Research About Dry Powder Inhalation Of Phage Against Drug-resistant Tuberculosis Infection Disease

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.