Preparation And Preliminary Pharmacological Evaluation Of S-allylmercaptocysteine Cyclodextrin Complex For The Treatment Of Pulmonary Fibrosis

Pulmonary fibrosis(PF)is one of the chronic and progressive interstitial lung disease,which is characterized by diffuse interstitial inflammation and fibrosis,destruction of alveolar structure,and eventually loss of lung function.The pathogenesis of pulmonary fibrosis is not clear because of its complex etiology.At present,the known pathogenesis includes inflammatory response,oxidative stress imbalance,epithelial mesenchymal transition(EMT),abnormal proliferation of myofibroblasts and transforming growth factor betal(TGF-?1)signal pathway overexpression and so on;there still have high mortality because the deficiencies in traditional clinical treatment methods,especially in the middle and late stage of pulmonary fibrosis.Therefore,it is necessary to develop a therapeutic drug to inhibit the development of lung fibrosis.S-allylmercaptocysteine(SAMC)is a kind of water-soluble organic sulfur garlic derivative,has been considered for many pharmacological effects,such as anti-inflammatory,anti-oxidation,anti-concer,and immune regulation.Based on the pathogenesis of pulmonary fibrosis and the pharmacological mechanism of SAMC,this paper explored the preliminary therapeutic effect and mechanism of anti-fibrosis effect of SAMC.In addition,because the water solubility of SAMC can not reach the concentration in vivo and in vitro,it is necessary to carry out solubilizer screening to improve its solubility.The main contents of this study include the following three parts:(1)Preparation and evaluation of new preparation of SAMC;(2)The study on anti-fibrosis effect and its signal pathway of new preparation of SAMC in vitro;(3)The study on the treatment effect and mechanism of new preparation of SAMC on pulmonary fibrosis mice.The research contents of SAMC new preparation include the establishment of HPLC methodology,the selection of solubilizer and formulation,the optimization of preparation process and the preliminary performance evaluation.We first established HPLC methodology of SAMC and used it in prescription screening.The study of SAMC solubilizer screening showed that the solubilization effect of cyclodextrin excipients could meet the requirements of this research.The best inclusion material hydroxypropyl-?-cyclodextrin,was selected from three different cyclodextrin derivatives by phase solubility method.Single factor screening and orthogonal experiment were used for inclusion,and the best formulation of S AMC-HP-?-CD was obtained as follows:HP-?-CD:SAMC was 5:1,the best preparation condition is magnetic stirring at 40? for 3 h.The formation of SAMC-HP-?-CD inclusion complex was confirmed by differential scanning calorimetry(DSC)and infrared spectroscopy(FT-IR).Finally,we studied the effect of inclusion on the dissolution and release of SAMC-HP-?-CD.The study of anti-fibrosis effect and signal pathway of new of preparation of SAMC in vitro consists of three parts:the establishment of pulmonary fibrosis cell model,the evaluation of anti-fibrosis effect and the study of signal pathway.We used TGF-?1 to stimulate the model of the transformation of fibroblast(HFL-1)to myofibroblast(FMT).We got the optimal concentration of TGF-?1 stimulation was 5 ng/mL and the optimal time was 48 h by using the expression of ?-SMA as an indicator.The expression of ?-SMA protein was significantly reduced after administration of SAMC-HP-?-CD,which indicated that SAMC-HP-?-CD could inhibit the transformation of fibrocytes.The expression of TGF-?1 and its downstream proteins,P-Smad2 and P-Smad3 were decreased after administration of SAMC-HP-?-CD,and it would inhibit the process of FMT by inhibiting TGF-?1/Smad2/3 signal pathway.The study on the prevention and treatment effect and mechanism of new preparation of SAMC on pulmonary fibrosis mice mainly includes the establishment of mice model of pulmonary fibrosis;the evaluation of anti-fibrosis effect in vivo;the study on the mechanism of anti-fibrosis effect of new preparation of SAMC.Finally,we established the PF mice model by intraperitoneal injecting BLM.After administration of SAMC-HP-?-CD by gavage,we observed the morphological changes of lung tissue by HE staining and Masson staining;counted the inflammatory cells in BALF and measured the level of inflammatory factors and the content of oxidation related factors in lung homogenate to determine the prevention and treatment of SAMC-HP-?-CD in PF mice.At the same time,Western blot and Immunohistochemistry were used to study the regulation mechanism.The results indicated that SAMC-HP-?-CD could reduce the infiltration of inflammatory cells and the aggregation of collagen fibers in the lung tissue,and inhibit the secretion of pro-inflammatory factors like TNF-? and IL-6.It can inhibit the production of ROS,increase the level of GSH and SOD,decrease the level of TGF-?1 in BALF and the level of HYP in lung tissue to protect mice avoid BLM injury.In terms of molecular mechanism,SAMC-HP-?-CD can inhibit the production of collagen in mouse tissues and the progress of fibrosis by restraining the expression of ?-SMA,Col ? and col ?proteins;it plays an anti-fibrosis role by inhibiting TGF-?1/Smad2/3 pathway and activating Nrf2/HO-1 pathway.In conclusion,the project first selected a suitable dose form to improve the solubility of SAMC,carried out the formulation selection and process optimization,and carried out the characterization and preliminary performance evaluation.It is shown that the new SAMC preparation could inhibit TGF-?1 signal pathway to achieve its anti-fibrosis effect to inhibit the process of FMT in vitro.It was confirmed that the new SAMC preparation can reduce the expression of inflammatory cytokines,oxidative stress response and the expression of fibro related factors in the lung tissue of mice,and its mechanism is to regulate the TGF-?1 and Nrf2 signaling pathways in vivo.