Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Anticoagulants are used as the first line therapy for management and prevention of thrombotic disorders. Thrombin and factor Xa have been the prime targets for regulation of the coagulation cascade. In this work, a small library of 17 benzofuran derivatives were synthesized and screened against thrombin and factor Xa. The derivatives that displayed inhibitory potential were docked on the exosite-II of factor Xa using a docking protocol that was developed in our research group. These compounds were based on the β-5 structural unit found in the oligomer -'CDSO3‘, which was prepared in our lab and was found to inhibit both thrombin and factor Xa by an exosite-II mediated allosteric disruption of the catalytic triad.The results revealed that these β-5 like derivatives are inhibitory against thrombin and factor Xa, although their potency is weak. Thrombin and factor Xa appear to recognize different structural features suggesting a significant selectivity in recognition. Furthermore, a slight preference for the benzofuran scaffold was observed with factor Xa. Probing the mechanism of inhibition using Michaelis-Menten kinetics reveal that these compounds display uncompetitive inhibition of these proteases and the mechanism of inhibition is allosteric. Docking of these compounds on factor Xa were done using GOLD (Genetic algorithm for ligand docking) and the results, explain the observed inhibition profile. The computed docked poses also give an idea of the residues on the exosite-II of factor Xa critical for inhibition. The molecules studied here are radically different in terms of structure and mechanism of inhibition from any other ligand described in literature. This represents an opportunity to discover novel molecules with a possibly different pharmacological and toxicological profile.

Direct factor Xa inhibitors (FXaI) account for most oral anticoagulant use. FXaI-associated bleeding events are common and are associated with substantial morbidity and mortality. Nonspecific hemostatic therapies such as prothrombin complex concentrates (PCC) are often administered for FXaI-associated bleeding. The mechanism by which these agents improve hemostasis in the setting of direct oral anticoagulation is unclear. Thrombin generation assays may effectively measure the effect of anticoagulation reversal among FXaI-treated patients when bleeding cessation would otherwise be challenging to measure. To build a research program on the utility of thrombin generation assays to measure both the impact of direct oral anticoagulation and anticoagulation reversal, we completed a review of the literature with narrative synthesis and carried out a pilot study to determine the feasibility of a full scale prospective observational study of TGA responses among patients receiving PCC for FXaI-associated major bleeding or needing urgent surgery.

Biliary excretion is an important elimination route for many drugs and metabolites. For such compounds, it is important to know the extent of excretion and drug exposure in the bile, e.g., for the risk assessment of drug interactions, liver toxicity and the effects of genetic variants. In this thesis, duodenal aspiration of bile was performed in healthy volunteers and complemented with experiments in an in vivo model in pigs to increase the understanding of the intestinal and hepatobiliary disposition of two direct thrombin inhibitors. The compounds investigated, ximelagatran and AZD0837, are both prodrugs that require bioactivation to exert their pharmacological effect. Upon co-administration with erythromycin and ketoconazole, respectively, altered plasma exposure to ximelagatran and AZD0837 and their respective metabolites has been observed. The main objective of this thesis was to characterize the biliary excretion of the compounds, and investigate whether this elimination route explains the observed drug-drug interactions. High plasma-to-bile AUC ratios were observed, in particular for ximelagatran, its active metabolite melagatran, and AR-H067637, the active metabolite of AZD0837. These high ratios indicate the involvement of active transporters in the biliary excretion of the compounds, which is important since transporters constitute possible sites for drug interactions. The effects of erythromycin and ketoconazole on the plasma exposure of the prodrugs and metabolites were confirmed in both the pig and the clinical studies. The changes seen in plasma for ximelagatran and its metabolites were partly explained by reduced biliary clearance. Inhibited CYP3A4 metabolism likely caused the elevated plasma levels of AZD0837, whereas reduced biliary clearance was seen for AR-H067637 suggesting an effect on its excretion into bile. In summary, the studies led to mechanistic insights in the hepatobiliary disposition of ximelagatran and AZD0837, and demonstrate the value of combined clinical and animal studies for the investigation of the biliary drug excretion.

The design of biomaterials, historically initiated and developed by physicians and engineers, in the last decades has slowly shifted toward a more biochemical based approach. For the replacement, repair and regeneration of tissues scientists are now focusing on materials that stimulate specific biological response at the molecular level. These biomaterials have already shown interesting applications in cell proliferation, differentiation, and extracellular matrix production and organization when the material modifications are designed to elicit specific interactions with cell integrins. In the present work we propose the application of this strategy for the development of blood-compatible materials. We first identified, in the coagulation cascade a key enzyme that constitute a valuable biological target for the development of anti-thrombogenic compounds. Piperazinyl-amide derivatives of N-alfa-(3-trifluoromethyl-benzenesulfonyl)-L-arginine were synthesized as graftable thrombin inhibitors. These inhibitors provided a spacer arm for surface grafting and a fluorine tag for XPS (X-ray photoelectron spectroscopy) detection. The possible disturbance of biological activity due to a variable spacer-arm fixed on the N-4 piperazinyl position was evaluated in vitro against human alfa-thrombin, in silico by molecular modelling and via X-ray diffraction study. Selected inhibitors, having inhibition potency in the mM range, were grafted on polyesters surface via wet chemistry and photochemical activation treatments. Wet chemistry surface grafting was performed by specific hydroxyl chain-ends activation and resulted in bioactive molecules fixation of 20-300pmol/cm2. The photochemical grafting was performed using a molecular clip providing an aromatic azide, for nitrene insertion into a polymer, and an activated ester for grafting of tag compounds. This grafting technique resulted in a dramatic increase in fixed bioactive signals (up to nmol/cm2). The material blood-compatibilization induced by the surface fixation of the inhibitors, was measured by a static blood clot weight measurement test. The wet chemistry grafting technique resulted in moderate blood-compatibilization while by the photochemical grafting method important decrease in surface blood clot formation was observed. In the latter case, the blood response to material contact was found to be strongly affected by the polyester surface photo-degradation induced by the activation treatment.

The design of biomaterials, historically initiated and developed by physicians and engineers, in the last decades has slowly shifted toward a more biochemical based approach. For the replacement, repair and regeneration of tissues scientists are now focusing on materials that stimulate specific biological response at the molecular level. These biomaterials have already shown interesting applications in cell proliferation, differentiation, and extracellular matrix production and organization when the material modifications are designed to elicit specific interactions with cell integrins. In the present work we propose the application of this strategy for the development of blood-compatible materials. We first identified, in the coagulation cascade a key enzyme that constitute a valuable biological target for the development of anti-thrombogenic compounds. Piperazinyl-amide derivatives of N-alfa-(3-trifluoromethyl-benzenesulfonyl)-L-arginine were synthesized as graftable thrombin inhibitors. These inhibitors provided a spacer arm for surface grafting and a fluorine tag for XPS (X-ray photoelectron spectroscopy) detection. The possible disturbance of biological activity due to a variable spacer-arm fixed on the N-4 piperazinyl position was evaluated in vitro against human alfa-thrombin, in silico by molecular modelling and via X-ray diffraction study. Selected inhibitors, having inhibition potency in the mM range, were grafted on polyesters surface via wet chemistry and photochemical activation treatments. Wet chemistry surface grafting was performed by specific hydroxyl chain-ends activation and resulted in bioactive molecules fixation of 20-300pmol/cm2. The photochemical grafting was performed using a molecular clip providing an aromatic azide, for nitrene insertion into a polymer, and an activated ester for grafting of tag compounds. This grafting technique resulted in a dramatic increase in fixed bioactive signals (up to nmol/cm2). The material blood-compatibilization induced by the surface fixation of the inhibitors, was measured by a static blood clot weight measurement test. The wet chemistry grafting technique resulted in moderate blood-compatibilization while by the photochemical grafting method important decrease in surface blood clot formation was observed. In the latter case, the blood response to material contact was found to be strongly affected by the polyester surface photo-degradation induced by the activation treatment.

No description available.