Development Of Activated Protein C Like Enzyme, A New Drug For Treating Severe Sepsis

Severe sepsis is a devastating disorder with high morbidity and mortality. 30-50% of severe sepsis patients will die despite advances in critical care. The economic burden of severe sepsis is considerable; in the United States, approximately 750,000 new sepsis cases occur each year, of which at least 225,000 are fatal, with additional cost of about $22800 per patient. Key elements of evolving sepsis include release of proinflammatory cytokines, diffuse endovascular injury, and procoagulant host responses induced by organ dysfunction. Followed by advanced understanding of the mechanisms underlying the septic response from 1980's, various drugs that were aimed at interrupting specific steps in the septic cascade were under development. More than 80 clinical trials have been done. However, with just one exception (activated protein C), the results were disappointing.Recombinant human activated protein C was approved by FDA and launched on November 21, 2001. Then it is also launched in Australia, Germany, France, etc. In several post-marketed clinical trials, it was found that activated protein C can significantly reduce severe sepsis mortality.Snake venom is a mixture containing many different biologically active proteins and peptides. A number of these proteins interact with components of the human hemostatic system. Hemostatically active components are distributed widely in the venom of many different snake species. The venom components can be grouped into a number of different categories depending on their hemostatic action, such as enzymes that clot fibrinogen, protein C activator, prothrombin activator, factor V activator, factor X activator, platelet aggregation inhibitor (disintegrin), thrombin inhibitor, etc. Based on the fact that activated protein C was developed successfully and snake venoms contain many different biologically proteins, this work was proposed that an activated protein C "me-too" drug (activated protein C like enzyme, APCL) will be developed from snake venom.To determine which species contain APCL among 8 Chinese snake venoms, anticoagulant activity was examined by activated partial thromboplastin time (APTT) assay, and amidolytic activity was measured with activated protein C specific chromogenic peptide substrate (Pefachrome Pea). The result showed that the venom from Agkistrodon halys might contain APCL.A protein was obtained from the venom of Agkistrodon halys by applying anion-exchange and gel filtration chromatography, which has the abilities of prolonging significantly human plasma APTT and generating strongly amidilytic activity for Pefachrome Pca. This protein appears to be a single component determined by both SDS-PAGE and size exclusion chromatography detections. The molecular weight of APCL is 15.3 kD by SDS-PAGE. Its p1 determined by isoelectrofocusing was 4.80. The result of hydrolyzing Pefachrome Pca showed that the dynamic constants Km and Vmax were 3.16 X 10-5 mol L-1 and 1.16X10-4 mol L-1min-1, respectively. All these data verified that the venom from Agkistrodon halys contains positively APCL.A mouse model of sepsis was established by injecting intravenously lipopolysaccharide (LPS). To evaluate whether it can reduce the mortality of septic mice, APCL was administered into the experimental mice. The result showed that APCL could significantly reduce the mortality of septic mice, which indicated that it might have the potential effects in treatment of sepsis like activated protein C.Because APCL was purified from snake venom, it suggested that there were the disadvantages of immunogenicity and proteolytic degradation when used in human. APCL was modified by using activated PEG reagent so as to obtain the PEGylated APCL, which immunogenicity and proteolytic degradation were significantly decreased while remained voluminous activity.