Peptides that mimic glycosaminoglycans: high-affinity ligands for a hyaluronan binding domain.

BACKGROUND: Hyaluronan (HA) is a non-sulfated glycosaminoglycan (GAG) that promotes motility, adhesion, and proliferation in mammalian cells, as mediated by cell-surface HA receptors. We sought to identify non-carbohydrate ligands that would bind to and activate cell-surface HA receptors. Such analogs could have important therapeutic uses in the treatment of cancer, wound healing, and arthritis, since such ligands would be resistant to degradation by hyaluronidase (HAse). RESULTS: Peptide ligands that bind specifically to the recombinant HA binding domain (BD) of the receptor for hyaluronan-mediated motility (RHAMM) were obtained by screening two peptide libraries: (i) random 8-mers and (ii) biased 8-mers with alternating acidic side chains, i.e. XZXZXZXZ (X=all-L-amino acids except Cys, Lys, or Arg; Z=D-Asp, L-Asp, D-Glu, or L-Glu). Selectivity of the peptide ligands for the HABD was established by (i) detection of binding of biotin- or fluorescein-labeled peptides to immobilized proteins and (ii) fluorescence polarization of FITC-labeled peptides with the HABD in solution. HA competitively displaced binding of peptides to the HABD, while other GAGs were less effective competitors. The stereochemistry of four biased octapeptides was established by synthesis of the 16 stereoisomers of each peptide. Binding assays demonstrated a strong preference for alternating D and L configurations for the acidic residues, consistent with the calculated orientation of glucuronic acid moieties of HA. CONCLUSIONS: Two classes of HAse-resistant peptide mimetics of HA were identified with high affinity, HA-compatible binding to the RHAMM HABD. This demonstrated that non-HA ligands specific to a given HA binding protein could be engineered, permitting receptor-specific targeting.

A hyaluronic acid-taxol antitumor bioconjugate targeted to cancer cells.

A cell-targeted polymeric prodrug prepared from Taxol and chemically modified hyaluronic acid (HA) was evaluated in vitro. Herein we report four results in support of the selective uptake and targeted toxicity of the HA-Taxol prodrug. First, a fluorescently labeled HA-Taxol (FITC-HA-Taxol) was synthesized and used to demonstrate cell-specific binding and uptake using flow cytometry and confocal microscopy. Second, the selective cytotoxicity of FITC-HA-Taxol allowed direct correlation of uptake with selective cytotoxicity. Third, the rapid uptake and selective cytotoxicity of HA-Taxol bioconjugates could be blocked by either excess HA or by an anti-CD44 antibody, but not by chondroitin sulfate (CS). Finally, the release of free Taxol from HA-Taxol in human plasma or in cell culture media revealed that the free drug was hydrolytically released from the bioconjugate by cleavage of the labile 2' ester linkage. Taken together, these data support the notion that the targeted cytotoxicity of HA-Taxol bioconjugates requires receptor-mediated cellular uptake of the bioconjugate followed by hydrolytic release of free Taxol.

Control of enzymatic degradation of hyaluronan by divalent cations.

Enzymatic degradation of hyaluronan (HA) by testicular hyaluronidase (HAase, hyaluronate 4-glucanohydrolase) requires inclusion of mono- or divalent cations in the reaction mixture. Most divalent cations activated HAase with equal potency; however, Cu2+ suppressed degradation, and Ca2+ showed a concentration-dependent regulation of size of the oligosaccharide products. Careful selection of HAase assay parameters is critical for discovery of novel HAase inhibitors and for preparation of controlled-size oligosaccharide fragments.

Controlled chemical modification of hyaluronic acid: synthesis, applications, and biodegradation of hydrazide derivatives.

Controlled modification of the carboxylic acid moieties of hyaluronic acid with mono- and polyfunctional hydrazides leads to biochemical probes, biopolymers with altered physical and chemical properties, tethered drugs for controlled release, and crosslinked hydrogels as biocompatible scaffoldings for tissue engineering. Methods for polyhydrazide synthesis, for prodrug preparation, for hydrogel crosslinking, and for monitoring biodegradation are described.