Σχεδιασμός και σύνθεση πεπτιδίων με πιθανή αντιθρομβωτική δράση

Abstract

Thromboembolic diseases comprise one of the major causes of hospitalization and mortality worldwide. Anticoagulant therapy has historically consisted of heparins for the treatment of acute thrombosis and vitamin K antagonists for long-term or chronic treatment. Though effective if appropriately dosed and monitored, these traditional agents have shortcomings that stem mainly from their nonspecific mechanisms of action and variable pharmacodynamics. New anticoagulant drugs target specific steps in coagulation. Anticoagulant strategies that inhibit thrombogenesis focus on blocking initiation of coagulation, preventing thrombin generation by attenuating the propagation of coagulation, or by reducing fibrin formation by inhibiting thrombin. During the blood coagulation process, the tissue factor complex activates factor IX with Josso pathway. Factor VIII which is activated by the produced thrombin and combined with phospholipids (derived from activated platelets), calcium ions and IXa forms the intrinsic tenase complex, that activates factor X. It is considered that, the binding surface of factor VIIIa to factor IXa is localized in A2 and A3 domains of factor VIIIa that interact with various regions of factor IXa. A3 domain of the light chain of FVIIIa contains a high affinity binding site. Studies with synthetic peptides targeting A3 domain have shown that this binding site is localized in sequence 1811-1818. Probable binding sites of A2 domain of factor VIIIa with factor IXa include sequence 558-565 and the area around Asp712. According to the previews, it has been chosen to synthesize peptide analogues of the FVIIIa region, which has been determined as the interaction region with factor IXa, in order to study their function as inhibitors of thrombosis. The selection of peptide analogues that were synthesized, was made in order to study the influence of factors that affect the conformation of the molecules as well as the effect of the side chains of the amino acids in the biological activity of the peptides The peptides were synthesized by the solid phase technique. For the protection of the functional groups the Fmoc/tBu method was used, while the carbodiimides method was used for the coupling of amino acids. Completion of the coupling reactions was determined by the Kaiser test and thin layer chromatography (TLC). The reaction products were purified by reverse phase HPLC and identified by RPHPLC and Electron Spray Mass Spectrometry (ESI-MS). Initially, we synthesized the sequence 1811-1818. Following, we replaced Glu1811 with Asp. There were synthesized peptide analogues with N-terminal aminο group either free or protected with acetyl-group. Then, we replaced Tyr1815 with Ile as well as Phe1816 with Ile to investigate the role of aromatic amino acids in the biologic functionality of the peptides. There were synthesized peptide amides with N-terminal aminο group either free or protected with acetyl-group. Experiments that were conducted to evaluate the biological activity of the analogues included the activated partial thromboplastin time (APTT) and the prothrombin time (PT) clotting assays, inhibition of blood coagulation factor VIII assay and platelet aggregation assay. The measurement of APTT is generally used as a screening procedure for the evaluation of the intrinsic coagulation pathway. The prolongation of it against the normal values indicates inhibition of the coagulation process. All peptides presented a tendency to prolongate of APTT, with peptide Ac-Glu-Thr-Lys-Thr-Tyr-Phe-Trp- Lys-NH2 (ΡΑ6) presenting the most important activity (prolongation 6,10sec). Experiments concerning PT did not show any significant prolongation, providing evidence that their function is mainly localized on inhibiting the formation of the intrinsic tenase complex and not on the extrinsic tenase complex. Additionally, this fact enhances the possible functionality of the analogues in treatment of thrombophilia without causing side effects like bleeding, since the inhibition is selective and does not affect all the coagulation mechanisms. As far as the inhibition of blood coagulation factor VIII assay is concerned, the results showed significant activity (41%) of analogue PA6 opposite to the rest of the analogues that showed a low inhibition tendency (3-13%). This assay is indicative of the influence of the peptides in the FVIIIa and more specifically in the possible inhibition of the formation of the intrinsic tenase complex. This finding is in agreement with the results of the APTT measurement. Furthermore, experiments took place in vitro where the effect of the analogues on platelet aggregation was studied. When collagen was used as an agonist, the analogues did not present any antiplatelet activity, while the ADP is concerned, even though the analogues cause a small inhibition of aggregation, this does not considered important enough. Afterwards, encapsulation experiments of PA6 analogue in hydrogel took place. This analogue was chosen because it showed the most significant biological action among those that were tested. The encapsulation of PA6 in hydrogel of cross linked PVA was attempted for the preliminary study of bead production suitable for drug release. PVA was chosen as the encapsulation polymer due to its non-toxicity, fast bead formation and their attributes. As cross linking agents, boric acid and glutaraldehyde were used. The water hydrogel uptake and entrapment efficiency of PA6 in hydrogel beads were determined. Also, studies of the peptide release from the beads in water, were conducted and the release times as well as the diffusion coefficient were estimated. The release times varied between 50 and 80 min in case of boric acid polymers, while in case of glutaraldehyde the release time was 120 min, which was the highest value that was observed. The diffusion coefficient that characterizes the release kinetics of the peptide in water was determined. The conclusion was that the values vary in a narrow window, which shows that the bead porosity is slightly different in all the cases that were studied. The encapsulation results were satisfactory since durable and elastic beads were produced that show specific peptide release kinetics, which indicates that these beads can be used for controlled release of the biologically active peptide.