The Preclinical Pharmacokinetics Study Of Steroid EAD And Peptide HYD-PEP06

Objective:17-Ethinyl-3,17-dihydroxyandrost-5-ene?EAD?is an agent designed for the treatment of acute radiation syndrome?ARS?.It is derivative from androst-5-ene-3?,17?-diol?5-AED?by bringing in an acetenyl at C-17 position.As the result of whole blood count showed,the effect of EAD was better than5-AED at the time of post irradiation in enhancing the scalar of white blood cell,platelet,red blood cell,neutrophil,hemoglobin and lymphocyte in peripheral blood.Given its vital role played in the prevention and mitigation of ARS,the development of a sharp,sensitive and robust liquid chromatography tandem mass spectrometry?LC-MS/MS?method to monitor the metabolism of EAD in vivo was crucial.HYD-PEP06,an anti-carcinoma peptide,is designed as angiogenesis inhibitor to block the supply and metabolism of nutrition.It is based on Endostar which is the first anti-angiogenic endostatin in the world.The motif of RGD is attached to the N-terminal.The patent of HYD-PEP06 has been accepted in China.It is on the road to clinical trial declaration.It is the aim of this research to evaluate the preclinical pharmacokinetic characteristics of HYD-PEP06 in animals,so as to support for the study of administration route,toxicology,as well as the clinical experiment.Methods:In the study,we chose liquid chromatography tandem mass spectrometry to study the preclinical pharmacokinetics characteristic of EAD and HYD-PEP06.The most important thing was the establishment and optimization of the LC-MS method to determine the concentration of EAD and HYD-PEP06 in biological matrix.Results:1.The establishment and validation of an LC-MS/MS analytical method for determination of EAD in biological matrixA new method was constructed and validated for the determination of EAD with the internal standard of 5-AED.The blood samples were precipitated with methanol,centrifuged,from which the supernatant was separated on UPLC with C18 column and eluted in gradient with acetonitrile and Milli-Q water both containing 0.1%formic acid.A good linearity was obtained with R>0.99 for EAD within its calibration ranging from 5 to 1000 ng·mL^-11 with a lowest limit of quantification?LLOQ?of 5 ng·mL^-1.Inter-and intra-day precision of three levels of quality control?QC?samples were within the range of 4.35%¹2.69%and 4.38%¹3.89%.The accuracy varied from-6.48%to 0.01%.Samples were stable under the circumstances encountered in the experiments.No carryover,no matrix effects.The method was simple,accurate and robust applied to determine the concentration of EAD in Wistar rat after a single oral administration of EAD at the dose of 100 mg·kg^-1.2.Pharmacokinetic study of EAD in wistar ratsEAD was administrated orally at the dose of 100 mg·kg^-1.Primary pharmacokinetics parameters were drafted by non-compartment analysis?NCA?.The area under the plasma concentration-time curve?AUC?was?178.03±71.72?ng·h·mL^-1.The volume of distribution?V/F?was?1590.79±702.88?L·kg^-1,illustrating that EAD was distributed into a wide range of organs and tissues.The body clearance?CL/F?was?527.49±207.55?L·h^-1·kg^-1.The pharmacokinetics parameters presented in this study would be useful for further study on EAD in vivo in the future.It was up to the maximum concentration at 2h after administration.The elimination half-life time(T_1/2)was?2.15±0.87?h.The data manifested a double-peak phenomenon during 8-12 h after a single oral administration of EAD.The first peak climbed to the top at about 2 h,while the second peak appeared at about 8-12 h which was much lower than the first one.It was supposed to be attributed to biabsorption,which was possibly due to entero-hepatic circulation?EHC?.3.The establishment and validation of an LC-MS/MS analytical method for determination of HYD-PEP06 in biological matrixA simple,sensitive,specific LC-MS/MS method was established and validated for determination of HYD-PEP06 in rat blood.There was no adsorption of HYD-PEP06 during the transfer between polyethylene vials.There was formic acid in mobile phase and needle washing solution to raise the response and get over the effect of carryover.In addition,in biological matrix,peptides often undergo rapid degradation in presence of different metabolic enzymes to obtain extreme short half-life time.Therefore,a preliminary investigation on stability and metabolism of HYD-PEP06 using LC-MS/MS was performed.Through basic acid/base/organic solvent stability test,HYD-PEP06was found to be more stable in acid environment.However,remarkable degradation was found in the whole blood.Acid,antioxidant and enzyme inhibitors could not prevent its rapid degradation in blood.While,in low temperature,bathing in ice,it is stable in 30 minutes.Pre-treatment of the samples was protein precipitation by adding in organic solvent.A good linearity was obtained over the blood concentration range 10²000ng·mL^-1of HYD-PEP06 in rat blood.The precision of intra-assay and inter-assay for HYD-PEP06 was evaluated by analysis of variance with the results of4.81%⁹.42%and 3.13%⁴.87%,the accuracy was-5.06%⁸.54%.The recovery of HYD-PEP06 was 57.35%⁶1.41%.Meanwhile,there was no matrix effect,no dilution effect or carryover effect.It was stable during preparation and analysis.After validation,it was proved to be satisfied with the requirement.4.Pharmacokinetic study of HYD-PEP06 in ratsFollowing intravenous infusion administration with different doses of HYD-PEP06(3.3,30,90 mg·kg^-1)to rats,the blood samples were collected at designed times after dosing.All collected blood samples were prepared and determined by method described above.While AUC_last were?702.63±126.47?h·ng·mL^-1,?7932.19±3606.62?h·ng·mL^-1,?21104.82±4396.20?h·ng·mL^-1,separately.And the ratio increasing trend between AUC?1:11.3:30.0?and dose?1:9:27?was of positive correlation.As the results shown,the pharmacokinetic behaviors of HYD-PEP06 in wistar rats were complied with linear kinetics within the examined doses range.1min after administration,concentrations of HYD-PEP06 in blood were determined as C_max?11252.5±1607.4?ng·mL^-1,?126987.5±45697.2?ng·mL^-1,?442125.0±96828.9?ng·mL^-1.The elimination half-live times(T_1/2)were?1.35±0.07?min,?2.84±0.10?min,?8.10±1.00?min,respectively.5.Bioavailability of HYD-PEP06 in wistar rats after subcutaneous administrationThe bioavailability of HYD-PEP06 in wistar rats after subcutaneous administration was?3.15%±0.94%?.After administration,the concentration in blood reached the maximum at 5min.HYD-PEP06 was found still remained in blood 60min after administration.Conclusions:1.The simple,accurate and robust method was applied to determine the concentration of EAD in wistar rats after a single oral administration of EAD at the dose of 100 mg·kg^-1.It was supposed to be attributed to biabsorption,which was possibly due to entero-hepatic circulation?EHC?.2.The method to determine the concentration of HYD-PEP06 in wistar rats was established.After validation,it was proved to be satisfied with the requirement.As the results shown,the pharmacokinetic behaviors of HYD-PEP06 in wistar rats were complied with linear kinetics within the examined doses range.The bioavailability of HYD-PEP06 in wistar rats after subcutaneous administration was?3.15%±0.94%?.