The Stability And Metabolism Of Protamine Peptide Analog In Vitro

Protamine sulfate,a highly basic protein with more 60%arginine isolated from various fish sperms,is routinely used as heparin antidote.It was widely used as an antagonist of heparin in extracorporeal circulation surgeries,such as cardiopulmonary bypass and hemodialysis.But protamine sulfate has many adverse effects which include obvious arterial hypotension,pulmonary hypertension and allergic reactions.Thus far,protamine sulfate is the only antidote approved by the U.S.Food and Drug administration and CFDA to reverse heparin activity.Therefore,we synthesized a peptide hydrochlorate with fifteen arginine residues as a protamine substitute?abbreviated as R15?,the molecular weight is 2.362 kDa.A preliminary pharmacodynamic evaluation concluded that R15 has similar ability of antagonism to heparin as protamine sulfate.The method for the study of protamine mimetic peptides in biological matrices and solutions had not yet been developed.We aimed to establish high performance liquid chromatography method for quantifying protamine peptide analog in rat blood.The stability of API?Active Pharmaceutical Ingredient?and stock solutions of protamine mimetic peptide was investigated.And further studied the metabolic stability in vitro and related metabolic enzymes of protamine mimetic peptide by using the technology.The metabolic pathways of protamine mimetic peptides were to provide reference for further chemical modification of candidate compounds and pre-clinical research,to reduce the risk of new drug development.1.The stability of of protamine mimetic peptideObjective:To establish a high performance liquid chromatography method for determining the content of protamine mimetic peptide in stock solution.The stability of API of protamine mimetic peptide was investigated referring to technical guidelines for the study of the stability of combined drugs?raw materials and preparations?.And to study the stability of stock solutions of protamine mimetic peptide by using the UPLC method.Methods:HPLC method was adopted.To study the stability of R15 stock solution sample placed at 4?for 15 days.The high temperature condition was 20?±5?,the high humidity condition was 92.5%RH,and the intensity of light illumination was 4500Lx±500 Lx.Results:The linear range of protamine mimetic peptide was 20⁵00?g·mL^-1,the recovery rate was 100.9%,RSD was 0.6%,repeatability RSD was 0.2%,and protamine mimetic peptide stock solution was stable at 4?for 15 days.The protamine mimetic peptide drug substance was stable at 20?±5?,92.5%RH,and 4500 Lx±500 Lx.Conclusion:Protamine mimetic peptides have good stability,and the indicators are in compliance with the guiding principles.The stability of the API of the protamine analogue peptide provides a scientific reference for the synthesis of it and storage conditions such as light.The stock solution is stable at 4?for 15 days.Subsequent studies of protein mimetic peptide provide a basis for stability.2.Development and validation of UPLC method for determining protamine peptide analog in rat bloodObjective:A selective,sensitivie,accurate and reliable UPLC method was established for determination of protamine peptide analog in biological samples,and completed a complete methodological study.To provide necessary technical conditions for the further study of protamine mimetic peptide.Methods:UPLC method was adopted.The conditions such as elution gradient,mobile phase,and precipitant were investigated.With external standard method,blood samples were precipitated with 10%trifluoroacetic acid-acetonitrile-water.The isolation was performed in a 300?C18 column using a multi-step gradient with a mobile phase consisting of water:acetonitrile,including 0.05%trifluoroacetic acid and the flow rate was0.3 mL·min^-1 with 3?L injection volume.The detection wavelength was 210 nm and column temperature was 55?.Results:In Wistar rats'blood,a good linearity was obtained over the concentration range 25¹000?g·mL^-1 of protamine peptide analog.The lower limit of quantification was 25?g·mL^-1.The precision of intra-assay and inter-assay for protamine peptide analog were evaluated by analysis of RSD with the results of 1.8%⁵.2%and 3.3%¹2.4%,the accuracy was 3.4%⁷.0%,all within 15%.The recovery rate of R15C was 99.4%¹00.4%.The protamine analogue peptide is stable within 10 min of untreated whole blood samples under an ice bath or zero degree centigrade.The treated whole blood samples were stable at 4?for 24 hours or in the sample warehouse for 24 hours.Conclusion:The method is specific,sensitive,accuracy and reliable.The method meets the guiding principles for biological sample analysis method and Chinese Pharmacopoeia which could be applied to the determination of proteoglycan mimetic peptide concentration in rat blood.To provide necessary technical conditions for the study of metabolic stability in vitro and related metabolic enzymes of protamine mimetic peptide.3.The stability of protamine peptide analog in vitro and identification of metabolitesObjective:The metabolic stability of the protamine analogue peptide was screened in the rat whole blood,liver homogenate,kidney homogenate,blood of beagle dog and human.The half-life of the protamine analogue peptide in the whole blood of different species and the metabolism of rat liver and kidney homogenate in vitro was obtained and the elimination ratio of different species was compared.The types of proteolytic enzymes involved in the metabolism of R15 in rat blood,liver and kidney homogenates.Methods:Substrate elimination method was used to incubate protamine analogue peptide with different biological substrates at 37?.Analyze the peak time of R2^R15arginine peptide to judge metabolites of R15 in rat blood.The concentration of protamine analogue peptide and the identification of metabolites were analyzed by UPLC.Results:R15 was metabolized in the whole blood,liver homogenate and kidney homogenate of rats.The half-life of it in rat blood,liver and kidney homogenate was 15.6min,27.4 min and 9.1 min respectively.The half-life of it in beagle dog and human blood was 11.9 min and 21.0 min respectively.Three metabolites M1,M2 and M3 were found in proteoglycine mimetic peptides in rat whole blood.The metabolites were identified by UPLC method,M1,M2,and M3 were identified as R14,R13,and R12 arginine polypeptide fragments,respectively.Conclusion:The degradation rate of protamine analogue peptide in different tissues of rat was different,the fastest is rat kidney,and rat blood is faster than rat liver.It was eliminated most quickly in kidneys.So R15 was mainly metabolized by the kidney.The three metabolites of rat whole blood are R14,R13,and R12,respectively.4.The study on metabolic peptidase of protamine peptide analogObjective:To indicate the types of proteolytic enzymes that inhibited in the metabolism of R15 in rat blood,liver and kidney homogenates.Methods:Substrate elimination method was used to select appropriate proteolytic enzyme concentrations for incubation with R15 in vitro.The concentration of protamine analogue peptide and the identification of metabolites were analyzed by UPLC.Results:The inhibitory effect of EDTA protease inhibitor which was put in the experimental group was strongest,about 92.7%and inhibited the production of metabolites M1,M2 and M3.Inhibition ratio of Pepstatin group was 41.3%,inhibition ratio of Benzamidine HCl was 33.0%,and the production of metabolite R12 was inhibited of pepstatin and Benzamidine HCl group.The inhibition ratio of protease inhibitor group such as AEBSF,Leupeptin and Trypsin was low,which suggested that it may be involved in the metabolism of R15.There was no statistical difference between Aprotinin,Chymostatin,Phosphoramidon,Rivaroxaban and control group,so they can not effect the metabolism of R15.The inhibitory effect of EDTA in the experimental group of rat liver homogenate and kidney homogenate which the inhibition ratio was 90.2%,81.3%,respectively.The inhibition ratio of the N-Ethylmaleimide and DL-thiorphan protease inhibitor group was 86.5%and 41.2%,respectively in the liver homogenate.Conclusion:Through the influence of various proteolytic enzymes in different biological substrates,it was concluded that protamine analogue peptide are mainly metabolized by metal carboxyl peptidases.Cysteine carboxypeptidase plays a role in the conversion of R15 in rat liver.Serine protease and cysteine protease inhibite the metabolite R12 during the protease screening.