The Study On Baicalin/Ambroxol Lung Inhalation Preparation

Pulmonary fibrosis,as a sequelae of severely ill patients with COVID-19,has a high incidence,and the treatment effect isn’t obvious from all aspects.The pathogenesis of pulmonary fibrosis is currently unclear.Medical practitioners believe that pulmonary fibrosis is caused by factors such as inflammation,immune response,and lung injury.Pirfenidone and nintedanib are currently two approved oral preparations for the treatment of pulmonary fibrosis,but the therapeutic effects of the drugs are limited and both have serious adverse reactions.Therefore,it is urgent to find therapeutic drugs and dosage forms that are effective,safe and effective.Baicalin（BA）is an important active ingredient in Scutellaria baicalensis Georgi.It has a wide range of pharmacological activities,such as anti-inflammatory,anti-oxidation,protection of myocardial ischemia and liver protection,anti-tumor,immune regulation and other modern pharmacological effects.It can be used clinically for pneumonia,hepatitis,respiratory infection,cerebral ischemia and tumors,etc.Ambroxol（AH）is a kind of airway lubricating and expectorant drug,which has anti-inflammatory,anti-oxidation and promoting the release of alveolar surfactant.It is commonly used in the treatment of chronic respiratory diseases such as bronchial asthma,emphysema,tuberculosis,postoperative cough and so on.The combination of BA and AH can relieve cough and phlegm,improve lung function,delay and treat pulmonary fibrosis.However,the very low solubility of BA results in low oral absorption and availability,which limits its application.Dry powder inhaler for lung administration is a new drug delivery technology,which is composed of insoluble and unstable drugs and excipients with moisture-proof,good fluidity and no irritation.The preparation has smaller particle size and larger surface area,and also improves the solubility of the drug.In this project,a baicalin/ambroxol dry powder inhaler（BA/AH-DPI）was prepared by a co-spray drying method,and the prepared dry powder particles were systematically studied,including the particle size and shape of the dry powder particles,moisture,and hygroscopicity,Crystal morphology and in vitro aerodynamics,as well as preliminary in vivo pharmacodynamic evaluation and pharmacokinetic properties.The specific content includes the following parts.1.Preparation and quality evaluation of BA/AH-DPIThis chapter mainly established the content determination methodology of BA and AH,and investigated the specificity,repeatability,precision and stability of the method.The results showed that the method was simple,rapid,reproducible,and the two drugs were stable.This chapter adopts nano spray drying method to prepare BA/AH-DPI particles.The particle size of the method is uniform,and the method is simple and feasible.And the process of adding different proportions of L-Leucine（L-leu）was investigated.Using a series of physical and chemical properties and quality evaluation such as its preparation yield,particle size and morphology,moisture,hygroscopicity and in vitro aerodynamics as indicators to screen out the optimal prescription ratio.The measured results of the laser particle size analyzer show that the particle size of the prepared particles meets the requirements（D90<5μm）,the distribution is uniform,and the system is stable.Particle size results more intuitively observe the shape of DPI particles are spherical with wrinkles on the surface.The differential scanning calorimeter（DSC）and X-ray diffractometer（XRD）results show that the DPI powder has a crystalline form to an amorphous form during the preparation process.The hygroscopicity results show that as the content of L-leu in the prescription increases,the moisture content in the dry powder decreases and the hygroscopicity decreases.The results of in vitro aerodynamics show that the addition of L-leu can increase the fluidity of the micropowder and increase the fine particle fraction（FPF）;when the proportion of L-leu is 20%,the FPF is the largest and the fluidity is better.Through the above screening,the best prescription ratio is selected（20%L-leu）,which lays the foundation for the follow-up preliminary pharmacodynamics and pharmacokinetics in vivo.2.Pharmacodynamics of BA/AH-DPI in vivoThis chapter establishes a rat idiopathic pulmonary fibrosis model and randomly divides it into normal group,sham operation group,bleomycin（Bleo）group,Bleo+BA-DPI group,Bleo+AH-DPI group,Bleo+BA/AH-DPI group,the Bleo+tail vein injection group and the Bleo+pirfenidone group were administered continuously for 28 days.By testing the lung function of rats compared with the Bleo group,the parameters TI,TE,TV,EV,RT,PEF,PIF and AV in the Bleo+BA/AH-DPI group were significantly different（p<0.01）;HE staining of lung tissue in Bleo group showed typical fibrotic appearance,inflammatory cells were infiltrated by inflammatory exudate,while Bleo+pirfenidone group and Bleo+BA/AH-DPI group showed less pathological changes of lung tissue and smooth surface Plump,with few bleeding points;compared with the Bleo group,the lung coefficients of the Bleo+pirfenidone group and the Bleo+BA/AH-DPI group were different（p<0.01 or p<0.05）;Determination of lung bronchoalveolar lavage fluid（total protein content,IL-8,IFN-γ,IL-4,IL-6,IL-1βand TGF-β1）inflammatory factors are all different（p<0.01 or p<0.05）;There are also differences in inflammatory factors and oxidative stress in lung tissue homogenate（HYP,SOD,MDA,LDH）and serum（MPO）（p<0.01 or p<0.05）.The tail vein injection and pirfenidone were used as a control to prove the feasibility and rationality of BA/AH-DPI pulmonary administration.The exploratory research on the combination of BA and AH proved that compared with the pulmonary administration of BA-DPI and AH-DPI,the combination of BA/AH-DPI has a more prominent therapeutic effect on idiopathic pulmonary fibrosis.There is no difference in the treatment of pirfenidone（p>0.05）,which confirms the feasibility of the combination of the two drugs and has far-reaching significance in clinical treatment for further application.3.Pharmacokinetics of BA/AH-DPI in lung administrationIn this chapter,a LC-MS/MS method for the determination of BA/AH in rat plasma and lung tissue was established.Other components in plasma and lung tissue have no interference with BA/AH and internal standard detection,and linearity,precision,accuracy,recovery rate,and matrix effect meet the requirements.The above method was used to detect the contents of BA and AH in the plasma and lung tissues of the rats in the tail vein injection group and the pulmonary administration group at 6 hours,and the relevant data were brought into the Pharsinght Winnonlin software to fit the non-compartmental model to calculate the pharmacokinetics Learn related parameters.Statistical analysis was performed using Graph Pad Prism 8 software,and the difference in pharmacokinetic parameters between different groups was investigated by T test method.The results showed that BA reached the maximum plasma concentration in plasma at about 9 min after pulmonary administration（p<0.01）,and compared with tail vein injection,the half-life in vivo was significantly prolonged（p<0.01）,and the average retention time in the body was significantly extended（p<0.01）,so as to achieve the effect of continuous absorption.The half-life is relatively large,the absolute bioavailability reaches 60.7%,and the bioavailability is high.After pulmonary administration,the plasma concentration of AH reached its peak in the plasma about 4 min,the drug retention time in the body was slightly longer（p>0.05）,and the absolute bioavailability reached 53.5%.After pulmonary administration,the AUC_0-6h of BA in the lung tissue was（11136.06±1703.71）h·ng/m L,while the AUC_0-6h in the lung tissue of the tail vein injection group was（344.81±175.48）h·ng/m L,with significant difference（p<0.01）.Compared with tail vein injection,pulmonary administration significantly improved the bioavailability in the lung tissue of BA,and the clearance rate in the lung tissue was lower（p<0.01）.Compared with tail vein injection,AH has a relatively longer residence time in lung tissue（p>0.05）,AUC_0-6his significantly higher（p<0.01）,half-life is prolonged（p>0.05）,and clearance in vivo The rate is reduced（p<0.01）.It can be seen that the DPI particles prepared by spray drying used for pulmonary administration can significantly improve the lung bioavailability of BA and AH,and have certain lung targeting properties,which bring potential prospect for the treatment of lung diseases.