Table of Contents:
Peptides are recognized for being highly selective and efficacious; simultaneously, they are relatively safe and well-tolerated. As a result, there is an increased interest in peptides in pharmaceutical research and development. Meanwhile, over 60 peptide-based drugs have been approved by the FDA and approximately 140 peptide therapeutics are currently being evaluated in clinical trials.
However, many peptide and peptidomimetic drugs suffer from rapid clearance in vivo.
This phenomenon can be reduced by increasing the respective size through oligomerization or
covalent conjugation with polymers. The objective of this doctoral thesis was to develop
a new and convenient strategy for drug oligomerization, in which peptidomimetic thrombin inhibitors are to be incorporated into the liposomes. For this purpose, two types of benzamidine-derived thrombin inhibitors were used which were initially modified with palmitic acid to enable their incorporation into liposomes. Type I inhibitors contain an L-configurated trifunctional amino acid (lysine) in P3 position and inhibit thrombin in the low nanomolar range, while possessing negligible potency against related proteases such as factor Xa, plasmin, uPA, plasma kallikrein and matriptase. The inhibitor moiety was covalently coupled to a palmitic acid residue, either directly to the P3-side chain or via a short bifunctional ethylene glycol spacer. The second type of inhibitors with D-cyclohexylalanine in P3-position is less specific and inhibits besides thrombin also factor Xa, although in a significantly weaker way. Type II inhibitors have been modified at their sulfonyl residue in P4 position.
For both inhibitor-type series, strong thrombin-inhibitory potencies were obtained in enzyme kinetic studies, showing that the modification was very well-tolerated. The palmitoylated molecules were directly added to the lipid mixture (DPPC, cholin) which was used for liposome preparation.
The inhibitory potency of the obtained liposomes was determined by enzyme kinetic studies. Furthermore, the physicochemical properties of the liposomes and their anticoagulant activities were characterized in plasma. The obtained liposomes possess strong thrombin inhibitory potency in enzyme kinetic measurements as well as anticoagulant activity in plasma. Their strong potency and positive ζ potential indicate that large amounts of the benzamidine-derived inhibitors are located on the surface of the liposomes. The liposomes prepared from inhibitor containing L-lysine-residue in P3 position, elongated with ethylene glycol linker, show excellent inhibitory potency (IC50 = 0.55 nM), whereas for the vesicles with the compound lacking the spacer a strongly reduced activity were determined (IC50 = 81 nM). This result indicates the importance of the flexible spacer between the inhibitor-head and the surface of the liposomes to improve the accessibility for the target enzyme. Moreover, employing a long flexible spacer would facilitate the binding of several thrombin molecules to inhibitor-loaded liposomes, resulting in enhanced inhibitory effects.
In case of complications in the drug treatment, it is highly important to have a specific tool in order to reverse the action of drugs. In a previous study conducted by Olson etal., a chimeric anticoagulant containing both an indirect factor Xa inhibitor (fondaparinux analog) and a direct thrombin inhibitor (α-NAPAP analog) has been prepared. Furthermore the Lys-side chain of this compound was additionally coupled to biotin, which enables a neutralization of their anticoagulant activity by injecting avidin.
To adapt this antidote strategy, bifunctionalized liposomes were prepared containing the inhibitor, along with the additional biotinylated compound Me-(CH2)14-CO-Tota-Biotin on their surface. However, it was only possible to achieve a partial neutralization of the thrombin inhibitory potency by the addition of avidin. A stronger neutralizing effect was observed for free biotinylated inhibitors.
In this doctoral thesis described strategy could be also applied to other drug molecules which suffer from rapid renal clearance and tolerate modification with a linker coupled to a suitable fatty acid. Although we did not perform any pharmacokinetic studies, we assume that incorporation of these inhibitors in to liposomes should increase their half-lives in circulation relative to that of the unmodified peptidic drug molecules.