Study On Chitosan Microspheres As Pulmonary Sustained Delivery Systems And Their Pharmacodynamics

Asthma is a serious commonly chronic airway disease which do harm to health of people, in the past two decades, the prevalence and the mortality rates of asthma are increasing in all countries throughout the world, which has been paid much more attention not only by the specialist of respiratory, but also by the WHO and the government of many countries, so how to manage asthma effectively has become a public health problem. Theophylline (TH) is one of the most important drugs for treatment of asthma and recent studies indicate that it has anti-inflammatory effects. Thus, great attentions have been shown due to the new effect on treatment of asthma. Unfortunately, since its short half-life (6 h), conventional dosage forms have to be administered 3-4 times a day in order to avoid large fluctuations in plasma concentrations, which lead to poor patient compliance. The large fluctuations of plasma TH concentration lead to gastrointestinal and cardiovascular adverse effects. Moreover, its therapeutic index is narrow (10-20μg/ml). The therapeutic effects of TH require plasma concentration of TH at least 5-10μg/ml and toxic effects are frequent above 20μg/ml. Sustained release dosage forms can overcome these drawbacks, but long-term oral therapy can't avoid the side effects of gastrointestinal, cardiovascular and central nervous system's disturbances because its elimination half-life varies widely between patients. Hence a long-term TH therapy also leads to serious management problems in asthmatics. Despite the occurrence of numerous new sustained release products of TH, an optimal therapeutic use continues to evolve. Inhalation of aerosolized sustaining release of drugs in the lung can prolong drug action, reduce side effects and improve patient compliance. Therefore new sustained microparticles have been paid much attention by applying biomaterial and the techniques of pharmaceutics and clinical respiratory. Chitosan (CTS) is a cationic natural copolymer of glucosamine, which is abundant biodegradable biomaterial. It has been widely used in several pharmaceutical formulations as sustained release carrier systems such as beads, gels, films, sponges and microspheres for its many unique properties such as low toxicity, biocompatibility, biodegradability and good physicochemical properties. Furthermore, CTS has been used as a drug carrier to attain the desirable drug release profile and enhance dissolution rate of low water soluble drugs. Recently, CTS has become a good candidate for using in pulmonary delivery because its effect on the lung epithelium is reversable. It can bind with mucosal surfaces due to its cationic nature, which leads to bioadhesion and a reduced mucociliary clearance. In addition, CTS has another dramatic effect in terms of improving drug absorption (5～10 fold) by opening the intercellular tight junctions of the lung epithelium. In this paper, TH is used as model drug, while CTS andβ-cyclodextrin (β-CD) are used as drug carriers. The main aim is to study the characterization of TH/CTS/β-CD micropheres with different formulations made by spray drying as pulmonary sustained drug delivery systems. The microspheres are characterized by a series of physicochemical and powder properties, biocompability, degradability and pharmacodynamic effect.The TH/CTS/β-CD microspheres are prepared by spray drying method. The characteristics of TH/CTS/β-CD microspheres are carried out by Scanning Electron Microscopy (SEM), Laser diffraction, Fourier Transform Infrared Spectroscope (FT-IR) and UV-Vis Spectrophotometer. The result of SEM shows spherical microspheres with smooth or slightly wrinkled surfaces. The microspheres B have a narrower particle size distribution with the diameter between l and 10μm. FT-IR spectroscopy reveals that hydrogen bonds are formed between TH and CTS orβ-CD. The drug entrapments significantly increase from 13.33% to 35.70% with an increase of the ratio of drug/polymer. The encapsulation efficiencies are from 85.16% to 91.40%. The in vitro release of TH from microspheres is slower at pH 6.8 than that at pH 1.2 and also relate to the swelling ability, especially to the ratio of drug/polymer. The microspheres B have a prolonged release pattern with the release rate of 60.20% (pH 6.8) within 8 h. The results of permeation test reveale that the microspheres are easy to penetrate the membrane.The microspheres are stability under the experimental condition. The microspheres B have the low rate of moisture uptake (0.3071 mg/h) and low tap density (0.44 g/cm3).In order to overcome the disadvantage of acid soluble of chitosan, three kinds of carboxymethyl chitosan(CMCS)/β-CD microspheres loaded TH are successfully made by spray drying method and the characteristics are investigated. The microspheres obtained after spray drying are found to be spherical shaped with smooth or wrinkled surface. The mean particle size is between 3.39 and 6.06μm. The micropheres demonstrate high product yield (43.7-50.2%), high drug loading (13.7-38.1%), and high encapsulation efficiency (86.9-92.8%). The results of FT-IR indicate that there are interactions of TH with CMCS matrix. The TH/CMCS/β-CD microspheres are stability under the experimental condition which have the low rate of moisture uptake (0.0538～0.1864 mg/h) and tap density (<0.44 g/cm3). The in vitro release of TH from microspheres is slower at pH 1.2 than that at pH 6.8 and also related to the ratio of drug/polymer. The microsphere F has a prolonged release pattern (pH1.2).To study the biocompability of the microspheres, the tests such as the cell toxicity experiment, hemolytic test, acute cell toxicity experiment (LDH and protein in BALF), micronucleus test and muscle implantation test have been investigated. The results demonstrate that the microspheres have no toxicity and don't cause hemolysis. The micronucleus ratio of the microspheres is 0.99‰, being less than 3‰. The result of medullary micronucleus test is reported negative. The ciliary movement time of TH/CTS microspheres, TH/CTS/β-CD microspheres, and TH/CMCS/β-CD microspheres are 493.00,512.33 and 514.33 min,and the ciliary beat frequencies are 92.4%,96.0% and 96.4%.This result shows that the microspheres have no significant toxicity to palate mucosa. This result suggests that the microspheres can effectively reduce the ciliotoxicity and possess better adaptability. The total contents of protein and LDH in BALF of pulmonary delivery group are not significantly different from that of normal group. The wounds are free from suppuration and necrosis after muscle implantation in all periods. The results of implantation show that the microspheres have no effect on the hematopoisis, hemoglobin and have no toxicity in the liver and kidney. The inflammations of muscle tissue are not significantly different from that of operative suture, therefore, the TH/CTS/β-CD microspheres possess good biocompability and can be applied as pulmonary sustained release systems.In vitro degradation was performed in PBS buffer solution and with enzyme solution. Microspheres are found to be degraded by enzymatic hydrolysis and buffer solution, which indicate that enzymatic hydrolysis is faster than that in buffer solution. The degradation rate of MS A is faster than that of other microspheres (MS B,MS C and blank microspheres). Morphology of SEM of microparticles with PBS and enzymic solution develope rough surfaces and become irregularly shaped and porous after 12 weeks. In addition, microparticle aggregation is also observed both in PBS and enzyme solution. Microparticles degrade faster in vivo and show similar morphology after 8 weeks, which the TH/CTS/β-CD microspheres also degrade into irregular, sheet, porous shape, and the diameters are smaller than 5μm. The degradation rate of TH/CTS/β-CD microspheres has no different from that of the TH/CTS microspheres.The objective of this experiment is to study the influence of TH/CTS/β-CD microspheres on the allergen induced asthmatic rat model and the asthma-hypertension rat model, which also compare with the effect of TH solution. The quantity of WBC and its classification in blood is detected by automatic blood analyzer and in BALF is smeared sedimentation of the cell before stained with Wright-Giemsa and count differential leukocyte number. The LDH in BALF, ALT,AST and Cr in blood is detected by automatic clinical analyzer. The contents of protein in BALF are measured by protein quantification kit-rapid. To investigate the change of inflammation in the lung tissue and the blood pressure in asthma–hypertension rat model, the middle lobe of right lung is fixed in 10% formalin, then stained with hematoxylin-eosin and the blood pressure is measured. The total of WBC and Eos in blood and BALF of asthmatic group are higher than that of normal group (p < 0.05). Compared with the asthmatic group, the total of WBC and Eos in blood and BALF from DXM group is significantly different from asthmatic group (p < 0.05), the decrease tendency is found. The total of WBC and Eos in blood and BALF results of TH solution group, the TH/CTS/β-CD microspheres group (delivery to the lung), the TH/CTS/β-CD microspheres group (po group), and asthma hypertension group (po) are all significantly different from asthmatic group (p < 0.05), which are lower than that of asthmatic group, furthermore, there are no significant difference between these groups (p>0.05). The result of histological changes in asthmatic model reveal obvious allergic inflammation such as dissepiments of bronchioles and small vessels becoming thicker, epithelial damage and the tissue edema compared to normal group. The lung tissue of DXM group seem normal except a few inflammatory cell infiltration. The lung tissue of TH solution group seem normal too except a few inflammatory cell and Eos infiltration. In the TH/CTS/β-CD microspheres group (delivery to the lung), the TH/CTS/β-CD microspheres group (po group), and asthma hypertension group (po), the lung tissue also seem normal, but still a few inflammatory cell infiltration and tissue edema, which are signicantly different from asthmatic group. The routes of pulmonary delivery and po have no damages to the liver and kindney, and also have no effect on the blood pressure. There is no effect on the LDH and contents of protein in BALF. In short, the TH/CTS/β-CD microspheres can significantly reduce the quantity of Eos and the total of inflammatory cell in blood and in BALF and can inhibit airway inflammation, whose efficiency is similar to dexamethasone. This study indicates that the TH/CTS/β-CD theophylline/chitosan/β-cyclodextrin microspheres applied as pulmonary delivery is feasible.