Study On Long-acting Staphylokinase And Its Pharmaceutical Properties Based On Eight-arm PEG And Arabinogalactan-PEG Modification

Staphylokinase(SAK)is useful for the treatment of coronary thrombosis and acute myocardial infarction due to its high fibrinolytic activity.However,the serum half-life of SAK is very short and requires repeated administration to maintain its physiological effects,which renders great pain to patients.In addition,SAK suffers from strong immunogenicity and poor stability.These problems limit the clinical application of SAK.Polyethylene glycol(PEG)modification and polysaccharide(PS)modification can prolong the serum half-life of SAK and reduce its immunogenicity.But these modifications can reduce the bioactivity of SAK.In order to solve the above problems,a recombinant SAK was used,which was connected with Gly-Gly-Cys at its C-terminus and lacked the first 10 amino acids of native SAK.The immunogenicity of the recombinant SAK was lower than native SAK and it can be modified at the site far from its bioactive domain.Eight-arm PEG was used to modify SAK,and the effects of polymerization on pharmacokinetics and bioactivity of SAK were investigated.In addition,PEG with low molecular weight was used as a linker to mediate the AG-modified SAK,and the effects of modification on immunogenicity,pharmacokinetics and bioactivity of SAK were investigated.Eight-arm PEG-MAL 10 kDa was used for site-directed conjugation with the C-terminal of SAK.Two linear PEGs(m-PEG-MAL 10 kDa and MAL-PEG-MAL 10 kDa)were used for conjugation with SAK,which acted as the control groups.Three PEGylated SAKs were prepared and purified.The results showed that conjugation with the three PEGs did not alter the secondary structure of SAK,but slightly perturb the tertiary structure of SAK.After conjugation with PEG,the hydrodynamic volume and thermal stability of SAK increased significantly.This prolonged the in vivo half-life and improved other pharmacokinetic parameters.SAKp-PEG exhibited the largest hydrodynamic volume and the best pharmacokinetic properties due to polymerization of SAK.The large hydration shell of PEG around SAK could sterically shield the bioactive domain of SAK,thereby decreasing the bioactivity of SAK.SAKp-PEG could maintain up to 90%bioactivity of SAK.PEG could also shield the epitope of SAK,thereby decreasing the immunogenicity of SAK.However,the reduction of immunogenicity was offset by an increase in molecular weight due to polymerization of SAK.Compared to SAK,SAKp-PEG exhibited slightly enhanced immunogenicity.In addition,SAKp-PEG showed no apparent toxicity to the cardiac,liver and renal functions of mice.Eight-arm PEG was used to conjugate with a protein drug,leading to greatly prolonged half-life and high bioactivity of the protein drug.Two heterobifunctional PEGs with low molecular weights were used as the spacer arm to conjugate AG with SAK.SAK-P2K-AG and SAK-P5K-AG were thus prepared.SAK-AG,SAK-P2K and SAK-P5K were prepared as the control groups.The results showed that conj ugation with AG and PEG did not alter the secondary structure of SAK.Compared with PEG,AG exerted a stronger shielding effect on SAK,resulting in the lowest fluorescence intensity,the lowest bioactivity and the significantly low immunogenicity of SAK-AG in the five modified products.The PEG linker between SAK and AG could decrease the steric shielding effect of AG on SAK.Compared with SAK-AG,SAK-P2K-AG and SAK-P5K-AG both showed significantly increased fluorescence intensity,improved bioactivity and decreased immunogenicity.Conjugation with AG and PEG could synergistically decrease the immunogenicity of SAK without triggering the production of anti-AG and anti-PEG IgG.After conjugated with AG and PEG,the hydrodynamic volume of SAK increased significantly,leading to prolonged half-life in vivo and improved other pharmacokinetic parameters.SAK-P5K-AG exhibited the largest hydrodynamic volume and the best pharmacokinetic properties.Conjugation with AG and higher molecular weight PEG could synergistically increase the hydrodynamic volume of SAK and improve the pharmacokinetic properties.In addition,conjugation with AG-PEG showed no apprant toxicity to the heart,liver and renal functions of mice.The modification strategy of this study could be effective for the development of new long-acting protein drugs.