title: Comparison of antithrombin activity of the polysulphate chitosan derivatives in in vivo and in vitro system.
abstract: In order to choose the proper method for evaluating the antithrombin activity in samples of chitosan polysulphate (CP) with different polymerization degrees and sulphation degrees, we estimated the ability of direct anticoagulants to depress the coagulability of recalcified sheep blood using the third international heparin standard (A1 - in vitro system) and determined such activity on pharmacodynamic curve (A2 - in vivo system). The curve admits the kinetics of CP elimination to be nonlinear in case of intravenous injection to rabbits, as it is observed in heparin: Ct = C(o)exp(-K(e)lt), where Ct is the CP concentration at the time moment t; C(o) is the CP concentration at the injection moment; Kel is the elimination constant. Besides, it is assumed that there is a linear approximation of the anticoagulant effect on the dose, which finally makes it possible to calculate the specific activity A2: T = KTCt+T(in), where T is the time of clot formation at different time intervals after CP injection; T(in) is the time of clot formation prior to CP injection. T value was assessed in two tests: blood coagulation time (BCT) and activated partial thromboplastin time (APTT). No correlation was observed between A1 and A2. At the same time, the values of Kel and the period of semi-elimination, with the use of the biospecific cetylpyridinium chloride electrophores for the quantitative determination of CP in rabbit's blood taken at different time intervals after injection, showed a close correlation (r = .94, P < .05) between the same parameters, obtained with the help of the rectilinear pharmacodynamic plot in BCT test. Thus, experimentally, it was proven that the assumption of the CP nonlinear elimination and the CP effect-dose dependence was true, which is necessary for A2 calculation. Relatively low molecular weights (MW 61-82 kDa, polymerization degree 188-252 ) and high sulphation patterns (sulphur amounts 15.6-16.9%, sulphation degree 1.58-1.86) were slowly cleared and there was more antithrombin activity (30-52 IU/mg). We recommend the use of in vivo system for evaluating the antithrombin activity of the CP derivatives.
authors.affiliation: Haematological Scientific Centre, Moscow, Russia.
authors.forename: N N
authors.initials: NN
authors.lastname: Drozd
authors.forename: A I
authors.initials: AI
authors.lastname: Sher
authors.forename: V A
authors.initials: VA
authors.lastname: Makarov
authors.forename: L S
authors.initials: LS
authors.lastname: Galbraikh
authors.forename: G A
authors.initials: GA
authors.lastname: Vikhoreva
authors.forename: I N
authors.initials: IN
authors.lastname: Gorbachiova
mesh_terms: D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D001780:Blood Coagulation Tests; D002686:Chitin / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000493:pharmacokinetics / Q000494:pharmacology*; D048271:Chitosan; D005343:Fibrinolytic Agents / Q000008:administration & dosage / Q000493:pharmacokinetics / Q000494:pharmacology*; D006490:Hemostatics / Q000494:pharmacology; D006493:Heparin / Q000592:standards; D008657:Metabolic Clearance Rate; D008954:Models, Biological; D011479:Protamines / Q000592:standards; D012756:Sheep; D013463:Sulfuric Acid Esters; D013917:Thrombin / Q000037:antagonists & inhibitors
chemical_list: D005343:Fibrinolytic Agents; D006490:Hemostatics; D011479:Protamines; D013463:Sulfuric Acid Esters; D002686:Chitin; D006493:Heparin; D048271:Chitosan; D013917:Thrombin