C. D. Ebert

Immobilized heparin: Spacer arm effects on biological interactions

Heparin immobilized to polymer surfaces via different length diaminoalkane spacer arms was evaluated for anticoagulant activity and for platelet interactions. The anticoagulant activity of the immobilized heparin, as determined by APTT assays, was found to increase with increasing spacer arm length. Variations in spacer arm length produced no affect on platelet retention or PF 4 release for heparin immobilized materials. To investigate immobilized heparin-adsorbed plasma protein interactions, XPS analysis of heparinized surfaces, before and after plasma contact, was conducted. Immobilized heparin was not able to penetrate adsorbed plasma protein layers with any spacer arm length evaluated, indicating that immobilized heparin does not directly interact with platelets.

Covalently bound conjugates of albumin and heparin: Synthesis, fractionation and characterization

Covalently bound conjugates of human serum albumin and heparin were prepared as compounds which could improve the blood-compatibility of polymer surfaces either by preadsorption or by covalent coupling of the conjugates onto blood contacting surfaces. The conjugates (10-16 weight % of heparin) were obtained by a condensation reaction between albumin and heparin using 1-ethyl-3-(dimethylaminopropyl)-carbodiimide. Unreacted albumin and heparin were removed by diethyl-aminoethyl (DEAE)-cellulose and Cibacron Blue Sepharose chromatography respectively. The activity of the heparin component incorporated in the albumin-heparin conjugates (Ac) was compared with that of the heparin used for the synthesis of the conjugates (Anat) by thrombin time, inhibition of Factor Xa and the activated partial thromboplastin time (APTT) assays. The Ac/Anat ratio for the above assays was as follows: Thrombin time 1.25, Factor Xa inhibition 0.5. and APTT 0.5. Gel filtration chromatography showed broad-molecular weight distributions. The conjugates were fractionated using immobilized antithrombin III (ATIII). High ATIII and low ATIII affinity conjugate fractions showed the same behavior as ATIII fractionated heparin with respect to thrombin times and Factor Xa inhibition.

Mucosal delivery of macromolecules

Low molecular weight heparin (LMWH), a glycosaminoglycan of approximately 6000 molecular weight, is currently used in the prevention of postsurgical thrombosis and in the treatment of deep vein thrombosis. Current dosing regimens entail subcutaneous injections of 2500–5000 anti-factor Xa units per day. Transbuccal delivery of LMWH may provide significant advantages over the current injectable dosage forms. The kinetics and extent of LMWH absorption from prototype buccal dosage forms were evaluated. Based on pharmacokinetic analysis, over 3000 anti-factor Xa units (i.e., 20 mg) could be delivered from a single application. These results demonstrate the feasibility of administering macromolecular drugs, such as LMWH, via the buccal route.

Interaction of antithrombin III with preadsorbed albumin-heparin conjugates

The adsorption of antithrombin III (AT III) onto polystyrene surfaces preadsorbed with albumin or albumin-heparin conjugates was studied using a two step enzyme immuno assay. When AT III-buffer solutions were used, the highest adsorption values were measured on high affinity albumin-heparin conjugate pretreated surfaces. Less AT III adsorption was found on nonfractionated albumin-heparin conjugate preadsorbed surfaces. AT III adsorption could also be detected on low affinity conjugate and albumin coated surfaces. When AT III was adsorbed from plasma or plasma dilutions with buffer, only AT III on surfaces preadsorbed with high affinity or nonfractionated albumin-heparin conjugate was found. These results demonstrate that the heparin moiety of the conjugate is directed to the solution phase whereas the albumin moiety contacts the polystyrene surface.

Antithrombotic Agent Releasing Polymers

The use of pharmacologically active agents with antithrombotic effects combined with blood contacting polymers provides a novel approach towards the improvement of blood materials interactions. Antithrombotic agents can be physically combined in a polymer solution producing a monolithically dispersed delivery device. This monolithic system contains both dissolved and undissolved active agent and is capable of diffusional release of the active agent at the surface of the polymer. Through proper design, use of this technique provides optimal therapeutic levels of selected antithrombotic agents which can be delivered to a desired site of activity while minimizing unwanted systemic side effects.