RESUMEN
The dipeptide Mtr-Asp-D-Adf-Pip 10 represents a potent thrombin inhibitor. In comparison to NAPAP, 10 exhibited improved tolerability and a longer half-life in vivo, i.e., 20 +/- 5 min. We have coupled aminopolyethyleneglycolmonomethylether of various molecular weights to the carboxyl moiety of 10 and evaluated their biological properties. First, Mtr-Asp-OBut was coupled to the amino group of the PEG employing TOTU as an activating agent. This was followed by the removal of the OBut protecting group and coupling of D-Adf-Pip using TOTU as well. The PEG-bound thrombin inhibitors showed inhibition constants vs. thrombin in the subnanomolar range, i.e., they were more active than the parent molecule 10. Moreover, the pegylated inhibitors exhibited a longer lasting effect in vivo. In rats the half-life of Mtr-Asn (PEG10000-OMe)-D-Adf-Pip 14 was determined to be 63 min. Mtr-Asn(PEG10000-OMe)-D-Adf-Pip 14 showed a half-life of 120 min in pigs. It could be concluded that these PEG-bound thrombin inhibitors may be employed as versatile drugs for parenteral administration in treating thrombotic disorders.
Asunto(s)
Antitrombinas/síntesis química , Dipéptidos/síntesis química , Fenilalanina/análogos & derivados , Polietilenglicoles/química , Animales , Antitrombinas/química , Antitrombinas/farmacocinética , Antitrombinas/farmacología , Sitios de Unión , Coagulación Sanguínea/efectos de los fármacos , Dipéptidos/química , Dipéptidos/farmacología , Femenino , Espectroscopía de Resonancia Magnética , Estructura Molecular , Ratas , Ratas EndogámicasRESUMEN
Thrombin inhibitors have been thought to play a pivotal role in myocardial infarct and stroke incidences and their aftermath. Quite some time ago potent synthetic thrombin inhibitors became known based on peptide derivatives D-Phe-Pro-Arg and benzamidine. One of them, fairly well characterised was beta-naphthylsulphonylglycyl-D,L-4-amidino-phenylalanylpiperidi de (NAPAP). NAPAP was prone to being administered intravenously due to its short plasma half life. Drawbacks to this compound such as effects on histamine release and blood pressure may have obstructed its clinical use. Long half life and oral bioavailability would be desirable for prophylactic treatment of thrombotic disorders. We have used NAPAP as a template for new synthetic compounds to improve some characteristics of its profile. For screening purposes we have investigated fairly simple surrogate parameters, aspects that were considered to contribute to pharmacological effects. Potency was correlated to thrombin inhibition, side effects were addressed by specificity toward thrombin as well as reduction in basicity, and plasma half life was considered to be modulated by plasma stability of the compound. Oral bioavailability would be affected by instability during the passage through the gut wall. Chemical introduction of a carboxylic group and exchange of the naphthyl group for 4-methoxy-2,3,6-trimethylphenyl led to a compound that when compared to NAPAP, exhibited a 4-fold increase in thrombin inhibitory activity and a 3-fold increase in trypsin specificity. Plasma stability decreased to 22 h, however, sufficient enough not to play a major role in plasma half life. Gut homogenate stability of the compound has not changed. The potency increase did not translate into a reduction in IC50-values for the coagulation assay aPTT and TT, in contrast to the IC50-values for thrombin-induced platelet aggregation.
Asunto(s)
Antitrombinas/farmacología , Dipéptidos/farmacología , Piperidinas/farmacología , Trombina/antagonistas & inhibidores , Antitrombinas/síntesis química , Antitrombinas/química , Antitrombinas/farmacocinética , Sitios de Unión , Coagulación Sanguínea/efectos de los fármacos , Dipéptidos/síntesis química , Dipéptidos/química , Dipéptidos/farmacocinética , Diseño de Fármacos , Inhibidores del Factor Xa , Fibrinolisina/antagonistas & inhibidores , Humanos , Mucosa Intestinal/metabolismo , Cinética , Estructura Molecular , Piperidinas/síntesis química , Piperidinas/química , Piperidinas/farmacocinética , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Serina Proteinasa/síntesis química , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacocinética , Inhibidores de Serina Proteinasa/farmacología , Relación Estructura-Actividad , Trombina/metabolismo , Tripsina/metabolismoRESUMEN
The specific thrombin inhibitors r-hirudin and a synthetic peptide (I) D-FPRP(G)4-NGDFEEIPEEYL were compared in in vitro tests. r-hirudin proved to be the superior compound with respect to inhibition of amidolytic small substrate turnover that is catalysed by soluble and immobilised thrombin as well as to inhibition of fibrinogen activation. In an in vitro clot model significantly higher molar concentrations of peptide I are needed to achieve fibrin bound thrombin inhibition equivalent to that of r-hirudin. Stable complexes consisting of thrombin and hirudin oppose labile complexes containing the synthetic peptide. The latter leads to a regaining of thrombin activity with subsequent additional fibrin accretion. Analyses of the mixtures of thrombin and peptide I display a time dependent release of amino-terminal D-FPR peptide (III) exhibiting, similar to the residual fragment (peptide II), only weak inhibitory activity. Peptide I and the carboxy-terminal fragment induce, within a certain concentration range, an increase in thrombin activity and clot growth.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Hirudinas/análogos & derivados , Hirudinas/farmacología , Fragmentos de Péptidos/farmacología , Trombina/antagonistas & inhibidores , Secuencia de Aminoácidos , Fibrina/metabolismo , Hirudinas/síntesis química , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Proteínas Recombinantes/síntesis química , Proteínas Recombinantes/farmacología , Relación Estructura-Actividad , Trombina/metabolismo , Factores de TiempoRESUMEN
Magnetic resonance (MR) spectroscopy of an EDTA or citrate plasma sample (also referred to as "Fossel test" after Fossel, who first described it) started out as a promising method for fast and simple diagnosis of malignancies. Although the results of our own studies of the head and neck area confirm this tendency, we believe--and so do meanwhile also other investigators--that the clinical use of this test in its present form is not feasible because of lacking specificity.