A Study On Preclinical Bioactivity And Content Assay Of Recombinant Protein Drug

Human Brain Natriuretic Peptide(hBNP),also known as B-type Natriuretic Peptide,is an endogenous polypeptide secreted by the ventricles as a compensatory function during heart failure.Through natriuresis,vasorelaxation,and inhibition of renin-angiotensin-aldosterone and sympathetic nervous system,it can reduce cardiac preload and postload,improve hemodynamics of patients with congestive heart failure(CHF),and thus play a role in maintaining cardiac function.Recombinant BNP drugs have been used for first-line treatment of acute heart failure in China and abroad.In addition,due to significant changes in BNP concentrations in patients with heart failure,the detection of plasma BNP levels has also been listed as an important indicator of clinical diagnosis and prognosis evaluation.This study mainly focuses the following three parts.1.A double-antibody sandwich Enzyme-Linked Immunosorbent Assay(ELISA)method was established to determine the drug concentration in plasma after hBNP administration.The method was optimized and the methodological verification was completed.At present,this approach has been used for preclinical animal pharmacokinetic studies of hBNP.2.hBNP binds to a specific receptor and activates guanylate cyclase and then increasethe intracellular level of cyclic guanosine monophosphate(cGMP).As a second messenger,cGMP can exerts a biological effect in the cell.Therefore,measuring the intracellular cGMP changes can reflect the biological activity of hBNP.This research cooperated with the China Institute of Food and Drug test to establish a competitive enzyme-linked immunosorbent assay(ELISA)method by constructing a monoclonal cell line that stably transduces the hBNP receptor gene,and completed the method validation.This method has been used for the detection of hBNP activity in the present study.The method has been applied for detection of biological activity of rhBNP products.3.A New Zealand rabbit model of acute heart failure was established by infusing pentobarbital sodium into New Zealand rabbit ear veins.New Zealand rabbits with successful heart failure were given high,medium and low-dose self-prepared recombinant h BNP to determine hemodynamic parameters.By measuring the hemodynamic parameters and the activities of plasma creatine kinase and lactate dehydrogenase,a preliminary study was conducted on the efficacy of the on-going products in animals.The results showed that the recombinant hBNP had the same therapeutic effect as the original drug.The results of this study laid the foundation for the successful completion of the preclinical research and development of rhBNP.