Characterization of the Hepatic Disposition of Lanoteplase, a Rationally Designed Variant of Tissue Plasminogen Activator in Rodents

Abstract

ABSTRACT:Lanoteplase is a recombinant mutant of tissue-type plasminogen activator (t-PA) that was developed with an aim to overcome the drawback of rapid systemic elimination of t-PA. In this study, we examined the disposition profile of lanoteplase in vivo and the kinetics of receptor-mediated endocytosis (RME) of this recombinant t-PA in vitro to kinetically characterize the mechanism(s) underlying its tissue distribution and elimination. Integration plot analysis of the initial-phase tissue distribution in rats revealed a much lower uptake clearance (CL uptake ) of lanoteplase in the liver than that of t-PA. Rate constants for cell surface binding, internalization, and degradation of lanoteplase were also lower than those for t-PA in primary cultured rat hepatocytes. These results suggest that the improved stability of lanoteplase in vivo could be accounted for by the delay in the RME of this recombinant protein.The CL uptake in the liver decreased with coadministration of lactoferrin, a ligand for the low-density lipoprotein receptor-related protein (LRP) and the asialoglycoprotein (ASGP) receptors in normal mice, and in lrpap1 (؊/؊) mice, which have a hereditary deficiency of LRP; In contrast, CL uptake was not affected by mannose, whereas that of t-PA decreased with both ligands and in the lrpap1 (؊/؊) mice. Thus, the hepatic disposition of lanoteplase seems to be mediated by common specific receptors for t-PA, including LRP and the ASGP receptors, whereas the mannose receptor seems to be only minimally involved in the disposition of lanoteplase.Tissue-type plasminogen activator (t-PA) is a serine protease composed of 527 amino acids, containing five structural domains (the finger domain, the epidermal growth factor domain, the kringle 1 domain, the kringle 2 domain, and the serine protease domain) and three sugar chains at amino acid positions 117, 184, and 448. Because of its potent thrombolytic activity, wild-type t-PA has been widely used as a therapeutic thrombolytic agent for myocardial infarction (Llevadot et al., 2001;Collen and Lijnent, 2004), and it has recently been approved for the treatment of stroke. However, even in such emergent clinical care situations, t-PA must be administered by intravenous infusion because of its short half-life of 3 to 4 min. Consequently, several types of recombinant t-PA proteins have been developed to prolong the plasma retention time and thereby make a rapid bolus administration of t-PA possible.Lanoteplase is one of the mutant forms of t-PA, which was developed with the recombinant DNA technology to achieve such an aim.Lanoteplase was produced by removing the epidermal growth factor domain, a part of the finger domain, and by substituting Gln for the Asn residue at position 117, resulting in a removal of the sugar chain. Despite such a major structural modification, lanoteplase still exhibited a potent thrombolytic activity at much lower dose than t-PA both in vivo and in a rabbit model of jugular vein thrombosis (Furuya et al., 1999). In addition, the efficiency...