Pulmonary Biopharmaceutics Study Of Chuankezhi Injection And Icariin Phospholipid Complex

Chuankezhi injection(CKZ)is a traditional Chinese medicine(TCM)refined from two traditional Chinese herbs,i.e.Yin-Yang-Huo and Ba-Ji-Tian and it has been utilized for the treatment of respiratory diseases,such as pneumonia,recurrent respiratory infection and asthma.As an injectable formulation,repeated injection of CKZ often leads to low patient compliance.As a consequence,CKZ has been clinically used off-label as a nebulization therapy for treating respiratory diseases in recent years.However,little is known about the aerosolization performance and pulmonary fate of the inhaled CKZ.Furthermore,the active ingredients in CKZ exhibit rapid metabolism and elimination in vivo.Thus,we need to find the ingredient in CKZ with long-acting anti-inflammatory effect and prolong the lung retention of this ingredient through preparation of phospholipid complex.Therefore,this work aimed to evaluate the aerodynamic characteristics of nebulizer generated aerosols and to compare the properties of pulmonary pharmacokinetics and anti-inflammation effects of CKZ after intratracheal and intravenous administration.In addition,considering that icariin represents the major active component of CKZ,this work also intended to prepare icariin-phospholipid complex(IPC)for sustained nebulization delivery that enabled excellent inhalability,improved lung exposure and prolonged duration of action.First,the nebulization performance of CKZ was evaluated in vitro based on the aerodynamic particle size distribution and aerosol output.The results showed that both CKZ and diluted CKZ solutions could be aerosolized using a Pari LC Sprint Star nebulizer to generate stable aerosols and the D(50)values of the aerosols from either CKZ or diluted CKZ solution were between 3.47 and 3.51 μm whereas the MMAD values of the major ingredients were almost identical,ranging from 3.24 μm to 3.26 μm with the fine particle fractions being between 71.5%and 76.5%.The delivery efficiency in the percentage of nominal dose under adult breathing pattern ranged from 28.7 ± 2.02%to 31.8 ± 1.32%whereas those under child breathing pattern were between 18.7± 1.38%and 21.9± 0.32%.Second,the pulmonary biopharmaceutics of CKZ was evaluated based on the concentrations of the main active ingredients of CKZ in plasma,bronchoalveolar lavage fluids(BALF)and lung tissues whereas the pulmonary anti-inflammatory efficacy were tested using LPS-induced pulmonary inflammation mice model as indicated by the total cells counts,and the levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in BALF.Following intratracheal instillation of CKZ,epimedin A,B and C,and icariin,were absorbed into the bloodstream with the mean absorption time varying from 101.8 min to 271 min,and their absolute bioavailabilities ranging from 26.4%to 104%.The instillation of CKZ increased the lung to plasma concentration ratios by 25.5-718 folds compared to intravenous administration,leading to improved and prolonged local anti-inflammatory effects.Third,CKZ was separated into non-flavonoid and flavonoid fractions and each fraction and the major flavonoid compounds were subjected to pulmonary antiinflammatory efficacy evaluation.The results showed that flavonoids were mainly responsible for the anti-inflammatory effects and both epimedin C and icariin exhibited dose-and time-dependent anti-inflammatory efficacy.Finally,icariin was found to react with soybean phospholipid to form supramolecular IPC,which was able to self-assemble into nanoparticle suspension.The suspension was stable during autoclaving and nebulization processes with its aerosols generated by a commercial nebulizer showing excellent aerodynamic properties and delivery efficiency.In vitro studies showed that the formation of complex sustained drug release,improved lung affinity and slowed lung dissociation.Following intratracheal administration to mice,the drug distribution in lung epithelial lining fluid(ELF)also demonstrated in vivo sustained release.In addition,compared to free icariin,IPC improved the drug exposure to lung tissues and immune cells in the ELF by 4.61-fold and 39.5-fold,respectively,leading to improved and prolonged local anti-inflammatory effects up to 24 h in lipopolysaccharide-induced acute lung injury in mice.In summary,this work revealed that CKZ exhibited excellent nebulization performance.Compared to intravenous administration,intratracheal administration of CKZ could prolong the pulmonary drug retention and improved lung to plasma ratio,leading to prolonged duration of anti-inflammatory effect.Icariin-phospholipid complex was suitable for sustained nebulization delivery that enabled excellent inhalability,improved lung exposure and prolonged duration of action.