, Helen N, Rebecca I, et al. Enhanced Dispersibility and Deposition of Spray-dried Powders for Pulmonary Gene Therapy. Drug Targeting,2003,11(7),425-432
[38] Tom HJ, Eric T, Jaap G, et al. Stabilizing formulations for inhalable powders of an adenovirus 35-vectored tuberculosis (TB) vaccine (AERAS-402). Vaccine,2010,28(2010)4369-4375
[39] Li HY, Seville PC, Williamson IJ, et al.The use of amino acids to enhance the aerosolisation of spray-dried powders for pulmonary gene therapy. Gene Med,2005,7:343-353
[40]国家药典委员会.中华人民共和国药典(二部).北京：中国医药科技出版社,2010,附录XH；附录IL