| This is the first attempt to develop an azithromycin (AZM) dry powders for inhalation, is mainly used for local treatment of pneumonia. There are many advantages:AZM is of a broad-spectrum antibiotics and it is the first choice for the treatment of community acquired pneumonia. The immune adjustment of AZM can improve lung function and reduce inflammation & lung injury; AZM is of time dependent antibiotics, which ensures the safety and effective. High local concentration in lung not only can reduce the dosage, the bacterial resistance and side effects, but improve the patient compliance.At first, HPLC method and UV method (sulfuric acid color method) were developed to determine the content and in vitro release of AZM, separately. The stability, specificity and reproducibility of the method were good. A quality evaluation system including two types of index, physicochemical properties (i.e. hygroscopicity, moisture content, morphology, particle size and distribution, density and flowability) and aerosol characteristics (emitted dose and respirable fraction), was established. Scanning electron microscopy, laser diffractometry, thermogravimetry and Twin Stage Impactor were performed to characterize the manufactured powders.The stability of AZM solution was investigated before the preparation of AZM dry powder inhalation (DPI). The observed rate obtained by measuring the remaining intact AZM was shown to follow pseudo-first-order kinetics. The maximum stability of AZM was shown at an approximate pH 6.3. Bronchoalveolar lavage (BAL) was employed to extract the enzyme on rat lung surface. More than 80% of bronchoalveolar lavage fluid (BALF) was recovered. AZM was stable for 8.0 h in BALF at 37℃. The enzyme in lung had no effect on lactone bond of AZM to ensure the effectiveness of local treatment. The pH of lung is about 7.30～7.56, AZM is stable at least for 32 h in lung tissue homogenate. And, AZM were stable in SLF, Ringer and ACD, which were benefit for the release of AZM in vitro and microdialysis study in vivo. Taking stability, solubility and irritant into the consideration, the pH of AZM solution for spray was selected as pH 7.0.Different carriers such as suga、sugar alcohols、amino acid carrier F68, and pH regulators were investigated in the experiment. Based on the selected carriers and pH regulators, Central composite design (CCD) was used for conducting the experiments. Finally, the feedstock solution containing 6.0 g AZI,1.82 g Mannitol,1.35 g L-leucine and 38 mg F68 was diluted to 400 mL and then spray-dried under certain condition. Subsequently, multi-index evaluation was performed to optimize the preparation process of AZM DPI. The extreme difference demonstrated that the factors affecting the quality of DPIs following the order of feed flow rate>atomizing pressure>air flow>inlet temperature. According to the Z-score results, the statistical optimization of the spray drying variables was:inlet temperature 120℃; atomizing pressure 190 kPa; air flow 0.7 m3 min-1 and feed flow rate 4.0 mL min-1.The optimized AZM DPI was prepared under the optimum formulation and preparation process, which had a volume average diameter of 5.66μm, an aerodynamic diameter of 3.82μm, a repose angle of 36.0°, a moisture content of 2.27%, and a RH of 43.3%. The product could be quickly atomized to form a uniform smoke aerosol with good emptying rate of more than 90% and an in vitro deposition of 51.04%. Air flow rate and sample loading had no influence on in-vitro deposition. Meanwhile, the result of X-ray powder diffraction and the analysis of the powder surface revealed that AZM DPI was amorphous and leucine was rich on the surface of the AZM DPI.The sustained AZM albumin microspheres were prepared by thermal denaturation. All the quantity of albumin, the time of thermal treatment, the temperature of thermal treatment affect in-vitro release of AZM albumin microspheres. Considering microsphere drug loading, stability and release characteristics in vitro, BSA5 and BSA5-120℃-24h were used for further study in vivo. After thermal denaturation, there is no change for the volume average diameter and the angle repose of BSA5-120℃-24h microspheres, but the content increased slightly, tap density and aerodynamic particle size decreases slightly.AZM PLGA porous microspheres were prepared by double emulsion-solvent volatilization method. The resulting product is fluffy and very light, and bulk density (ρb), tap density (ρtap) and Carr’s Index were 0.07 g cm-3,0.11 g cm-3 and 33%, separately. The PLGA porous microspheres had a volume average microsphere diameter of 18.15μm, particle aerodynamic diameter of 6.02μm. Meanwhile, good atomization performance was obtained. The emptying rate was greater than 90%, drug loading was 18.1%, and encapsulation efficiency was about 54.3%. The in vitro release of AZM PLGA porous microspheres was similar with BSA5-120℃-24h in general, but the characteristic of sustained release was more obvious and burst release in early stage was improved compared to BSA5-120℃-24h.A simultaneous blood and lung microdialysis coupled with UPLC-MS/MS method was established for in vivo analysis of AZM. In vivo recoveries of the probes determined by retrodialysis were 5.57% and 5.88% for blood and lung, respectively. The local pharmacokinetics of AZM DPI and BSA5 in ELF after trachea] administration was investigated. For comparison, the pharmacokinetic behavior of AZM solution after intravenous administration was also investigated. The values of AUCELF/AUCBlood obtained from AZM DPI and BSA5 were 42.30±30.8 and 11.10±4.59, respectively, which was significant different from that of solution,0.087±0.59(p<0.05). Significant pulmonary Loction for both AZM DPI and BSA5 were obtained. And the apparent ELF bioavailabilities were 161.6 and 156.2, respectively. Pulmonary administration increased the AZM concentration in ELF and reduced the AZM concentration in blood, so it was helpful for the local treatment of pneumonia. Furthermore, the pharmacokinetic behaviors in ELF of AZM DPI, BSA5, BSA5-120℃-24h and porous PLGA microspheres were investigated and compared. Compared with AZM DPI, MRT of BSA5-120℃-24h was significantly prolonged. It increased residence time in ELF (p<0.05) to some extent, but AUCELF/dose and Cmax/dose were significantly reduced (p<0.05). As for AZM porous PLGA microspheres, it increased residence time in ELF significantly, reduced clearance and apparent volume of distribution, subsequently increased the unit dose of AUC and Cmax (p <0.01). An obvious sustained release effect was shown. AZM was easily uptaked by macrophages and accumulated in macrophages, while some small particles (especially particle size less than 5μm) were also easily phagocytized. It had a great impact on the pharmacokinetic of AZM in the ELF.A slightly stimulation for lung tissue was observed for AZM DPI, BSA5-120℃-24h and AZM PLGA porous microspheres, respectively. There were no edemas in lung slices of each group. All showed a uniform sponge-like. However, there were slight neutrophil infiltrations in lung slices of two rats at 4.0 h after tracheal administration of AZM porous PLGA microspheres, which caused mild inflammation. While the immune adjustment of AZM can improve lung function and reduce inflammation & lung injury. Therefore, AZM DPI, BSA5-120℃-24h and AZM porous PLGA microspheres were safe to some extent. |
PDF Full Text Request