| Tuberculosis, especially for multi-drug resistant tuberculosis(MDR-TB), has already become a worldwide disease. And clinical mortality of pulmonary tuberculosis is next to HIV in infectious disease. Since Rifampicin was invented, there is no new drug for tuberculosis. Modified of existing dosage form and administration route has been present research focus, lung targeting drug delivery get more attention. Compared with other administration methods, Interventional administration with bronchoscopy is a safety and mature technology, and can accurately control dosage. Drugs is delivered to local focus of pneumonophthisis with bronchoscopy, especially for bronchial tuberculosis, MDR and chronic fibrocavernous pulmonary tuberculosis. But for existing drugs, time of effective concentration is short, drug-loaded substrate can't be absorbed and implanted to distal bronchus. It is time to develop a new dosage form that will be a exclusive drug for Bronchoscopy therapy, can degrade and be absorbed, and is safety for pulmonary delivery.In this study, drug carrier is sodium alginate, model drug is rifapentine, preparation method is electrostatic drop generation for manufacture of rifapentine-sodium alginate microspheres which could be used in lung. Characterization characteristics, endosomatic and exosomatic sustained release effect, safety will be investigated and evaluated.PART ONE Preparation of rifapentine-sodium alginate microspheresObjective: To prepare sustained release rifapentine-sodium alginate microspheres which could be used in lung, and establish determination methond of rifapentine with high performance liquid chromatography(HPLC), and investigate influence factors in preparation process, and optimize formulation and technology.Methods: Drug carrier is sodium alginate, model drug is rifapentine, preparation method is electrostatic drop generation for manufacture of rifapentine-sodium alginate microspheres. Formulation and technology was screened by means of orthogonal test. Characterization characteristics was investigated with light microscope and scanning electron microscope(SEM). Determination was measured with laser particle size instrument.? Entrapment efficiency and drug loading was determined with HPLC. Stability was investigated in environment of 4℃, 25℃and 40℃in 6 months. Testing that rifapentine-sodium alginate microspheres whether can be aspirated or injected by 7#needle or catheter.Result: The optimum formulation and technology is that mass fraction of sodium alginate is 2%, mass ratio of sodium alginate and rifapentine is 1:1, mass fraction of Cacl2 is 2%, crosslinking time is 2h, voltage is 8kv, advance speed of constant flow pump is 100ml/h. Average particle size of rifapentine-sodium alginate microspheres is 99.77μm. Average drug loading is 22.17±0.61% and Average entrapment efficiency is 83.06±1.31%. Microspheres can be injected by 7#needle or catheter. Except that rifapentine-sodium alginate microspheres was saved in 40℃environment for 6 months, others is no changes in the properties.Conclusion: Rifapentine-sodium alginate microspheres is successfully prepared, characterization characteristics, Entrapment efficiency and drug loading are accorded with requirement.PART TWO In vitro evaluation of rifapentine-sodium alginate microspheresObjective: To investigate sustained release effect of rifapentine-sodium alginate microspheres in vitro.Methods:Release test was done by basket method in vitro. 10mg rifapentine-sodium alginate microspheres were put in reagent bottle, 1000ml phosphate buffer of pH7.2 were release medium, rotational speed was 100r/min, temperature was 37±0.5℃, 5.0mL sample were took to test concentration. Same volume phosphate buffer was supplemented into reagent bottle.Result: As mass fraction of sodium alginate or Cacl2 increased, mass ratio of sodium alginate and rifapentine decreased and crosslinking time prolonged, drug release rate was slower. Release time of rifapentine-sodium alginate microspheres is 15d in vitro.? Accumulative release rate was 59.3% in 5d. Drug release was slowly stationary in 5d-15d. Accumulative release rate was 99.7% in 15dConclusion: Rifapentine-sodium alginate microspheres have better sustained release effect in vitro.PART THREE Safety evaluation of rifapentine-sodium alginate microspheres used in lungObjective: To observe solubility, permeability and absorption of rifapentine-sodium alginate microspheres used in lung of new zealand white rabbits and beagle dogs.Methods:60 rabbits are randomly divided into 3 groups, 20 in each group, control group injected 2ml physiological saline into left lung, blank microspheres group injected 2ml blank microspheres(20mg) plus physiological saline into left lung, drug microspheres group injected 2ml drug microspheres(20mg) plus physiological saline into left lung. 60 dogs are randomly divided into 3 groups, 20 in each group, control group injected 5ml physiological saline into left lung, blank microspheres group injected 5ml blank microspheres(300mg) plus physiological saline into left lung, drug microspheres group injected 5ml drug microspheres(300mg) plus physiological saline into left lung. For rabbits and dogs, body temperature and weight were detected; general conditions were observed; blood routine, T lymphocyte subsets and cytokine (IL-1β, IL-6, IL-8, IL-10, TNF-α) were detected after injecting 1d, 7d, 14d; all animals were sacrificed after injecting 14d, and observed lung gross anatomy. Lung tissue of local injecting were evaluated by HE staining and immunohistochemistry (IL-1β, IL-6, IL-8, IL-10, TNF-α). For dogs bronchial mucosa was observed by bronchoscopy after injecting 5d and 10d. Respiratory rate, maximal expiratory pressure and minimal inspiratory pressure were observed by respiratory record instrument before injecting to after injecting 60min. Blood gas analysis were done after injecting 1d, 7d, 14d. Lung tissue of local injecting were observed by SEM and TEM(transmission electron microscope).Result: Compared with control group in rabbits and dogs after injecting blank or drug microspheres, asphyxia and cough didn't be found; general conditions were normal; body temperature and weight, blood routine and T lymphocyte didn't show significantly diversity(P>0.05); lung gross anatomy didn't show abnormal; histopathological examination showed that bronchiole didn't obviously changed by comparing with control group's, and alveolus had complete structure and spacing of alveolus was not incrassation; expression of IL-1β, IL-6, IL-8, IL-10 and TNF-αdidn't show significantly diversity in lung tissue of local injecting (P>0.05).For rabbits after injecting 1d, content of IL-1β, IL-6, IL-8, IL-10 and TNF-αof control group, blank microspheres and drug microspheres group were higher than before injecting in blood; blank microspheres and drug microspheres were higher than control group (P<0.05). After injecting 7d, IL-1β, IL-6, IL-8, IL-10 and TNF-αof control group didn't show significantly diversity compared with before injecting(P>0.05); IL-8 and IL-10 of blank microspheres and drug microspheres didn't show significantly diversity compared with before injecting(P>0.05); IL-1β, IL-6 and TNF-αof blank microspheres and drug microspheres were higher than control group(P<0.05). After injecting 14d, except that IL-1βof blank microspheres group were higher than before injecting(P<0.05), others didn't show significantly diversity compared with before injecting(P>0.05). In dogs variations of cytokine were similar to that in rabbits.For dogs bronchial mucosa didn't show abnormal, and no hyperemia, edema, induration and necrosis were found after injecting 5d, 10d by bronchoscopy observation. Respiratory rate, maximal expiratory pressure and minimal inspiratory pressure didn't show significantly diversity in control group, blank microspheres and drug microspheres group after immediate injecting(P>0.05), but show significantly diversity after injecting 15min(P<0.05), didn't show significantly diversity after injecting 30min(P>0.05). Blood gas analysis didn't show significantly diversity in control group, blank microspheres and drug microspheres group after injecting 1d, 7d, 14d(P>0.05). SEM and TEM didn't show abnormal in in control group, blank microspheres and drug microspheres groupConclusion: Rifapentine-sodium alginate microspheres has better adhesion, and no influence on normal respiration, no irritation to bronchial mucosa, and can be degraded and absorbed. Safty and feasibility of Rifapentine-sodium alginate microspheres used in lung are preliminariely confirmed.PART FOUR In vivo evaluation of rifapentine-sodium alginate microspheresObjective: To establish determination method of rifapentine concentration in blood and lung tissue. To investigate sustained release effect of rifapentine-sodium alginate microspheres in vivo.Methods:60 dogs were injected 5ml drug microspheres(300mg) plus physiological saline into left lung. 5 dogs were randomly selected after injecting 6h, 12h, 24h, 36h, 48h, 4d, 6d, 8d, 10d, 12d, 14d and 16d. And those were sacrificed to collect blood, lung tissue of local injecting, lung tissue of 1cm far from local injecting, lung tissue of 3cm far from local injecting and contralateral lung tissue. Rifapentine concentration was determined with HPLC.Result: Rifapentine concentration of blood and lung tissue were respectively up to 9.7μg/ml and 24.6μg/ml after injecting 12h. Rifapentine concentration of blood was down to 0.6μg/ml after injecting 72h. Rifapentine concentration of lung tissue of local injecting was down to 0.5μg/ml after injecting 336h. Rifapentine concentration of lung tissue of 1cm far from local injecting was down to 0.5μg/ml after injecting 144h. Rifapentine concentration of lung tissue of 3cm far from local injecting was down to 0.8μg/ml after injecting 48h. Rifapentine concentration of contralateral lung tissue was down to 0.9μg/ml after injecting 36h.Conclusion: Rifapentine-sodium alginate microspheres has sustained release effect in vivo. Rifapentine concentration of lung tissue of local injecting is higher than that of blood, and can maintain 336h.
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