It was found that when dendrimer GS-7340 purchase was bound, neutralisation of anticoagulant activity occurred ( Fig. 4); heparin spiked (0.8 IU/ml) in human plasma was inactive at a 1:1

heparin:dendrimer mass ratio. This further confirmed the result obtained by MB spectroscopy which showed that the maximum association between heparin and dendrimer also occurred at the 1:1 mass ratio. The new conformation of heparin is considered to abolish the negative charge and make it inaccessible to AT-III and the coagulation proteases (i.e. prothrombin III and factor Xa), a process, which inactivates anticoagulant activity. Heparin dendriplexes demonstrated no anticoagulant activity in vitro, however it was considered of interest to determine if the behavior of such macromolecular complexes could allow activity Caspase cleavage in complex biological systems; events such as partitioning between the dendrimer and heparin binding peptides may occur, as the binding of heparin to heparin binding peptides has been reported in plasma following intravenous administration [14]. Other effects that could occur in vivo include an interaction with extracellular matrices after subcutaneous injection and/or interactions with intracellular

components following cellular uptake (e.g. susceptibility to endosomal escape following endocytosis). To investigate whether heparin was able to regain anticoagulant activity in vivo, rats were injected subcutaneously Histamine H2 receptor with free or complexed heparin. Heparin anticoagulant activity in platelet-free rat plasma was estimated at 0, 1, 3, 6 and 24 h post-injection using the antifactor Xa assay. The heparin calibration curve in platelet-free pooled rat plasma was plotted ( Fig. 5A) and the active heparin concentration in the plasma determined ( Fig. 5B). It can be seen ( Fig. 5B) that heparin injected subcutaneously at a dose of 10 mg/Kg was therapeutically

active up to 6 h post-administration and complete loss of its activity was observed at 24 h time point. However, heparin dendriplexes at a 1:3 heparin:dendrimer mass ratio (10/30 mg/Kg) (a ratio above maximum association), resulted in an inactive complex in vivo; this lack of activity compared with the oral heparin-treated groups. At autopsy, gross observations were made on the injection site in animals treated with heparin and heparin dendriplexes. In animals injected with heparin there was evidence of subcutaneous hematoma formation (i.e. localized extravasated blood). There was no evidence of hemorrhage at the injection site in animals treated with heparin dendriplex ( Fig. 6). It was considered that the lack of anticoagulant activity in vivo in rats treated with heparin dendriplexes may have been due to the precipitation of the aggregates at the site of subcutaneous injection.