Prediction of the pharmacokinetics of succinylated human serum albumin in man from in vivo disposition data in animals and in vitro liver slice incubations
Introduction
Chemical modifications of plasma and milk proteins have been performed to increase the activity of their biological functions or to introduce entirely new pharmacological activities (Harmsen et al., 1995, Jansen et al., 1991, Meijer et al., 2001, Neurath et al., 1995, Swart et al., 1996b, Swart et al., 1999b). It was found that human serum albumin modified by succinic anhydride (Suc-HSA) or cis-aconitic anhydride yielded polyanionic proteins with potent anti-viral activities in particular against HIV. These negatively charged albumins (NCAs) were evaluated for their anti-HIV activity in vitro as well in vivo in various laboratories. The anti-viral activity was not only directed against HIV laboratory strains but also, although with higher IC50 values, against several primary clinical HIV isolates that differed in their syncytium-inducing capacity (Groenink et al., 1997). The NCAs, with the prototype Suc-HSA, act in an early phase of HIV replication, and they presumably interact with the adsorption/fusion event by binding to the viral glycoproteins gp120 and gp41 (Groenink et al., 1997, Kuipers et al., 1996, Kuipers et al., 1999, Meijer et al., 2001, Swart et al., 1996b).
The disposition of succinylated albumin was studied in detail in various animal species: mouse, rat and monkey (Kuipers et al., 1997, Swart et al., 1996c, Swart et al., 1996a, Swart et al., 2001). It was shown that succinylated albumin is eliminated from the blood by the liver via receptor-mediated endocytosis (Jansen et al., 1993, Swart et al., 1996a). Furthermore, these studies made clear that after intravenous and intraperitoneal injections of high doses, succinylated albumin can exhibit an apparent plasma half-life of several hours as a result of the saturable endocytotic elimination process (Swart et al., 1996c, Swart et al., 1996a). The compound distributes significantly to the lymphatic system (Swart et al., 1999a), a reservoir for viral particles (Haase, 1999), where it may reach the major target cells for HIV infection (Pantaleo et al., 1993). The concentrations attained in these tissues largely exceeded the minimal effective concentrations against HIV-1 as determined in vitro (Swart et al., 1999a). In preliminary experiments, succinylated albumin did not show acute toxicity in rats and monkeys, and did not provoke immunogenic reactions in rats (Jansen et al., 1993, Swart et al., 1996a). On the basis of these data, efficacy studies in an HIV-mouse model were initiated. These experiments showed that Suc-HSA is not only effective as an anti-HIV agent in vitro, but also can exhibit potent anti-HIV effects in vivo (Kuipers et al., 1997).
The elimination of succinylated albumin from the blood in monkeys occurred more rapidly than in rats. In order to enable pharmacokinetic and efficacy studies in man, a prediction of the pharmacokinetics of Suc-HSA in man is required. As mentioned above, succinylated albumin is mainly cleared by the liver. Liver slices have been used to study various organ functions including cellular uptake of glycoproteins and related drug targeting preparations (Olinga et al., 1994, Olinga et al., 2001). Studies with 125I-labeled succinylated albumin showed that this polyanionic protein is taken up by rat liver slices by a temperature-dependent process. Furthermore, an excess of non-labeled succinylated albumin blocked the uptake, suggesting the occurrence of a receptor-mediated uptake process (Olinga et al., 2001). These studies in slices confirmed the saturable kinetic patterns in vivo and supported the idea that the in vivo disposition of Suc-HSA in man might be predicted on the basis of in vitro liver slice uptake studies and in vivo disposition data in animals. Therefore, studies with liver slices from rat, monkey and man were performed to allow pharmacokinetic interspecies scaling.
The major objective of the present study was to predict the pharmacokinetics of Suc-HSA in man in vivo, in order to optimize the dosing regimen for future phase I clinical studies. A secondary objective was to describe a procedure for predicting pharmacokinetics in man using the available data from in vitro and in vivo studies.
Section snippets
Albumins
Human serum albumin (HSA) was obtained from Sanquin (Amsterdam, The Netherlands). The monomeric fraction was at least 95% pure as determined by Fast Protein Liquid Chromatography (FPLC) (Amersham Biosciences, Uppsala, Sweden).
Simian serum albumin (SSA) was isolated in our laboratory from blood plasma (Cohn fraction V) obtained from at least two donor animals, using ethanol fractionation techniques. The isolated albumin was solubilized in phosphate buffered saline (PBS), desalted by Amicon
In vitro estimation of Vm and Km of uptake in slices
The proteins used in the in vitro binding and uptake experiments were radiolabeled with 125I according to the tyramine cellobiose method. Following this labeling technique and exposure to endocytotic cell types, radioactivity remains stored in lysosomes and cannot be transported out of the cells (Zhong et al., 1992). Thus, the total uptake of the protein by the tissue can be determined. The binding and uptake of succinylated albumin was markedly higher at 37 °C compared to the binding at 4 °C,
Discussion
Suc-HSA is a potent HIV-inhibitor with possible application in man. To facilitate the assessment of dosing regimens for future phase I clinical studies, we predicted the pharmacokinetic properties of Suc-HSA in man from in vitro studies with liver slices from rat, monkey and man and in vivo pharmacokinetic studies in rat and monkey, using pharmacokinetic analysis and allometric interspecies scaling of the available data.
Because the liver was shown to be the main organ for clearance of these
Acknowledgements
This work was in part supported by research grants from the council for Health Research (RGO/PccAo) and from The Netherlands Centre Alternatives to Animal Use. Monkey livers were kindly donated by Dr. A. Osterhaus, Erasmus University Rotterdam, The Netherlands. Suc-HSA used in the in vivo rat experiments was kindly donated by Dr. A. Koenderman, Sanquin, Amsterdam, The Netherlands. The authors wish to thank Mrs. A. van Loenen-Weemaes, Mrs. I. Hof and Mr. B.P. Stok for technical assistance.
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