Unique overlap in the prerequisites for thrombin inhibition and oral bioavailability resulting in potent oral antithrombotics.

Despite intense research over the last 10 years, aided by the availability of X-ray structures of enzyme-inhibitor complexes, only very few truly orally active thrombin inhibitors have been found. We conducted a comprehensive study starting with peptide transition state analogues (TSA). Both hydrophobic nonpeptide analogues as well as hydrophilic peptidic analogues were synthesized. The bioavailability in rats and dogs could be drastically altered depending on the overall charge distribution in the molecule. Compound 27, a tripeptide TSA inhibitor of thrombin, showed an oral bioavailability of 32% in rats and 71% in dogs, elimination half-lives being 58 and 108 min, respectively. The thrombin inhibition constant of compound 27 was 1.1 nM, and in an in vivo arterial flow model, the ED(50) was 5.4 nmol/kg.min, comparable to known non-TSA inhibitors. A molecular design was found that combines antithrombotic efficiency with oral bioavailability at low dosages.

Two sensitive and rapid chromogenic assays of fondaparinux sodium (Arixtra) in human plasma and other biological matrices.

Fondaparinux is a synthetic selective inhibitor of factor Xa recently approved for thromboprophylaxis after major orthopedic surgery. Determination of its concentration gives valuable insight into specific pharmacokinetics or safety studies. The aim of the study was to develop direct, sensitive, precise and accurate assays of fondaparinux sodium in different biological matrices. Consistency with the recommended chromogenic assay for low molecular weight heparin required a similar method. However, recent data indicated some variability in the determination of anti-Xa level between commercial chromogenic assays. Consequently, we developed and validated two chromogenic methods (A and B) for assaying fondaparinux in plasma and other biological matrices. The assays are calibrated with fondaparinux, a pure chemical entity, and the result is expressed as amount (microg) of the fondaparinux calibrator. Results showed that precision was lower than 5.2% in plasma or plasma water and 13% in placental medium. The accuracy was lower than 7.6% in plasma or plasma water and 10.2% in placental medium. The lower limit of quantification in plasma was 0.042 microg/mL with automated Method A and 0.019 microg/mL with Method B. The assay was not affected by the source of the samples, the presence of blood cells, EDTA, citrate or repeated cycles of freezing and thawing. The two chromogenic assays calibrated with fondaparinux sodium reach the equivalence criteria for plasma samples and provide reliable and reproducible results.