Microspheres as a Carrier System for Therapeutic Embolization Procedures: Achievements and Advances.

The targeted delivery of anti-cancer drugs and isotopes is one of the most pursued goals in anti-cancer therapy. One of the prime examples of such an application is the intra-arterial injection of microspheres containing cytostatic drugs or radioisotopes during hepatic embolization procedures. Therapy based on the application of microspheres revolves around vascular occlusion, complemented with local therapy in the form of trans-arterial chemoembolization (TACE) or radioembolization (TARE). The broadest implementation of these embolization strategies currently lies within the treatment of untreatable hepatocellular cancer (HCC) and metastatic colorectal cancer. This review aims to describe the state-of-the-art TACE and TARE technologies investigated in the clinical setting for HCC and addresses current trials and new developments. In addition, chemical properties and advancements in microsphere carrier systems are evaluated, and possible improvements in embolization therapy based on the modification of and functionalization with therapeutical loads are explored.

Peptide Antagonists for P-selectin Discriminate between Sulfatide-Dependent Platelet Aggregation and PSGL-1-Mediated Cell Adhesion.

BACKGROUND: Membrane-exposed sulfatides are proposed to contribute to P-selectin-dependent platelet aggregation. Here, we demonstrated that P-selectin-mediated platelet aggregation on a collagen-coated surface under flow indeed depended on sulfatides and that this interaction differed considerably from the interaction of P-selectin with P-selectin Glycoprotein Ligand-1 (PSGL-1), which underlies leukocyte-endothelium adhesion. METHODS AND RESULTS: Upon platelet activation, sulfatides were translocated to the platelet surface to form focal hot-spots. Interestingly, P-selectin was observed to exclusively interact with liposomes with a sulfatide density higher than 21% (w/w), indicating that the binding profile of P-selectin for sulfatide-rich liposomes was dependent on sulfatide density. Sulfatide-liposome binding to P-selectin and sulfatide/P-selectin-dependent platelet aggregation was blunted by peptide antagonists, carrying the EWVDV motif within N-terminal extensions, such as CDVEWVDVSC (half maximal inhibitory concentration IC50 = 0.2 µM), but not by the EWVDV core motif itself (IC50 > 1000 µM), albeit both being equally potent inhibitors of PSGL-1/P-selectin interaction (IC50= 7-12 µM). CONCLUSIONS: Our data suggest that the sulfatide/P-selectin interaction implicates multiple binding pockets, which only partly overlap with that of PSGL-1. These observations open ways to selectively interfere with sulfatide/P-selectin-dependent platelet aggregation without affecting PSGL-1-dependent cell adhesion.

Design and validation of a specific scavenger receptor class AI binding peptide for targeting the inflammatory atherosclerotic plaque.

OBJECTIVE: Scavenger receptor A (SR-A) is abundantly expressed by macrophage and plays a critical role in foam cell formation and atherogenesis. In search of selective SR-AI antagonists, we have used affinity selection of a phage displayed peptide library on the synthetic extracellular domain of SR-AI. METHODS AND RESULTS: Phage selection led to an almost 1,000-fold enrichment of SR-AI binding phage, which bound avidly to human THP-1 cells. A 15-mer corresponding to the peptide insert of the major SR-AI binding phage (PP1) displaced phage binding to SR-AI. Peptides, docked to a streptavidin scaffold, were effectively internalized by macrophages in an SR-AI-dependent manner. The enriched phage pool and streptavidin bound PP1 exhibited marked uptake by hepatic macrophages in mice. Importantly, PP1 significantly increased streptavidin as well as particulate accumulation in advanced aortic plaques, and in particular intraplaque macrophage, of apolipoprotein E(-/-) mice. CONCLUSIONS: We have identified a novel peptide antagonist selective for SR-AI; this antagonist could be a valuable tool in SR-AI targeted imaging of atherosclerotic lesions.

Efficient degradation-aided selection of protease inhibitors by phage display.

In this report, we describe a novel phage display strategy for the identification of dedicated protease inhibiting peptides, based on degradation-aided enrichment of protease resistant phages. Phages were directly incubated with a range of phage-degrading proteases, after which non-degraded phages were used for the next selection round. For proteinase-K we identified after only four selection rounds a peptide (VLIMPVLLGIPLLC) that inhibits proteinase-K activity with an inhibition constant of 4 microM. In analogy, we identified a peptide capable of inhibiting substrate degradation by cathepsin-S (VWNCERITISRLIN), which showed functional inhibition of cathepsin-S induced sprouting of endothelial cells. We envision that the pursued strategy of degradation-aided selection of protease inhibitors (DASPI) represents an effective approach in the design of new protease inhibitors but also of new strategies to render gene and drug vectors protease resistant.

Blocking endothelial adhesion molecules: a potential therapeutic strategy to combat atherogenesis.

PURPOSE OF REVIEW: This review provides a concise update of the involvement of endothelial adhesion molecules in atherogenesis, an overview of current advances in the development of adhesion molecule blocking agents, as well as an insight into the potential of these molecules in cardiovascular therapy. RECENT FINDINGS: As endothelial adhesion molecules are deemed to play an important role in the development and progression of atherosclerotic lesions, they are interesting targets for therapeutic intervention in this process. In particular, P-selectin and vascular cell adhesion molecule 1 are widely considered to hold promise in this regard. Current research efforts centre on the design of agents that directly block the interaction of the receptor with its ligand (e.g. soluble P-selectin glycoprotein ligand 1, blocking antibodies, EWVD-based peptides) or that interfere with their synthesis (e.g. antisense oligonucleotides) or their regulatory control by nuclear factor kappa B or peroxisome proliferator-activated receptor gamma. Furthermore, adhesion molecules have been exploited as a target for the specific delivery of drug carriers (e.g. biodegradable particles with entrapped dexamethasone) or therapeutic compounds (e.g. dexamethasone) to the plaque. All approaches have been shown to be effective in blocking adhesion molecule function in in-vitro studies and in-vivo models for inflammation or atherosclerosis. SUMMARY: Although the field has achieved considerable progress in recent years, leading to the development of a number of interesting leads, final proof of their efficacy in cardiovascular therapy is eagerly awaited.

P-selectin as a candidate target in atherosclerosis.

P-selectin is of critical importance in early atherogenesis by initiating leukocyte rolling at the site of endothelial injury. In order to validate P-selectin as a candidate target for the development of anti-atherogenic strategies, we wanted to obtain quantitative information on P-selectin expression, and identify novel peptide-based lead structures that interact with P-selectin. P-selectin mRNA expression in the aortic arch and in other tissues of apoE-deficient (apoE-/-) mice was determined by real-time PCR technology. P-selectin mRNA expression of apoE-/- mice increased steadily with age to levels 14-fold higher than that of control animals. The onset and level of P-selectin expression correlated well with the extent of lesion development, and was more specific for atherosclerotic tissue as compared with other adhesion molecules. Phage display technology was used to obtain novel P-selectin antagonists. Phage display selections resulted in the isolation of a highly P-selectin-specific phage clone. Synthetic peptide-equivalents of this clone displaced the binding of the parent phage and antagonized the binding of a sialyl Lewis(x) analogue to P-selectin. In conclusion, P-selectin expression correlates with early and advanced atherosclerotic lesion development. P-selectin ligands, like the lead structure we have developed here, can therefore be considered as promising tools to identify, target or antagonize P-selectin function within the chronically inflamed arterial wall.

Rational optimization of a short human P-selectin-binding peptide leads to nanomolar affinity antagonists.

P-selectin plays an important role in the development of various diseases, including atherosclerosis and thrombosis. In our laboratory we recently identified a number of specific human P-selectin-binding peptides containing a Glu-Trp-Val-Asp-Val consensus motif, displaying a low micromolar affinity for P-selectin (IC(50) = 2 microm). In search of more potent antagonists for P-selectin, we have optimized the EWVDV pentapeptide core motif via a two-step combinatorial chemistry approach. A dedicated library of peptide derivatives was generated by introducing seven substituents at the N and C termini of the motif. In particular, pentapeptides with gallic acid or 1,3,5-benzenetricarboxylic acid substituents at the N terminus proved to be considerably more potent inhibitors of P-selectin binding than the parental peptide. After removal of the N-terminal glutamic acid from the core sequence, which appeared to be replaceable by a carboxamide function without loss of affinity, a second library was synthesized to map the chemical moieties within the gallic acid or 1,3,5-benzenetricarboxyl acid groups responsible for the enhanced P-selectin binding. Moreover, by varying the length and rigidity of the connective spacer, we have further optimized the spatial orientation of the N-terminal substituent. The combined use of phage display and subsequent combinatorial chemistry led to the design of a number of gallic acid- containing peptides with low nanomolar affinity for P-selectin both under static and dynamic conditions (IC(50) = 15.4 nm). These small synthetic antagonists, which are equally as potent as the natural ligand P-selectin glycoprotein ligand-1, are promising leads in anti-atherothrombotic therapy.

Specific inhibition of P-selectin-mediated cell adhesion by phage display-derived peptide antagonists.

P-selectin is a leukocyte adhesion receptor expressed on activated vascular endothelium and platelets that mediates leukocyte rolling and attachment. Because P-selectin is critically involved in inflammation, we used phage display libraries to identify P-selectin-specific peptides that might interfere with its proinflammatory function. Isolated phage contained a highly conserved amino acid motif. Synthetic peptides showed calcium-dependent binding to P-selectin, with high selectivity over E-selectin and L-selectin. The peptides completely antagonized adhesion of monocyte-derived HL60 cells to P-selectin and increased their rolling velocities in flow chamber experiments. Peptide truncation and alanine-scanning studies indicated that an EWVDV (single-letter amino acid codes) consensus motif sufficed for effective inhibition. Intriguingly, the apparent avidity of the peptides was increased 200-fold when presented in a tetrameric form (2 microM versus 10 nM), which is consistent with the proposed divalent interaction of P-selectin glycoprotein ligand 1 (PSGL-1) with P-selectin. As the EWVDV peptides inhibit the binding of an established glycoside ligand for P-selectin (sulfated Lewis A), it is conceivable that EWVDV interacts with or in close proximity to the actual carbohydrate recognition domain of P-selectin, without being a direct structural mimic of sialyl Lewis(x). These ligands are among the most potent antagonists of P-selectin yet designed. Their high affinity, selectivity, and accessible synthesis provide a promising entry to the development of new anti-inflammatory therapeutics and might be a powerful tool to provide important information on the binding site of P-selectin.

Targeting of peptides to restenotic vascular smooth muscle cells using phage display in vitro and in vivo.

Restenosis after angioplasty occurs in 30-40% of the treated patients. To develop a strategy to deliver drugs to restenotic lesions, we selected phages that bind to proliferating vascular smooth muscle cells (VSMC), from a random constraint 15-mer peptide phage display library. Phages were selected for binding to cultured primary aortic VSMC (in vitro biopanning) and selected for binding to denudated carotid arteries in mice (in vivo biopanning). In vitro biopanning did not result in a consensus sequence, but recurring FLGW and LASR amino acid motifs were identified. In vivo biopanning resulted in two consensus peptides 5G6 (CNIWGVVLSWIGVFPEC) and 5E5 (CESLWGGLMWTIGLSDC). Surprisingly, these two sequences were recovered after both in vitro and in vivo biopanning, but predominantly in vivo. Moreover, a strong recurring motif, IGR, was identified in the in vivo clones. The consensus phages 5G6 and 5E5 bind selectively to VSMC compared to other cell types. Furthermore, they bind preferentially to proliferating VSMC compared to VSMC that were growth arrested, and are effectively internalized by their target cells. The specific binding capacities of 5G6 and 5E5 phages suggest that these peptide sequences can be used for targeting of restenotic lesions, in which proliferating VSMC are the dominant cell type.

The effect of TGF-beta receptor binding peptides on smooth muscle cells.

TGF-beta1 is a potent regulator of vascular smooth muscle cell (VSMC) proliferation, migration, and extracellular matrix (ECM) synthesis. In this study, we selected two peptides, IM-1 and IM-2, that bind to the TGF-beta type II receptor (TGF-beta RII) using phage display. IM-1 and IM-2 bind to the TGF-beta RII, with a K(d) of 1 microM. Like TGF-beta, IM-1 induced VSMC chemotaxis and PAI-1 mRNA expression, as determined using Boyden chambers and real time quantitative PCR. In contrast, IM-2 had no effect on VSMC chemotaxis or PAI-1 induction. Induction of ECM synthesis, involving proteins such as osteopontin and alpha-smooth muscle actin, was determined by ELISA. Osteopontin expression was inhibited by both peptides, but TGF-beta-induced alpha-smooth muscle actin expression could only be inhibited by IM-1. In conclusion, IM-1 activity on VSMC is agonistic with TGF-beta, except for ECM synthesis, whereas the IM-2 peptide is antagonistic for some examined TGF-beta functions.

Uptake and processing of modified bacteriophage M13 in mice: implications for phage display.

Internalization and degradation of filamentous bacteriophage M13 by a specific target cell may have major consequences for the recovery of phage in in vivo biopanning of phage libraries. Therefore, we investigated the pharmacokinetics and processing of native and receptor-targeted phage in mice. (35)S-radiolabeled M13 was chemically modified by conjugation of either galactose (lacM13) or succinic acid groups (sucM13) to the coat protein of the phage to stimulate uptake by galactose recognizing hepatic receptors and scavenger receptors, respectively. Receptor-mediated endocytosis of modified phage reduced the plasma half-life of native M13 (t(1/2) = 4.5 h) to 18 min for lactosylated and 1.5 min for succinylated bacterophage. Internalization of sucM13 was complete within 30 min after injection and resulted in up to 5000-fold reduction of bioactive phage within 90 min. In conclusion, these data provide information on the in vivo behavior of wild-type and receptor-targeted M13, which has important implications for future in vivo phage display experiments and for the potential use of M13 as a viral gene delivery vehicle.