Qing Kai Ling Injection Class And Intermediate Goods Of Allergy And Intracellular Pharmacokinetics

Qingkailing Injection is composed of Radix, gardenia, honeysuckle, buffalo horn, mother of pearl, baicalin, acid and flavor hyodeoxycholic,el. eight major drugs, which is developed by Beijing University of Chinese Medicine, having an effective treatment of stroke, hepatitis, and viral brain inflammatory. In recent years, its clinical application is also extended to the treatment of acute poisoning, cancer, chronic renal failure and other diseases.At the same time, it produce more and more adverse drug reactions, which limits its clinical application wide-spreaded. It is found that most symptoms of an allergic reaction appeared at the first time within30min by its analysis of adverse reactions. All these characteristics suggests that maybe it is a kind of anaphylactoid reaction. The study found that the sensitization mechanisms of Qingkailing injection could be primarily non-IgE-mediated, stimulating mast cells and basophils degranulation directly, through the release of a large number of allergic media, and then induce a series of class-induced allergic reactions.Currently, there is no guidelines and detection model of allergy testing of medicine injections, it is an urgent need to establish a sensitive and reliable model of allergy medicine research so as to solve the problem of adverse drug reactions. According to the mechanism of anaphylactoid reactions, there is a need to simulate mast cells to release allergy mediators in vitro cell model. Rat basophilic leukemia cell line (RBL-2H3) is an ideal alternative models of MC, which can simulate its physiological and pathological reactions.Previous studies have showed the pharmacokinetics(PK) of traditional Chinese medicine using High Performance Liquid Chromatography (HPLC) in blood, urine and saliva. However, the latest data suggest that more and more drug concentrations in plasma or tissues, which is the most commonly used site, cannot completely explain the drug efficacy on the target organ, instead, in cells. And those commen analytical methods for the pharmacokinetics of the drug have many problems, such as complex sample preprocessing required, low sensitivity, complicated process and costly outlay.The main results were summarized as follows:1Influence of Qingkailing Injection and Intermediate Products on cell proliferation of RBL-2H3cellsObjective: To investigate influence of Qingkailing injection (QKLI) and intermediate products on cell proliferation of RBL-2H3cells, and apply in establishing a cell model for anaphylactoid reaction, which can be used further for monitoring implementation of the whole process of QKLI. Methods:RBL-2H3was treated with11volume by volume concentrations (the initial is1/4, and the final is1/4096) of QKLI and intermediate products, the cell morphology was observed with inverted microscopy, and the cell proliferation was examined by methyl thiazolyl tetrazolium assay. Results:Different concentrations of QKLI and intermediate products have different effects on morphology, proliferation and apoptosis of the RBL-2H3cells. Conclusion: MTT assay can be used in test of proliferation of RBL-2H3cells, as well as determination of IC50, which could be used for further study as a cell model for anaphylactoid reaction. To explore the relationship between activity of RBL-2H3cells and anaphylactoid reaction and guide adverse drug reactions assessment, as well as monitor the whole process of QKLI.2Experimental Studies in Anaphylactoid Reactions of QingKailing InjectionObjective:To investigate influence of Qingkailing injection (QKLI) and intermediate products on degranulation of RBL-2H3cells, and evaluate anaphylactoid reactions of QKLI. Methods: The cell proliferation was examined by methyl thiazolyl tetrazolium assay, and compute IC50by SPSS17.0. RBL-2H3was treated with5different concentrations of QKLI and IC50of intermediate products,45min later, the release rate of β-hexosaminidase was examined by substrate method, and release amount of histamine using ELISA assay. Results:Different concentrations of QKLI have no concentration-dependent effect on the degranulation of RBL-2H3cells, and the extent of the degranulation of RBL-2H3cells is greater in the highest concentration of QKLI, and biggest in the extracts from honeysuckle. Conclusion: The results showed that different concentrations of QKLI could cause degranulation of RBL-2H3cells in varying degrees in vitro. Active material are released, which promote the happening of anaphylactoid reactions. Moreover, honeysuckle is probably the principal component of sensitization. This study would offer foundation of further study for mechanism of anaphylactoid reaction.3Study of influence of CA by immunoaffinity chromatography column to knockout CA of QKLI and RDNI.Objective:To investigate influence of CA by immunoaffinity chromatography column to knockout CA from QKLI and RDNI. Methods:Preparation of CA immunoaffinity chromatography column, and knockout CA from QKLI and RDNI. Then examine the release rate of β-hexosaminidase by substrate method of experiment2. Results:immunoaffinity chromatography of CA was prepared, CA of QKLI and RDNI were knocked out successfully. The extent of the degranulation of RBL-2H3cells was lower when CA of QKLI and RDNI were knocked out. Conclusion: CA is a suspect factor of TCMI sensitization.4Pharmacokinetic analysis of Baicalin in Qingkailing Injection by monoclonal antibody-based icELISA in RBL-2H3cellsBaicalin(BAL), being the flavonoids is extracted from Radix Scutellaria, which is a frequently used traditional Chinese medicine for several medicinal properties, such as clearing away heat and promoting diuresis, cooling blood and clearing away toxic material.On the basis of these, we successfully developed (ic)ELISA assay for the measurement and pharmacokinetic analysis of BAL in cells. All the pharmacokinetic parameters were computed using Kinetica version5.0software. We found a linear correlation in the range of5ng/mL-320ng/mL with a regression equation was Y=-0.14871n(C)+1.2203and a regression coefficient of0.994. Precision and accuracy of the icELISA method evaluated by the variations between replicates from both well to well (intra-assay) and plate to plate (inter-assay) repeatability and precision was achieved, with relative standard deviation (RSD) within the normal range of10%. A mean recovery of90-100%was obtained, with RSD lower than10%. Stability studies indicated that BAL sample solutions were stable during sample preparation and analysis. The main pharmacokinetic parameters derived from the above experiments were as follows:in the low concentration group: mean maximum concentrations (Cmax),82.43ng?mL-1; time of maximum concentration (Tmax),90min; AUClast=5.11ug?mL-1, AUCextra=0.71ug?mL-1, AUCtot=5.82ug?mL-1, mean residence time (MRT)=74.64min, and in the high concentration group: mean maximum concentrations (Cmax),426.59ng-mL-1; time of maximum concentration (Tmax),90min; AUClast=36.21ug-mL-1, AUCextra=12.25ug?mL-1, AUCtot=48.46ug?mL-1, mean residence time (MRT)=91.07min.Our study shows that the validated icELISA method can be successfully applied to study the pharmacokinetics of BAL in cells.