| Background:Unfractionated Heparin(UFH)is an anionic polysaccharide with an average molecular weight of 15 to 19 kDa,which is mainly used for systemic anti coagulation and prevention of thrombosis during surgery.But UFH is often used with risks of bleeding and other adverse reactions.Protamine sulfate(PS)is a cationic protein mixture with molecular weight of 4000～6000 Da,which is the only approved heparin antagonist in clinic.PS is mainly extracted from the sperm of wild salmon species.This cationic protein binds to anionic heparin to form UFH-PS complex,which inactivates heparin.However,PS has many adverse reactions in clinical applications,such as systemic hypotension,pulmonary hypertension,and anaphylactic reactions.Although lots of work were made,the mechanism of PS adverse reactions is still unclear.In addition,there is no alternative heparin antagonists,therefore PS has been used with caution since 1939.Mild adverse effects of PS have been reported as high as 16%,and unknown pathologic effects may lead to increased postoperative morbidity and mortality.In addition,the unclear mechanism of PS adverse reactions also poses challenges to the development of new heparin antagonists.It was reported that the adverse reactions of PS may be caused by multiple factors.In clinical practice,PS is also used as an excipient for long-acting insulin to prolong the action of insulin and improve the compliance of insulin in clinical use.Because of the strong immunogenicity of PS,diabetes patients who use this dosage form for a long time often carry PS antibodies.Such patients are prone to adverse reactions of PS when encountering systemic anticoagulant surgery that requires massive use of PS.In this study,a series of polyarginine peptides were synthesized based on the rich arginine in PS.A small peptide(named R15)containing 15 arginine was screened out.This small peptide can neutralize UFH in vitro and in vivo.Preliminary safety studies suggest that R15 was low toxicity and no immunogenicity.R15 is prepared by a fully synthetic process with controllable quality,which is not limited by raw materials from natural sources and does not contain impurities in frog fish.The purpose of this study is to further study R15 and reveal the potential toxicity of R15.At the same time,compared with the potential advantages of PS,it lays a foundation for the further research and development of R15,and provides inspiration and ideas for the study of new heparin antagonists.The clinical dose of UFH is usually 300 U/kg,and the average heparin concentration in heparinized patients is 4 U/mL,which is referred to as the therapeutic concentration in this paper.Objective:PS,as the only clinically approved UFH antagonist,has many adverse reactions.Howerer,PS is prepared from fish sperm,so a strict quality standard cannot be established.The purpose of this study is to investigate the druggability of a synthetic polypeptide R15 as a potential PS substitute.According to the requirements of new drug research and development and the characteristics of PS,R15 is to be investigated from four aspects:pharmaceutical study,pharmacodynamics study,safety study and pharmacokinetics study.Part of the pharmaceutical study and pharmacodynamics study have been investigated during my master’s period,and this topic mainly investigated the preliminary safety and pharmacokinetics of R15.This study not only investigated the druggability of R15 as UFH antagonist,but also hoped to clearify the possible mechanism of clinical adverse reactions of PS(the mechanism of adverse reactions of PS has not been clearly explained so far).Methods and Results:The methods and results of this study are divided into the following four aspects:1.Quality control of R15This section aims at quality control of R15 to ensure drug stability and reliability in subsequent experiments.A batch of R15 was synthesized in our lab for this project.Elemental analysis was carried out on R15 using elemental analyzer,and the proportion of carbon,hydrogen,oxygen,and nitrogen in this batch of R15 was measured as follows:carbon(34.28%),hydrogen(6.66%),oxygen(13.01%)and nitrogen(26.73%).The HPLC was used to analyze R15 and the retention time was 7.077 min.No other impurities were found in the chromatogram.The molecular weight of R15 was determined by TOF,which was 2361.4(+H),consistent with the theoretical molecular weight of R15.2 Pharmacodynamics of R15This study aimed to determine the affinity and potency of R15 with verification in animals,to provide a reasonable concentration and dose for subsequent safety experiments.The affinity between UFH and R15 was determined by Biacore.The results of intermolecular affinity showed that the affinity between R15 and UFH was very strong,which was difficult to elute once combined with PS.The dissociation rate equilibrium constant KD of UFH-PS complex and UFH-R15 complex was 4.80×10-11 M and 6.82×1011 M,respectively.Three methods(APTT method,anti-FXa method and turbidimetric method)were used to detect the potency of R15 and PS.Among the three methods,the results of antiFXa and turbidimetry were the same,with PS potency of 150 U/mg and R15 of 170 U/mg.In vivo potency validation was performed in Balb/C mice and Wistar rats in subsequent studies.Results showed that R15 could completely neutralize heparinized rats and mice at neutralizing doses.3 Preliminary safety study of R15The purpose of this study was to explore the in vitro safety of R15:interaction of UFH to R15,effects on RBC,platelets,fibrin,complement and rat plasma.In vivo safety:The safety of single injection of R15 in Wistar rats was investigated,followed by safety study of multiple neutralization of heparinized Balb/C mice with R15,and by immunogenicity of UFH-R15 complex in guinea pigs.To observe the binding characteristics of PS,R15 and UFH,turbidity test,particle size analysis and intermolecular affinity determination were carried out,respectively.It was observed that,like the binding of PS,UFH and R15 combined to form aggregates,which reduced the optical density and became concentration dependent.The particle size analysis was consistent with the turbidity experiment results.With the increase of R15 concentrations,the particle size of the complex gradually increased.When the critical value was reached,the complex formed large aggregates for deposition.This study investigated the effect of R15 on erythrocytes.In hemolysis tests,PS and R15 did not cause hemolysis of erythrocytes at high concentrations(up to 5000 μg/mL).In the erythrocyte osmotic resistance assay,it was found that with the increase concentrations of PS and R15,the influence on erythrocyte membrane became larger,but no erythrocyte membrane deformation was found in the range of treatment concentrations.The effect of R15 on erythrocyte aggregation was observed under the microscope.It was found that erythrocytes tended to aggregate when PS or R15 concentration exceeded 500μg/mL,and erythrocytes tended to aggregate when PS or R15 concentration exceeded 500 μg/mL.To further investigate the erythrocyte aggregation under 500 μg/mL of R15,the rheometer was used to determine the whole blood of R15-treated rats,and no erythrocytes aggregation was detected.This study examined the effect of R15 on platelet aggregation and showed that PS and R15 had no effect on platelet aggregation at 10 times the therapeutic concentration.This study investigated the effect of R15 on plasma fibrin.With the increase of concentrations,both PS and R15 can accelerate the conversion of fibrinogen to fibrin in a concentration-dependent manner.Microscopic observation of fibrin fibers incubated with PS and R15 showed that PS and R15 at high concentrations(4 and 10 times of treatment concentration)increased the diameter of fibrin with rough surface.However,this phenomenon is not obvious in whole blood matrix.The present study investigated the complement activation ability of R15.UFH,PS and R15 can activate complement,and the activation ability is UFH>R15>PS.When PS,R15 was added into UFH to form UFH-PS complex and UFH-R15 complex,respectively,the complement activation ability of UFH,PS and R15 disappeared.However,if UFH is added in excess to form UFH-PS complex with excessive UFH,or UFH-R15 complex with excessive UFH,the complement activation ability is greatly enhanced.The UFH-PS complex can activate complement even at very low concentrations(1/20 of the therapeutic concentration).This phenomenon of complement activation by complex in heparin excess also occurred in UFH-R15 complex.However,the complement activation capacity of UFH-R15 complex(UFH excess)was weaker than that of UFH-PS complex(UFH excess),which was about 1/7-1/8 of that of UFH-PS complex(UFH excess).In this study,we investigated the effect of R15 on the coagulation function of rat plasma and found that the addition of PS and R15 could increase the APTT value of rat plasma.However,with the extension of incubation time,R15 could be gradually degraded in plasma and APTT value returned to normal.However,no significant change of APTT was observed in PS within 2 h,indicating that PS was more stable in plasma than R15.To further investigate the degradation behavior of R15 in plasma,the stability of UFHPS complex and UFH-R15 complex in plasma was determined by APTT and anti-FXA methods.It was found that with the extension of incubation time,UFH was gradually released from UFH-R15 complex,leading to the increase of the APTT value of plasma.However,UFH-PS complex did not release UFH within 4 h.In consideration of possible rerelease of heparin in clinical use,R15 was used to neutralize heparinized rats(300 U/kg,1000 U/kg)in animal study with monitor for heparin release within 4 h.The results showed that R15 could completely neutralize UFH,and no significant release of heparin was observed within 4 h.This inconsistence of results in vivo and in vitro may be related to simultaneous decomposition and excretion of heparin.Toxicity test of a single injection of R15(900 U/kg)in normal or heparinized rats was studied.It was found that PS and R15 injection could increase creatinine(CREA)and affect renal function in normal rats.In heparinized rats,PS and R15 injection also caused an increase in creatinine,but there was no statistical difference compared with the control group.There were no significant changes in complement level,coagulation function,and tissues and organs in each group.This study investigated the toxicity of R15 repeatedly neutralizing heparinized mice(300 U/kg;5 times,once a week).Hematological analysis of R15-treated heparinized mice was performed 1 h after treatment(3 times,every 1 week),and no significant abnormal blood routine was found(a slight decrease in RBC was observed).Hematological analysis was conducted 1 week after the completion of drug administration,and the hematological parameters of all mice were normal.As for the antibody detection,both PS-and R15-treated heparinized mice produced no antibody in each group.In this study,the immunogenicity of UFH-PS complex and UFH-R15 complex in guinea pigs was investigated.It was found that the UFH-PS complex-treated guinea pigs had obvious immunogenicity,and 8 out of 12 had antibodies.Guinea pigs immunized with UFH-R15 complex showed no antibodies.4 Establishment of PS and R15 pharmacokinetic methods and determination of its pharmacokinetic parametersThis section aims to establish methods for PS and R15 determination in vivo,and to determine pharmacokinetic parameters of PS and R15 in rats.This method can monitor the antagonistic ability of heparin at a certain time in animals.Because PS and R15 are easily metabolized in the body,the metabolized fragments still have heparin antagonistic activity,so this method is very suitable.This study investigated the changes of drug concentrations in rats after a single intravenous injection of PS(300 U/kg)and R15(300 U/kg,900 U/kg,2700 U/kg).It was found that PS was cleared rapidly in vivo,and the concentration of PS in blood was 12.67±1.96 μg·mL-1 minute after injection.The average half-life(T1/2)was 3.94±3.08 min.PS has a short half-life and is easily metabolized or excreted in the body.The apparent volume of distribution(VZ)was 268±151 mL·Kg-1.The VZ of PS was larger than the blood volume of rats(70 mL·kg)-1)which may be distributed in extracellular fluid.The elimination rate(CL)was 53.65±11.49 mL·min-1·Kg-1,indicating that PS can be easily eliminated in the body;Area under blood concentration-time curve(AUClast)was 35.89±7.07 min·g·mL-1.In vivo pharmacokinetic parameters of R15 in rats showed that the dose of R15 was 300 U/kg,900 U/kg,and 2700 U/kg,respectively,with a ratio of 1:3:9.The average elimination half-life(T1/2)were 51.93 min,91.13 min,163.12 min,respectively,with a ratio of 1:1.8:3.1.The elimination half-life of the drug decreased with the increase of the dose concentration.The average Cmax was 10.89[μg/mL,11.54 μg/mL,11.65 μg/mL,with a ratio of 1:1.1:1.1.This suggests that the drug will rapidly combine with substances in the blood and act as a reservoir for slow release.Area under the average curve(AUClast)was 632 min·μg·mL-1,1030 min·g·mL-1,1320 min·g·mL-1,with a ratio of 1:1.6:2.1,which is not linear.The average apparent volume of distribution(VZ)was 194 mL·kg-1,608 mL·kg-1,2618 mL·kg-1,with a ratio of 1:3.1:13.5,which is not linear.The elimination rate(CL)was 2.73 mL·min·1·Kg-1,4.72 mL·min-1·Kg-1,11.16 mL·min-1·Kg-1,with a ratio of 1:1.7:4.1,which is not linear.Conclusion:In this study,the safety and efficacy of R15,a novel heparin antagonist,were explored by a series of in vitro and in vivo methods with comparative study of PS.The results showed that R15,compared with PS,had similar affinity constants and potency with UFH,and could be closely bound with UFH.At the therapeutic concentration,PS and R15 did not affect erythrocytes,platelets and fibrin,nor did they have long-term effects on coagulation function,organ of the tested animals.In this study,an unreported phenomenon of complement activation was found:Complement activation of the UFH-PS complex in UFH excess occured at 1/20 therapeutic concentration or above.Though R15 showed similar phenomenon but requireed a higher concentration(R15half-activated/PShalf-activated=7.78).Further more,compared with PS,R15 has no immunogenicity,which improves the drug safety.At the same dose,R15 had a longer half-life than PS in rats,and no re-release of heparin was observed.In conclusion,according to the current results,R15 has better safety than PS and is very likely to be a substitute for PS.At the same time,this study laid solid data and theoretical support for the further development of R15,and provided methodological exploration for the subsequent development of protamine analogues. |
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