| Pulmonary arterial hypertension is an extremely malignant cardiovascular disease, which possesseshigh mortality rateandpoor prognosis. In recent years, new targeted drugs for pulmonary arterial hypertensionhavebeen used in clinicalgradually, which improve the life quality.Tadalafil is a kind of long-acting PED-5 inhibitor, in May 2009, the FDA approved tadalafil for the treatment of pulmonary arterial hypertension. In the typical pulmonary arterial hypertension targeted drugs, tadalafil was the only one thatis not affected by dietary, and tadalafil can significantly increase exercise tolerance of patients, improve the dilation of flow-mediated, retard the further deterioration of pulmonary arterial hypertension. Tadalafilhas poor water solubility, resulting in a low bioavailability. So changing the dosage form to improve their bioavailability and reducing side effects of long-term medication, have important clinical significance.Dry Powder Inhalation for pulmonary drug delivery could deliver drug to the lung directly, which exerts a local effect, significantly reduces the amount of drugs and the distribution in other parts of the organization, thereby improving the efficacy and reduce systemic side effects of drugs. However, the general inhalationcould easily lead to high local concentrations of drug,possessinga certain side effects to lung, and requires frequent inhalation thus leading to a poor patient compliance. Sustained release preparations could release drugcontinuously and extend the duration of action of drugs, thus reduces the frequency of administration, having a safe and effective therapeutic effect.In this study, biology biodegradable materialpoly (lactic-co-glycolic acid), (PLGA) was used as carrier, tadalafil PLGA microspheres(TDF-PLGA-MS) was prepared by emulsification - solvent evaporation, and the microspheres wasdried by freeze-drying, preparing powder inhalation for pulmonary inhalation. This research included:the preparation of tadalafil PLGA microspheres, the physical-chemical properties and micromeritics studies ofTDF-PLGA-MS powder, in vitro release and preliminary stability study, lungs pharmacokinetics and tissue distribution study of tadalafil microsphere in rats.1. The formulation and preparation process of TDF-PLGA-MSA method to measuring tadalifil contend has been established by UV spectrophotometry; With encapsulation efficiency and particle size as evaluation index, tadalafil microspheres was preparedby emulsification-solvent evaporation, in the base of univariate study, the formulation and preparation processwere optimized by orthogonal design experiment; TDF-PLGA-MSdried power was prepared by freeze-drying and lyophilization process and formulation were investigated.Study results showed thatthe good precision,high recovery rate, and the good linear relationship in 3.54～26.55μg/ml could meet the measurement requirements; Based on single factor investigation,theorthogonal design was used to optimize the prescription andprocesses of microsphere. The optimal formulation and process as follows:drug and lipid ratio was of 1:8, PLGA concentration was of 100mg/ml, an oil phase/aqueous phase volume ratio was of 1:4, the aqueous/dilute phase volume ratio was of 1:5, shear rate was 5000rpm, shearing time was 0.5min.The best technology for the lyophilization:1ml 1％mannitol solution used as a scaffold agent,-80℃ pre-frozen 12h, freeze-drying 24h, and then obtain the microspheres powder. Three batches reproducible results showed that prescription and technology showed good reproducibility.2.The physical-chemical properties and micromeritics studies ofTDF-PLGA-MS dried powerUsing the naked eye observed the power appearance, optical microscopy and scanning electron microscopy observed microspheres form; Laser particle sizer measured tadalafil microsphere size and distribution; The microsphere were separated by centrifugation, and its entrapment rate and drug loading were detected by UV. The pH value of dried microsphere was determined; In addition, the powder properties were investigated.The results showed that the microspheres were full of loose powder, the microspheres were evenly rounded ball, no adhesion between the microspheres at optical microscopy and scanning electron microscopy; Particle size distribution was well-distributed, and an average particle size before drying was (10.04 ± 0.09) μm, and average particle diameter after drying was (10.29 ± 0.25) μm, no significant difference; The entrapment rate and drug loading was (81.68 ± 0.55)％and (8.52 ± 0.06)%, respectively, the pH of the microsphere suspension was (6.95 ± 0.05), close to neutral. Powder properties investigation results showed that the moisture content of the microspheres was (1.99±0.23)％, angle of repose of powder microspheres was (35.90 ± 2.05)°, a bulk density was (58.64 ± 2.06) mg/ml, emptying rate was (95.41±1.22)％, aerodynamic diameter was of (2.80 ± 0.05) μm. The result showed powder flowability and powder properties were good,meeting pulmonary administration require.3.Characteristics oftadalafil microspheres in vitro release and preliminary stability studiesIn vitro releaseof TDF-PLGA-MS and TDF-Sol weredetermined by dynamic membrane dialysis and the preliminary stability of microspheres was detected by influencing factors test (high and low temperatures, high humidity, light).The results showed that the drug was released rapidlyfrom tadalafil solution (TDF-Sol)and cumulative releasepercentage reached 98.72％at 8h, but TDF-PLGA-MS release was relatively slow,cumulative release was 46.87％at 24h, at 10d was 84.06%, indicating there was a certain burst, a proper burst effect could reach effective blood concentrations quickly and thus play the role of treatment, and the prepared microspheres hada bettersustained release effect. Factors Experimental results show that TDF-PLGA-MS gathered adhesion and the drug content decreased under high temperatures, high humidity, and light conditions, therefore the microspheres should be stored at cool and dryconditions.4. Lungs pharmacokinetics and tissue distribution studyofTDF-PLGA-MS in rats.An HPLC method was established for the determination of drug concentration in plasma and differenttissues, and its methodological was visited. Tadalafil solution as a control group, thepharmacokinetics in the blood and lung, and distribution in various tissues were studied after the intratracheal instillation of tadalafilmicrospheres.Methodological study results showed that the method for the determination of drug concentration in plasma and the organizations were established by specific, high precision, and all the recoveries distributed between 85％and 115％, which could met the requirements for the determination of biological samples.The measuring results of the drug concentrationin lung showed thatwhen infusingTDF-PLGA-MS into lungs, the drug penetrated into the blood slowly, the drug concentrations in lung was 3.5 times of the TDF-Sol group,and the drug concentrations in lung at 10d was still higher than the concentration of the TDF-Solat 48h, indicating microspheres showed a good sustained release effect.; Lung pharmacokinetic parameter showed intratracheal instillation of tadalafil microspheres, the elimination phase half-life t1/2β and the mean residence time MRTo-∞ were 3.10 times and 3.96 times of the solution group, respectively, the lungs residual drug concentration Cmax and AUC values were 3.48 times and 16.36 times of solution group,respectively, indicating that tadalafil was detainedin the lungs for a long time after being encapsulated in microspheres and the drug released slowly,thus, improving significantly therapeutic effect ofpulmonary arterial hypertensionandreducing the frequency of administration.In addition, when infusing TDF-PLGA-MS into lungs, the drug concentration in lungs was always higher than the solution group, while drug concentration in plasma was relatively lower than the solution group, indicating TDF-PLGA-MS was effective in the treatment ofpulmonary arterial hypertensionand reduced the leakage of the drug into the bloodstream and reduce systemic side effects.The results of tissue distribution showed that the Re of tadalafil in heart, spleen, liver and kidneyswere 0.367,0.442,0.451 and 0.699, respectively, whereas the Re values in the lung was 16.358after intratracheal instillation of TDF-PLGA-MS, indicating microsphere had a low targeting to heart, liver, spleen and kidney, while in the lungs had a higher retention than solutions group, thus enhancing greatly the therapeutic effect of the drug on pulmonary arterial hypertension, reducing non-target organ toxicity side effects. It provides an important clinical application value for the long-term application of tadalafil.Conclusion:In this study, tadalafil microspheres used forpulmonary deliverywas preparedbyemulsion solvent evaporation;Preparation process is simple and reliable, reproducible, and the EE is relatively high. TDF-PLGA-MS dried power was prepared by freeze-drying technology, the physical-chemical properties were stable, micromeritics properties were good, which could meet the needs of pulmonary administration. The results in vitro release test showed that tadalafil was released from TDF-PLGA-MS slowly, sustained releasing up to 10d. Lung pharmacokinetics and tissue distribution experimentresults showed that TDF-PLGA-MS had a good sustained release effect and a long retentiontime in lung; besides, it could reduce the leakageof drug to blood and other tissues. In conclusion, tadalafil microspheres used forpulmonary delivery overcame the frequentadministrationand side effects of general formulation, improved therapeutic efficacy and patient compliance. |
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