RESUMEN
INTRODUCTION: The anticoagulant r-hirudin lepirudin is eliminated exclusively via the kidneys. We examined the C-terminal amino acid degradation of lepirudin by the proximal kidney tubulus cells in humans as well as the antithrombotic efficacy of the metabolites and quantified the metabolite portions. MATERIALS AND METHODS: In vitro metabolites of lepirudin were produced by adding 250 microg lepirudin to urine of three healthy volunteers and a concentration of 100 ml fresh urine to 1.5 ml and subsequent separation by high performance liquid chromatography. Anticoagulant activities of the mass spectrometrically identified metabolites were measured by ecarin clotting time and protein determination with bicinchoninic acid. In 10 healthy volunteers 1 mg lepirudin was administered intravenously, urine was collected during the following 2 h. The urine amount containing 50 microg lepirudin measured by ecarin clotting time was enzyme-inactivated and measured analogously to the in vitro samples. RESULTS: The in vitro generated metabolites were shortened amino acid by amino acid at the C-terminal end, up to five amino acids. Their anticoagulant activity was reduced to 92.6% (M64), 80.1% (M63) and 74.4% (M60,61,62) in comparison to lepirudin. Lepirudin (57.9 +/- 8.6%) was eliminated unchanged via the kidneys. Identical to the in vitro situation metabolite fragments were built in the distribution M64 = 8.1 +/- 5.7%, M63 = 21.1 +/- 6.5%, and M60,61,62 = 12.9 +/- 4.5%. CONCLUSIONS: Lepirudin is metabolized spontaneously in more than 10-fold concentrated urine. Metabolization of lepirudin takes place in the proximal tubulus cells as well. In vitro, the degradation takes place amino acid by amino acid, but in vivo even dipeptides and perhaps tripeptides are degraded.