Comparison of antithrombin activity of the polysulphate chitosan derivatives in in vivo and in vitro system

In order to choose the proper method for evaluating the antithrombin activi ty in samples of chitosan polysulphate (CP) with different polymerization d egrees and sulphation degrees, we estimated the ability of direct anticoagu lants to depress the coagulability of recalcified sheep blood using the thi rd international heparin standard (A(1) - in vitro system) and determined s uch activity on pharmacodynamic curve (A(2) in vivo system). The curve admi ts the kinetics of CP elimination to be nonlinear in case of intravenous in jection to rabbits, as it is observed in heparin: C-t = C(o)exp( - K-elt), where C-t is the CP concentration at the time moment t; C-o is the CP conce ntration at the injection moment; K-el is the elimination constant. Besides , it is assumed that there is a linear approximation of the anticoagulant e ffect on the dose, which finally makes it possible to calculate the specifi c activity A(2): T = KTCt + T-in, where T is the time of clot formation at different time intervals after CP injection; Ti,is the time of clot formati on prior to CP injection. T value was assessed in two tests: blood coagulat ion time (BCT) and activated partial thromboplastin time (APTT). No correla tion was observed between A(1) and A(2) At the same time, the values of K-e l and the period of semi-elimination, with the use of the biospecific cetyl pyridinium 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, e xperimentally, it was proven that the assumption of the CP nonlinear elimin ation and the CP effect-dose dependence was true, which is necessary for A( 2) calculation. Relatively low molecular weights (MW 61-82 kDa, polymerizat ion 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 anti thrombin activity (30-52 IU/mg). We recommend the use of in vivo system for evaluating the antithrombin activity of the CP derivatives. (C) 2001 Elsev ier Science Ltd. All rights reserved.