title: Disease-induced drug targeting using novel peptide-ligand albumins.
abstract: Small therapeutic oligopeptides (two to 12 amino acids), designed for interaction with cytokine and growth factor receptors, unfortunately, are rapidly removed from the body. Efficient glomerular filtration and carrier-mediated membrane transport processes are involved in their clearance. By coupling of such peptides to macromolecules, elimination via these pathways is prevented and exposure to the particular receptors can be largely improved. Some of these constructs undergo receptor-mediated endocytoses and can be used as carriers to deliver associated drugs to various cell types in the body. It has been shown that, in the case of neo-glycoprotein carriers, down-regulation of the receptors aimed at can occur in the diseased state. We therefore designed a new type of polypeptide carrier, homing on receptors that are known to be highly upregulated in the pathological target tissue. For this purpose we designed ligand peptides (minimized proteins) representing the receptor-recognizing domains of PDGF and collagen type VI, aimed at receptors that are highly expressed, particularly on activated hepatic stellate cells (HSC). This myofibroblast-type of cell largely contributes to connective tissue expansion during liver fibrosis. Drug carriers for the stellate cell have not been reported before.
  
  
  METHODS
  Cyclic octapeptide moieties (n10--12) with affinity for the two receptors were coupled to HSA (pPB-HSA and pCVI-HSA, respectively). Receptor binding experiments confirmed binding of these ligand peptides to their receptors in vitro.
  
  
  METHODS
  rat HSC were isolated and purified according to standard techniques. The cells were cultured for 2 days (quiescent phenotype) or for 10 days (activated phenotype). Cell cultures were incubated with the carriers and the binding (at 4 degrees C), uptake (at 37 degrees C), and degradation were determined with radioactive and immunohistochemical methods. The results were compared with data obtained with unmodified HSA.
  
  
  METHODS
  the organ distribution of pCVI-HSA and pPB-HSA was determined 10 min after i.v. injection of tracer doses in normal and fibrotic rats, 3 weeks after bile duct ligation. Hepatocellular distribution was scored after double-immunostaining of the liver sections with an antibody against the designated hepatic cell type in combination with anti-HSA IgG.
  
  
  RESULTS
  
  
  
  METHODS
  All three carriers preferentially bound to the activated rather than to quiescent HSC. Binding to cells was inhibitable by an excess of unlabelled pCVI-HSA, endocytosis was inhibitable by 2 mM monensin suggestive of lysosomal routing of the proteins, whereas pPB-HSA, at least partly, remained at the cell surface. Degradation products of the carriers were detected extracellularly after incubation with fibrotic rat liver slices during 2-h experiments.
  
  
  METHODS
  62+/-6% of the dose of pCVI-HSA accumulated in fibrotic livers at 10 min after injection, of which the major part was taken up in HSC. 48+/-9% of pPB-HSA accumulated in fibrotic rat livers and this carrier was also mainly taken up by HSC (5). Similar amounts of both constructs were taken up in normal rat livers, but predominantly in other cell types. The preferential homing to the stellate cells, only in the fibrotic liver is explained by the marked proliferation of this cell type as well as overexpression of the targeted receptors on these cells in the diseased state.
  
  
  CONCLUSIONS
  The in vivo results support the in vitro studies showing accumulation of these modified albumins in HSC in fibrotic rat livers and, in particular, in the stellate cells. The results demonstrate the specificity of the stellate cell targeting and imply applicability of pCVI-HSA as carriers for drugs that act intracellularly. In addition, pPB-HSA may be used to deliver drugs that act extracellularly, such as receptor antagonists. This concept may create new opportunities for delivery of conventional drugs that are not effective enough in vivo and/or display serious extrahepatic side-effects. Minimized proteins attached to soluble or particle type of macromolecules represent a novel carrier modality of which selective body distribution is induced by the disease process to be targeted. They can be utilized as receptor antagonists and at the same time can deliver therapeutic agents to the desired site of action (dual targeting).
authors.affiliation: Department of Pharmacokinetics and Drug Delivery, Groningen University Institute for Drug Exploration, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. g.dempsey@farm.rug.nl
authors.forename: D K
authors.initials: DK
authors.lastname: Meijer
authors.forename: L
authors.initials: L
authors.lastname: Beljaars
authors.forename: G
authors.initials: G
authors.lastname: Molema
authors.forename: K
authors.initials: K
authors.lastname: Poelstra
mesh_terms: D000418:Albumins / Q000493:pharmacokinetics*; D000818:Animals; D016503:Drug Delivery Systems; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects; D007728:Kupffer Cells / Q000187:drug effects; D008024:Ligands; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000473:pathology; D008103:Liver Cirrhosis / Q000188:drug therapy* / Q000378:metabolism*; D009842:Oligopeptides / Q000493:pharmacokinetics*; D051381:Rats; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D000418:Albumins; D008024:Ligands; D009842:Oligopeptides