WO2000045831A1 - Activites de modulation des peptides de l'heparine, et d'autres glycosaminoglycanes ou proteoglycanes - Google Patents

Activites de modulation des peptides de l'heparine, et d'autres glycosaminoglycanes ou proteoglycanes Download PDF

Info

Publication number
WO2000045831A1
WO2000045831A1 PCT/US2000/002853 US0002853W WO0045831A1 WO 2000045831 A1 WO2000045831 A1 WO 2000045831A1 US 0002853 W US0002853 W US 0002853W WO 0045831 A1 WO0045831 A1 WO 0045831A1
Authority
WO
WIPO (PCT)
Prior art keywords
mammal
peptide
heparin
effective amount
administered
Prior art date
Application number
PCT/US2000/002853
Other languages
English (en)
Inventor
James D. San Antonio
Angela Verrecchio
Barbara P. Schick
Original Assignee
Thomas Jefferson University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Thomas Jefferson University filed Critical Thomas Jefferson University
Priority to CA002361612A priority Critical patent/CA2361612A1/fr
Priority to EP00905964A priority patent/EP1156816A4/fr
Publication of WO2000045831A1 publication Critical patent/WO2000045831A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention generally relates to the field of peptide chemistry and to compositions of matter comprising peptides of various sequences and sizes and to methods of using said peptides with a strong affinity for glycosaminoglycans and proteoglycans, and more particularly to the various methods of using said peptides of various sequences and sizes as described below, wherein said peptides interact strongly with heparin, other glycosaminoglycans, or proteoglycans (PGs).
  • PGs proteoglycans
  • Glycosaminoglycans modulate enzyme activities (e.g., of antithrombin III or heparin cofactor II), regulate cell behaviors (e.g., cell adhesion, growth, and differentiation), and control the function of extracellular matrices (e.g. , diffusion of ions through basement membranes, and fibrillogenesis and lateral associations of collagens), largely through non-covalent interactions with proteins.
  • enzyme activities e.g., of antithrombin III or heparin cofactor II
  • cell behaviors e.g., cell adhesion, growth, and differentiation
  • extracellular matrices e.g. , diffusion of ions through basement membranes, and fibrillogenesis and lateral associations of collagens
  • Heparin-binding consensus sequences were discovered by Cardin and Weintraub, who surveyed amino acid sequences of known heparin-binding proteins, where they identified two potential consensus sequence motifs for heparin-binding, X- B-B-X-B-X or X-B-B-B-X-X-B-X, where X represents a hydropathic or uncharged amino acid, and B a basic amino acid. (Cardin, A. D. and H. J. R. Weintraub, Arteriosclerosis 9:21-32, 1989). For example, such consensus sequences were identified in proteins including apolipoprotein B-100, apo E, and vitronectin, to name a few.
  • Hynes, Cell Biol 103: 1635- 1648, 1986) or site-directed mutagenesis of a heparin-binding sequence in fibronectin (FN) (Barkalow, F. J. B. and J. E. Schwarzbauer, J Biol Chem 266:7812-7818, 1991) eliminates or diminishes heparin-binding affinity.
  • FN fibronectin
  • peptide mimetics of proposed heparin binding consensus sequences often fail to reveal the high affinities demonstrated by the native heparin-binding proteins. (Conrad, H. E, Hepa ⁇ n-Binding Proteins. Academic Press, 1998).
  • Proteins often contain multiple, distal heparin- binding sequences that may come into proximity upon protein folding or multimerization, hence enabling binding through cooperativity. It has thus been speculated that the three dimensional arrangement of multiple heparin-binding consensus sites within or between heparin-binding proteins, and/or the presence of novel heparin-binding sites may be responsible for high affinity heparin- or HS- interactions with native proteins. Others have proposed a necessary approximately 20 A distance between basic amino acids for heparin binding, regardless of protein tertiary structure. (Margalit, H., et al. , J Biol Chem 268: 19228-19231, 1993).
  • the heparin-binding domain of von Willebrand factor resembles the motif XBBXXBBBXXBBX, a palindromic sequence in which the spacing and clustering of basic residues is important for heparin binding.
  • a third novel sequence has been demonstrated to be sufficient for weak heparin-binding in thrombospondin: WSXW. (Guo, N. H., et al., J Biol Chem, 267: 19349-19355, 1992).
  • this sequence must be flanked by basic residues.
  • Other proteins including type I collagen (Sweeney, S. M.
  • the two heparin-binding consensus sequences identified in the FGFs were shown not to mediate heparin-binding (Wong, P., et al., J Biol Chem, 270:25805-25811, 1995; Thompson, L. D., et al. , Biochem, 33:3831-3840, 1994). Therefore, there are likely other as yet undefined protein characteristics that must confer heparin-binding potential. Of relevance is the recent use of phage display technology to identify such novel heparin-binding sequences. This approach has generated three distinct HSPG-binding antibodies (van Kuppevelt, T. H. , et al., J Biol Chem, 273 21:12960-12966, 1998).
  • GRRLKD heparin-binding consensus sequence
  • SRMNGCGAHQ and YYHYKVN heparin-binding consensus sequence
  • All three anti-HS antibodies showed specificity for heparin and HS but not for other GAGs. Additionally, the antibodies all reacted differently towards HS from various sources, which would suggest a specificity in recognition of discrete HS molecules.
  • GAG structure may also play a role in determining binding affinity and selectivity for proteins.
  • a classic example is the antithrombin-binding site on heparin, which is present on only about one third of heparin chains (Lam, L. H. , et al., Biochem Biophys Res Commun, 69:570-577, 1976), but which has a thousand-fold greater affinity for antithrombin III than the overall heparin structure (Lee, M. K., and A. D. Lander, Proc. Nat Acad Sci USA, 88:2768-2772, 1991).
  • HS GAGs Several other sequences or structural motifs have been identified in HS GAGs which underlie their binding interactions with basic fibroblast growth factor (bFGF) (Maccarana, M., et al., J Biol Chem, 268:23898-23905, 1993), lipoprotein lipase (Parthasarathy, N., et al., J Biol Chem, 269:22391-22396, 1994), and interleukin-8 (Lindahl, U., et al. , J Biol Chem, 273:24979-24982, 1998).
  • bFGF basic fibroblast growth factor
  • heparin displays high affinities for sequences with contiguous clusters of basic amino acids
  • HS displays high affinities for those sequences in which clusters of basic amino acids are separated by non-basic residues
  • binding preferences may relate to the increased spacing between sulfates found throughout HS as compared with the more densely sulfated heparin.
  • Heparin is capable of binding to a wide array of proteins, due to its high degree of flexibility and ability to "fit" itself into proteins.
  • heparin-binding sequences Because of the presence of heparin-binding sequences in many physiologically important proteins, there was a need for small peptides with high affinities for heparin or for heparin-like molecules (i.e. , PGs, or other GAGs), to use in a variety of applications to modulate the activities of native GAGs and PGs. Therefore, in the present invention peptides with high affinities for heparin or for PGs have been designed to include heparin-binding consensus sequences; however, in doing so it was necessary to take into account previous studies showing that short peptides of native proteins do not behave like the native proteins, due to conformation and size limitations, and a lack of cooperativity in binding to various ligands.
  • the basis for the design of the peptides of the present invention is the inclusion in their structure of multiple copies of sequences (including XBBXBX or XBBBXXBX, where X is a hydropathic amino acid and B is a basic amino acid), representing consensus sequences for heparin or PG-binding in natural proteins, and, in addition, may include the presence of a single cysteine residue preferably occupying, but not limited to, a position within a three residue distance of either the C- or N-peptide terminus, that promotes peptide dimer formation and greatly enhances peptide binding interactions with heparin.
  • Any of these peptides may also be constructed of either L- or D-, or combinations of L- and D-amino acid isomer forms, or containing any amino acid in the X position of any peptide. Any of these peptides may also be used as carriers and/or integral components of various pharmaceuticals or bioactive agents targeted to interact with cell surfaces expressing PGs or heparin-like molecules, or to tissues which express PGs as cell surface or extracellular matrix components.
  • Current approaches to design peptides which bind to heparin include Wakefield et al. (U.S. Patent No. 5,534,619, and U.S. Patent No. 5,919,761) and Harris et al. (U.S. Patent No. 5,877,153).
  • the Wakefield peptide sequences are patterned after naturally-occurring protamines.
  • the Harris et al. peptides are a series of single-chain and multi-chain peptides which incorporate arginines within a backbone of alanines.
  • the spacings of the arginines are based on the heparin-binding sequence of antithrombin III. All the Harris peptides have AE as their N-terminal amino acid sequence.
  • the present invention includes peptides based on the consensus sequences (XBBXBX) and (XBBBXXBX) determined by the analysis of a wide range of known heparin-binding proteins by Cardin et al (Cardin, A. D. , and H. J. R. Weintraub, Arteriosclerosis, 9:21-32, 1989).
  • the peptides designated in this application consist of as many as 6 repeating units of these sequences. These sequences are not found in protamine. In contrast to Wakefield et al.
  • the peptides of the present invention contain repeating motifs with groups of two and 1 basic residues separated by a single alanine, or three and one basic residues separated by alanine-alanine. While single copies of these general sequences are associated with the heparin binding sites in many proteins, peptides derived from these proteins which include single copies of these sequences and their native surrounding amino acids have insignificant binding affinities for heparin. Furthermore, some proteins contain the Cardin type consensus sequences, but these sequences were shown not to bind heparin, and many other proteins bind heparin yet do not contain such consensus sequences. Thus it is not intuitive to use these types of sequences as heparin-binding agents.
  • the sequences used by Harris et al. mimic those found in a naturally occurring protein in terms of spacing and grouping of the basic residues, with no internal repeating structures, but the single-chain peptides have relatively weak ability to interact with heparin. Substantial binding is found only when multi-chain structures are formed.
  • the present invention involves, for the most part, single-chain peptides with repeating Cardin sequences. These peptides have a strong capability for binding to both unfractionated heparin and low molecular weight heparin.
  • a further difference between the peptides in the Wakefield and Harris patents resides in the engineering of alpha-helical structure into the peptides. Some of their peptides have partial alpha-helical structure. In the present invention, peptides are not alpha-helical in the native state, but assume an alpha-helical conformation when bound to heparin. Thus, the peptides of the present invention may have more flexibility to conform to a variety of heparin sequences encountered in any of the therapeutic heparin formulations.
  • An additional aspect of the present invention is the N-terminal -peptide sequences of the proteoglycan serglycin, which contain a single full or partial Cardin site near the N-terminus and a cysteine residue three amino acids from the C-terminus. These peptides dimerize through their cysteine residues and thus form a strong heparin- binding unit.
  • Another feature of the present invention is the inclusion of cysteines near the C-termini of all the Cardin site peptides and the serglycin peptides to further enhance their heparin-binding functions.
  • the peptides of the present invention have a number of uses. One method of using these peptides is to promote cell attachment or adhesion to natural or synthetic surfaces.
  • Vascular diseases such as atherosclerosis, restenosis, and aortic aneurysms often result in permanent damage to blood vessels; typically, vessels become occluded as a result of vascular insult, causing decreased blood flow (Robbins, S. L. and R. S. Cotran, Pathological Basis of Disease. W.B. Saunders, Philadelphia. 598-613 pp. , 1979).
  • One approach to treatment of damaged vessels is surgical replacement of the diseased segment with an autologous or non-autologous native tissue graft (Zarge, J. I., et al., In Principles of Tissue Engineering, R. P. Lanza, et al., editors. Academic Press, Austin,. 349-364, 1997).
  • inert polymers composed of terephthalate (Dacron) or of expanded polytetrafluoroethylene (ePTFE) have been used to construct prosthetic vascular grafts (Zarge, J. I. , H. P., and H. P. Greisler, In Principles of Tissue Engineering, R. P. Lanza, et al., editors. Academic Press, Austin, 349-364, 1997), but these materials typically invoke an immune response. Synthetic grafts can react with serum proteins and blood cells that can promote thrombus formation and lead to pseudointimal hyperplasia. (Zarge, J. I. , H. P. , and H. P. Greisler, In Principles of Tissue Engineering, R. P.
  • Vascular replacement has been limited to large or medium size arteries where blood flow rates are high, outflow resistance is low, and as a consequence, the graft is less likely to become occluded by a thrombus. Conversely, small arteries are more prone to graft failure via thrombosis or hyperplasia because of lower flow rates and higher outflow resistance. An inappropriate infiltration of smooth muscle cells during the healing process can also result in vessel occlusion. Control of this immune response and smooth muscle cell infiltration could occur in a vessel lined with endothelial cells, which secrete factors inhibiting platelet and erythrocyte aggregation (Fantone, J. C, and P.
  • Endothelial cells carry a negative surface charge (Vargas, F. F., et al., Membrane Biochemistry. 9:83, 1990) that can inhibit platelet adherence, and they express a variety of GAGs on their surface that bind the anti-coagulant anti-thrombin III (Mertens, G. , et al., J Biol Chem, 267 (28):20435-20443, 1992).
  • Vargas and co- workers have shown that sulfated GAGs are the main carriers of surface charge on vascular endothelial cells, primarily as heparan sulfate (HS) and chondroitin sulfate PGs (Vargas, F. F. , et al.
  • PGs on endothelial cell surfaces include the syndecans and glypican (Mertens, G., et al., J Biol Chem, 267 (28):20435-20443, 1992).
  • the surface chemistry i.e., the predominance of its PG component
  • One goal of the present invention is to discover peptides with high affinities for endothelial cell surface PGs. Such peptides are used by covalently attaching them to synthetic vascular grafts, and in the presence of endothelial cells, promote their attachment to the graft surface, thereby increasing the probability of graft success.
  • peptides of the present invention are for heparin-and PG- binding as modulators of hemostasis via interactions with endothelial cells and as anti- heparin therapy in plasma.
  • These peptides of the present invention function as agents for neutralization of unfractionated heparin, low molecular weight heparin, or Orgaran (Organon, mixture of chondroitin sulfate/heparan sulfate/dermatan sulfate) overdose.
  • Protamine heparin antidote
  • Protamine can cause several serious side effects in patients, and although Protamine is effective in humans against unfractionated heparin, it is not effective against low molecular weight heparins or against Orgaran. Since Protamine is a natural product that is an undefined mixture of amino acids, its content is variable across different preparations, and thus dosage is uncertain, presenting problems in its clinical use.
  • the peptides of the present invention are useful for counteracting the actions of heparin and other anticoagulant glycosaminoglycans on thrombin and Factor Xa activity, and may affect other proteins as well.
  • Heparin is used routinely for anticoagulation.
  • the interactions of exogenously administered heparin with the proteins of the coagulation and fibrinolytic pathways have been summarized in detail (van Kuppevelt, T. H., et al., J Biol Chem, 273 (21): 12960-12966, 1998). These interactions are complex on many levels.
  • the best-characterized targets for heparin are the procoagulant proteins thrombin and Factor Xa, which are inhibited by AT III when heparin binds to AT III.
  • heparin acts at many sites. In some cases, the effect of heparin is anticoagulant and in other cases procoagulant. Some proteins, e.g. AT III, have heparin-binding consensus sites. However, the putative heparin-binding sequences are different for every known protein in these pathways, and the effects may depend on the 3-dimensional relationships of basic residues resulting from protein folding, rather than a short linear sequence, as is known for the binding of heparin to AT III (Lam, L. H., et al., Biochem Biophys Res Commun, 69:570-577, 1976). A tetrameric protein conformation of platelet factor 4 (PF4) is required for long-chain heparin binding (Lee, M.
  • PF4 platelet factor 4
  • Heparin is a complex mixture of polysaccharides. Some of the interactions require long-chain heparins (AT III for inactivation of thrombin and binding to thrombin, HC II, PF4, and thrombospondin) while others depend on or can function with low molecular weight heparin chains(AT III for inhibition of Factor Xa, vitronectin, TFPI) (van Kuppevelt, T. H., et al., J Biol Chem, 273 (21): 12960-12966, 1998). To further complicate the situation, specific sequences within the heparin chains may be required for interactions with the different proteins (van Kuppevelt, T.
  • heparin it is often necessary to reverse the effects of heparin when anticoagulation has reached a stage at which hemorrhage becomes a threat, notably after the routine use of heparin for anticoagulation during cardiopulmonary bypass, and in patients who develop an endogenous heparin-like coagulation inhibitor.
  • the most commonly used anti-heparin drug is protamine, a mixture of basic proteins from fish sperm nuclei, that contains a high concentration of the amino acid arginine. When injected into a person who has been treated with heparin, it complexes rapidly to the heparin, thereby neutralizing its activity. Protamine also has numerous side effects including pulmonary hypotension that are difficult to control and provide significant health risks to the patient.
  • Protamine is a poorly-defined and potentially variable product, dosage determination can be problematic. Importantly, Protamine has been shown to be ineffective for neutralization of low molecular weight heparins and the non-heparin glycosaminoglycan anticoagulant Orgaran. Well-defined heparin-or other GAG-binding peptides could be of considerable utility for reversing overdose of these specific anticoagulant preparations. Carson and co-workers (Munro, M. S., et al.
  • peptides are effective neutralizers of low molecular weight heparin (Enoxaparin, Lovenox) and Orgaran in vitro, and of Lovenox in vivo in rats, in accordance with their affinity constants for low molecular weight heparin in vitro.
  • the peptides described in this application may have important clinical applications, especially if they can be targeted to specific reactions in the relevant pathway and to specific classes of heparins.
  • Another use for the peptides of the present invention is to block the uptake and clearance of heparin by blocking uptake receptors on tissue, without binding to the circulating heparin itself, and thus prolonging the half-life in the circulation.
  • Such an agent would reduce the frequency of administration of the drug, as well as the amount needed. This could be especially useful for home-based therapy with low molecular weight heparin, which is administered by subcutaneous injection and is becoming the standard for post-hospitalization anticoagulation.
  • HSPGs heparan sulfate PGs
  • AT III Antithrombin III
  • TFPI tissue factor pathway inhibitor
  • TFPI binds to Factor Xa and this complex then interacts with the Factor Vila/tissue factor complex to inactivate both Factors Vila and Xa.
  • Adherence of TFPI to the endothelium via the HSPG protects against proteolysis of the heparin-binding C-terminal domain (Thompson, L. D. , et al. , Biochem, 33:3831-3840, 1994); without this domain, activity is lost.
  • Heparin-binding peptides such as those described in this study could behave similarly to platelet factor 4 (PF4) in that they could bind to the heparan sulfates on the endothelial surface.
  • a peptide onto the heparan sulfate chain in a reversible manner could protect the GAG from degradation by platelet heparitinase released by aggregating platelets at the site of a developing thrombus, leaving the GAG able to resume its antithrombotic function in a shorter time frame than would be required for resynthesis.
  • a peptide with a very high affinity for the AT Ill-binding sequences of endothelial heparan sulfate could block the binding and therefore the activity of AT III and provide a more favorable surface for clot formation, thus promoting wound healing.
  • the peptides of the present invention have affinity for heparin/heparan sulfate on cell surfaces and can be used as agents to promote healing, either by injection or by topical application. Injection or topical application of the peptides alone also might serve to assist wound-healing by dislodging ATIII and/or tissue factor pathway inhibitor (TFPI) from their binding sites and subsequently blocking these binding sites on the endothelium of broken blood vessels, thereby reducing the anticoagulant activity of the surface and enabling a clot to form.
  • TFPI tissue factor pathway inhibitor
  • contemporaneous injection or application of a mixture of heparin and a heparin-binding peptide could generate a molecular complex, or low affinity heparin sink, that will then transfer the heparin to proteins with greater heparin-binding affinities.
  • the peptides of the present invention can be used to bind and neutralize or activate, or otherwise modulate the actions of various PGs or GAGs, thereby influencing their growth- or differentiation-modulating activities.
  • heparin and heparin-like molecules such as cell surface HSPGs are known to inhibit smooth muscle cell proliferation, to potentiate the activities of growth factors like basic or acidic fibroblast growth factor on endothelial cells, and to inhibit or promote cell differentiation of smooth muscle cells, chondrocytes, and other cell types.
  • the peptides described here could be used to modulate the actions of heparin or endogenous heparan sulfate PGs, with significant consequences to cell growth and differentiation.
  • GAGs exhibit a wide variety of potent activities on cell growth, migration, differentiation, metabolism, and adhesion (Jackson, R. L., et al., Physiol Rev, 71:481- 539, 1991; San Antonio, J. D., and R. V. Iozzo, Encylc Life Sci, In Press, 1999).
  • One of the earliest reports of an effect of GAGs on cell growth reported that fibroblastic mouse L cells in suspension culture exposed to 50 ⁇ g/ml heparin were growth inhibited by seventy-three percent (Karnovsky, M. J., et al., Annals of the New York Academy of Science, 556:268-281, 1989).
  • vascular smooth muscle cells Several strong antiproliferative activities of GAGs on a variety of cell types have been reported since (San Antonio, et al., Connective Tissue Res, 37:87-103, 1998).
  • heparin antiproliferative action may involve displacement of HS or heparin-binding growth factors from cell surface receptors
  • VSMC heparin may also be internalized and act directly in the cytoplasm and nucleus (Karnovsky, M. J. , et al., Annals of the New York Academy of Science, 556:268-281, 1989).
  • vascular smooth muscle cells An important component of vascular diseases including atherosclerosis and restenosis is the pathological growth of vascular smooth muscle cells.
  • GAGs are strong regulators of VSMC growth they are potentially useful in treating these diseases.
  • the effect of heparin on VSMC growth in vivo was first discovered in experiments aimed at determining whether heparin may inhibit the response to injury cascade of accelerated atherosclerosis owing to its antithrombotic activity; a dramatic inhibition of VSMC proliferation by heparin was observed (Karnovsky, M. J., et al., Annals of the New York Academy of Science, 556:268-281, 1989).
  • the peptides described here are useful in neutralizing the antiproliferative activities of endogenous or exogenous heparins or heparan sulfates on vascular smooth muscle cells or other cell types.
  • the peptides may be used to neutralize endothelial cell-derived HSPG's during vascular wound healing.
  • peptides described here may prove useful as modulators of cartilage differentiation, especially in instances where cartilage tissue scaffolds are being constructed for autologous tissue transplants, e.g., for use in orthopedic surgical applications.
  • Tumor matrix stromas may play important roles in potentiating tumor growth and metastasis (Iozzo, R. V., Lab Invest, 73: 157-160, 1995). For example, increases in perlecan expression are seen during development of colon carcinomas and of malignant melanomas; its HS chains may potentiate growth factor activity and induce angiogenesis surrounding the tumor, thereby enhancing its growth (Nugent, M. , and R. V. Iozzo, Internat J Biochem. and Cell Biol, In Press, 1999).
  • the binding selectivity of HS chains for various members of the fibroblast growth factor family can be influenced by fine structural features such as the patterns of 6-O-sulfation and the abundance of sulfated domains (Lindahl, U., et al., J Biol Chem, 273:24979-24982, 1998).
  • a pathological role of tumor cell surface PGs has also been suggested. For example, Chinese hamster ovary cells carrying various mutations of PG synthesis were injected into nude mice and tested for their tumorigenic abilities.
  • PGs secreted by normal cells are proposed to play a key barrier function by inhibiting the migration of tumor cells across basement membranes.
  • tumor cells have been shown to secrete the enzyme heparatinase, which degrades the HS chains within basement membranes, thereby potentially enabling such malignant cells to breach the basement membrane, enter the circulation, and spread throughout the body (Katz, B. Z., et al. , Invasion and Metastasis, 14:276-289, 1994-5).
  • the peptides described here could thus be used as inhibitors of GAG hydrolase-mediated tumor metastasis.
  • Tumor angiogenesis is strongly inhibited by the heparin- binding protein endostatin (O'Reilly, M. S. , et al. , Cell. 88 (7):277-285, 1997), and in vitro, heparin is required to promote angiogenesis in response to growth factors (Jackson, C. J., et al., Exp Cell Res, 215:294-302, 1994).
  • the peptides described here could therefore function as inhibitors of growth-factor dependent angiogenesis in vivo, therefore inhibiting tumor growth.
  • Yet another application of the present invention is the targeting of drugs to cell surfaces of endothelium or other cell types which express PGs.
  • drugs to be targeted to endothelial cells could be complexed with the peptides described here, or the peptide sequences could be integrated into the drug, and then the drug could be administered to the systemic circulation.
  • the peptide component of the drug would mediate high affinity interactions with the endothelial cell surface, effectively delivering the drug for action at that site, or potentially promoting the cellular uptake of the drug.
  • endothelial cell surface charge is largely due to cell surface GAGs and PGs (Vargas, F. F. , et al., Membrane Biochemistry, 9:8, 1990), and the peptides described in this patent exhibit high affinity interactions with endothelial cell PGs, then the peptides can be applied as tools to deliver drugs to endothelial cells in vivo.
  • a drug which is designed to act on endothelial cells could be complexed with the peptides either covalently or non-covalently, and delivered to the systemic circulation.
  • the peptide component of the complex would facilitate high affinity interactions with the endothelial cell surface, thereby bringing the drug in contact with the endothelial cell surface to exert its activity there, or to facilitate its uptake by the endothelial cells.
  • Such a use for these peptides is not limited to endothelial cells, since many cell types in the body express distinct classes or types of PGs.
  • structural variants of GAGs and PGs may be expressed, thereby distinguishing these cell variants on a structural and functional level.
  • GAG hydrolases including some of the heparinases and heparatinases contain heparin-binding consensus sequences which they are proposed to use in binding to their GAG substrates.
  • the peptides described here could be used to inhibit this binding through competition, thereby inhibiting the activity of the enzymes.
  • heparin and heparan sulfates are used commonly in scientific investigations to characterize the structure and function of GAGs within tissue and cell preparations. Furthermore, these enzymes are important natural products as they are secreted by specific types of bacteria, are present in the venom of some poisonous snakes, and are secreted by normal human cells, and by human tumor cells, where they are proposed to promote tumor cell metastasis (Katz, B. Z., et al. , Invasion and Metastasis , 14:276-289, 1994-5; Sasisekharan, R. , et al., Proc Natl Acad Sci USA, 90:3660-3664, 1993). Since these enzymes contain heparin-binding consensus sequences that are proposed to mediate the interactions between the enzyme and their substrates, the peptides described here will serve as effective inhibitors of enzyme action for many in vitro and in vivo applications.
  • heparin-binding proteins have been shown to interact with specific sequences or domain structural features on heparins or heparan sulfates, including antithrombin III, lipoprotein lipase, and laminin.
  • the peptides described here may similarly exhibit binding preferences for distinct sequences in GAGs, making them useful as affinity matrices for the purification of specific GAG sequences for a variety of uses.
  • Heparin-binding proteins have been shown to interact with specific sequences or domain structural features on heparins or heparan sulfates, including ATIII (Lam, L. H.
  • the determinant on heparin necessary for AT-III binding was located on only about one third of heparin chains, and is a pentasaccharide sequence composed of a 6-O-sulfated glucosamine in the first position, a 3-O-sulfated central glucosamine, two N-sulfated glucosamines, and a carboxylated iduronic acid (Jackson, R.
  • Modulating means binding, neutralizing, activating, or modulating.
  • the present invention generally relates to peptides of various sequences and sizes with a strong affinity for glycosaminoglycans and proteoglycans.
  • the present invention also comprises the methods of using said peptides of various sequences and sizes, wherein said peptides interact strongly with heparin, other glycosaminoglycans, or proteoglycans (PGs).
  • PGs proteoglycans
  • Peptides of the present invention can be used to:
  • peptides may include endothelialization of synthetic vein graft surfaces, which is known to increase the chances for the long term success of the vein graft.
  • peptides can be covalently linked to synthetic or natural polymers used to construct vascular graft scaffolds, where they will interact strongly with endothelial cell surface PGs, thereby promoting endothelial cell attachment and thus graft colonization and success.
  • Peptides could also be linked to synthetic tissue culture surfaces, to promote rapid and strong attachment of cells expressing PGs.
  • heparin and PG Bind heparin and PG to modulate hemostasis via interactions with endothelial cells and as anti-heparin therapy in plasma.
  • These peptides function as agents for neutralization of unfractionated heparin, low molecular weight heparin, or Orgaran (Organon, mixture of chondroitin sulfate/heparan sulfate/dermatan sulfate) overdose.
  • Agents to promote healing by binding heparin/heparan sulfate on cell surfaces.
  • the peptides could be administered either by injection or by topical application.
  • contemporaneous injection or application of a mixture of heparin and a heparin-binding peptide could generate a molecular complex, or low affinity heparin sink, that will then transfer the heparin to proteins with greater heparin- binding affinities.
  • heparin and heparin-like molecules such as cell surface HSPGs are known to inhibit smooth muscle cell proliferation, to potentiate the activities of growth factors like basic or acidic fibroblast growth factor on endothelial cells, and to inhibit or promote cell differentiation of smooth muscle cells, chondrocytes, and other cell types.
  • the peptides described here could be used to inhibit the actions of heparin or endogenous heparan sulfate PGs, with significant consequences to cell growth and differentiation.
  • FIG. 1 Calculation of heparin-binding affinities of peptides containing heparin- binding consensus sequences.
  • Peptides containing single consensus sequences do not bind heparin with a measurable affinity; in contrast, significant heparin-binding is seen with peptides containing multiple heparin-binding consensus sequences and increases as a function of peptide Mr.
  • FIG. 2 Calculation of heparin-binding affinities of SG peptides.
  • R values for the migration of low Mr 125 I-tyramine-heparin through peptides containing sequences native to the mouse (YPARRARYQWVRCKP,-0 -) or human (YPTQRARYQWVRCNP, - •-) SG PG core proteins were determined from ACE gel electrophoretograms as detailed in Materials and Methods.
  • SG peptides displayed relatively strong affinities for heparin (KdS 200 and 900 nM for the mouse and human peptides, respectively), in comparison to peptides of similar size which contain multiple repeats of heparin-binding consensus sequences [e.g.
  • AAARRARAAAARAKA (- ⁇ -) displayed negligible heparin-binding affinity (Kd--. 75 ⁇ M) indicating the importance of the non-basic residues to heparin- binding.
  • YPARRARYQWVRCKP-heparin binding in the presence of ⁇ -ME (YPARRARYQWVRCKP+ ⁇ -ME, -X-), was decreased by over 20-fold 4 ⁇ M).
  • Replacement of cysteine by alanine in the mouse SG peptide (YPARRARYQWVRAKP, -D-) further reduced heparin-binding affinity (Kd ⁇ 36 ⁇ M).
  • FIG. 3 CD spectroscopy of (AKKARA)e in the presence or absence of low Mr heparin.
  • heparin addition (1: 0.25, -O-; 1: 0.50, -X- )
  • the peptide conformation is altered and at a 1: 1 peptide-heparin ratio (- ⁇ -) the peptide becomes ⁇ -helical with characteristic ⁇ -helical peaks at approximately 190, 207, and 222 nm.
  • heparin (1: 2, -D-, or 1: 4, -A-) disrupts this interaction, and the spectra resembles that of a protein in a random coil conformation. Spectra are heparin and/or blank (water) corrected.
  • FIG. 4 CD spectroscopy of (AKKARA) 2 in the presence or absence of low Mr heparin.
  • (AKKARA) 2 which binds heparin weakly, remains a charged coil in the presence of heparin (1 : 0.25, -O-; 1 : 0.50, -X-; 1 : 0.75, -"-; 1: 1, -D-).
  • FIG. 5 ACE analysis of the interactions between peptides containing heparin- binding consensus sequences and HUVEC PGs.
  • ACE gel images as obtained by a phosphorimager in which (A) EC PGs/GAGs or (B) heparin was fractionated through peptides.
  • A at least two populations of high affinity PG/GAG, seen as two bands of radiolabeled material migrating with different mobilities, is visible at peptide concentrations of _ ⁇ _ 50 nM.
  • FIG. 6 Affinity of (ARKKAAKA) 4 for HUVEC PGs and PG components.
  • the peptide was analyzed for binding affinity to HUVEC PGs/GAGs by ACE, and the Kd of the peptide-PG/GAG interactions were calculated from binding plots as detailed in Experimental Procedures. Similar affinities ( — 300 nM) were obtained for total PGs, for PG samples devoid of HS GAGs via nitrous acid treatment (NA) or heparatinase I digestion (H), and for PGs devoid of CS GAGs via chondroitinase ABC (ABC) digestion. Liberation of GAG chains from the core protein by borohydride reduction (BH) of total PGs caused a 3-fold reduction in affinity (KdS 1200 nM).
  • BH borohydride reduction
  • FIG. 7 Neutralization of Lovenox by Peptides in Vivo: Anti -Factor Xa Assay.
  • Absorbance @ 405 nm defines the heparin concentration in the plasma as a function of the amount of anti-Factor Xa activity.
  • the amount of Factor Xa activity is determined by the change in A os over 1 minute by chromogenic assay. The low point on each curve represents the highest amount of anti-Factor Xa activity, a function of the highest concentration of heparin obtained in the particular animal.
  • a os of 1.0 represents about 0.5U/ml of anti-Factor Xa activity
  • a o 5 of 0.5 represents about 1.0 U/ml anti- Factor Xa activity, based on standardization against Hepanorm low molecular weight heparin standards for the Stachrom Heparin kit.
  • Administration of the peptide results in formation of a peptide/heparin complex, thus reducing the amount of ATIII-heparin complex and therefore reducing Factor Xa activity, resulting in reduced breakdown of the dye and return to baseline of the A 4 os.
  • Rats were injected with Lovenox alone (Panel A) or with Lovenox followed by peptide three minutes after injection of Lovenox (arrow) (Panels B-J).
  • Peptides were administered at 2 mg/300 gm animal except where noted otherwise. Blood samples (0.1 ml) were obtained for anti-Factor Xa analysis immediately before the injection of Lovenox, at 30-second intervals after the injection until 10 minutes, then at 1 minute intervals until 15 minutes, and at 5- minute intervals until 30 minutes.
  • ARRRAARA ARRKAAKA
  • AKKRAAKA AKKRAAKA
  • ARAARRRA ARAAKRKA
  • GKKKGGRG GKKKGGRG
  • GKKKGGRG GKKKGGRG
  • GRGGKRRG GRGGKKRG
  • ARRKAARA-ARRKACRA ARCAKKRA-ARAAKKRA-ARAAKKRA ARRAKA-ARRAKA-ARRCKA AKCKRA-AKAKRA
  • this patent will also cover inclusion of the D- isomer forms of amino acids in place of the L-forms, or inclusions of any combinations of D- or L-isomer forms to create reagents resistant to proteolytic degradation for in vitro and in vivo applications.
  • This patent will also include peptides which incorporate multiple copies of the heparin-binding consensus sequences, but which are not necessarily arranged as concatamers, e.g., two such peptide may be ARKKAARAAAAAAAAARKKAARA or ARKKAARAAAAAAAAAAAAAAAAARKKAARA Embodiments of this invention
  • the present invention relates to a number of different peptides of various sequences and sizes, including pharmaceuticals or bioactive agents composed of the peptides complexed with or incorporated into delivery vehicles such as salts, solutions, solvents, and/or carriers, and/or covalently linked to other bioactive agents.
  • delivery vehicles such as salts, solutions, solvents, and/or carriers, and/or covalently linked to other bioactive agents.
  • the peptides may be used as pharmaceutical salts of agents including, but not limited to alkali metal salts, organic carboxy lie or sulfonic acids, or inorganic acids, etc.
  • Acceptable carriers for the peptides may include any of a variety of diluents, solvents, time release polymers, fillers, or binders, etc, and formulated into dosage forms such as pills or injectable solutions, etc.
  • This patent also includes any of the peptides derivatized with functional groups and/or linked to other molecules to facilitate their delivery to specific sites of action, to potentiate their activity, or complexed covalently or non-covalently to other pharmaceuticals, bioactive agents, or other molecules. Such derivatizations must be accomplished so as to not significantly interfere with the heparin- or PG-interactive properties of the peptides.
  • Carriers and derivatizations must also be designed or chosen so as not to exert toxic or untoward activities on animals or humans treated with these formulations.
  • Functional groups which may be covalently linked to the peptides may include, but not be limited to, amines, alcohols, or ethers.
  • Functional groups to be covalently linked to the peptides to increase their in vivo half lives may include, but not be limited to, polyethylene glycols, small carbohydrates such as sucrose, or peptides and proteins.
  • the peptides may also be synthesized by recombinant DNA techniques with expression vectors for use in biological systems, such as bacteria, yeast, insect, or mammalian cells. Methods are well known in the art.
  • Radiolabeled Heparin- Whole heparin from pig intestinal mucosa (Sigma) was tyramine end-labeled and radiolabeled with Na 125 I (Amersham, Pharmacia Biotech, Inc. , Piscataway, NJ) to an average specific activity -. l.O x 10 7 CPM/ ⁇ g as described ⁇ .
  • Peptides were then mixed 1: 1 with 2% agarose/ 1 % CHAPS, 3-[(3 -cholamidopropy l)dimethy lammonio] - 1 -propanesulfonate , (Boehringer Mannheim, Indianapolis, IN), and loaded into wells of a 1 % agarose gel. Radiolabeled heparin or HUVEC PGs were then loaded in a slot on the anode side of the gel, and electrophoresed through the peptide-containing wells, towards the cathode.
  • ⁇ -mercaptoethanol ⁇ -ME
  • ⁇ -ME ⁇ -mercaptoethanol
  • Binding analysis of peptides to enzymatically or chemically degraded PGs was carried out by ACE as detailed, except that PG samples included 6 M urea to denature any residual enzymes.
  • Radiolabeling and Isolation of Total HUVEC PGs and GAGs- Exponentially growing, subconfluent HUVEC were labeled with 35 ⁇ Ci/ml 35 S-Na 2 SO (ICN Pharmaceuticals, Costa Mesa, CA) in normal culture media minus heparin for 12h. Culture media and cell layers were harvested separately. After removal of the media, cells were washed with 2.0 ml PBS plus Ca 2+ -Mg 2+ .
  • PG samples were resuspended in 100 ⁇ l enzyme buffer [chondroitinase buffer: 50 mM Tris-HCl, 30 mM sodium acetate, pH 8.0, 0.1 mM pepstatin A, 0.5 mg/ml BSA, 10 mM NEM, 1 mM PMSF, and 5 mM EDTA; heparatinase buffer: 50 mM Tris-HCl, 5 mM calcium acetate, pH 7.0, 0.5 mg/ml BSA, and 1 mM PMSF]. Samples were digested with 0.05 U/ml chondroitinase ABC at 37°C for 3h.
  • Circular dichroism spectroscopy- Circular dichroism (CD) spectra were recorded at 22 °C using a JASCO J-500C spectropolarimeter interfaced to a 486 PC.
  • the path length of the CD cells was 0.5 mm, and the CD was expressed in terms of
  • peptides containing two copies of the consensus sequence exhibited weak but detectable affinities for heparin ( ⁇ 6 ⁇ M), and peptides of higher molecular weight containing 4-6 copies of a consensus sequence showed a marked increase in heparin-binding affinity (40-150 nM) (Fig. 1).
  • the heparin-binding affinity of both the 6-mer and 8-mer tandem-repeat peptides reached a plateau as peptide length approached 30 amino acids [(AKKARA)5,
  • tandem-repeat peptides which confer their high affinity heparin-binding characteristics
  • peptides containing variants of one of the consensus sequences first tested, (ARKKAAKA) 3 were synthesized. These included those in which alanines were replaced by other hydropathic residues, the spacings between consensus sequences were altered by removal or addition of alanine residues, or the potential of the peptides to form stable ⁇ -helices was inhibited by including proline residues at various positions.
  • Two peptides were synthesized in which the spacings between adjacent consensus sequences were altered. Both increasing (ARKKAAKA-AAAA- ARKKAAKA-AAAA-ARKKAAKA) or decreasing (ARKKAAKA-RKKAAKA- RKKAAKA) the distance between consensus sequences resulted in decreased heparin- binding affinity (Kd ⁇ 250 and 450 nM respectively). Inclusion of prolines also decreased the heparin-binding affinity, the degree of which was influenced by their position and number.
  • heparin-binding affinity decreased to 360 nM when prolines were present in each tandem repeat in place of an alanine: (ARKKPAKA) 3 ; however, a weaker affinity was obtained when a single proline was substituted in the center of a series of three heparin-binding consensus sequences (ARKKAAKA- ARKKPAKA-ARKKAAKA, e 730 nM, Table I).
  • Other peptides synthesized and studied include sequences native to the mouse
  • Intrinsic CD of the peptides shows that they do not adopt ⁇ -helical conformations.
  • CD spectra for the ⁇ -helical conformations peptides were analyzed by CD in the presence of the non-polar solvent TFE.
  • Non-polar solvents are known to increase the degree of ⁇ -helicity of a peptide in solution by enhancing hydrogen bonding and electrostatic interactions (Adler, A. J. , and G. D. Fasman, J Phys Chem, 75: 1516- 1526, 1971).
  • CD of (AKKARAK at 0.1 mg/ml containing 0, 10, 20, 30, 40, and 50% TFE (v/v) was measured.
  • the peptide assumes an ⁇ -helical conformation with classic ⁇ -helical peaks at 206 and 220 nm and a cross over at 197 nm (data not shown).
  • cell layer-associated and secreted 5 S-SO 4 -radiolabeled PGs were purified by extraction with urea, and those PGs retained on DEAE after a 0.1 M NaCl rinse were studied for their binding to (ARKKAAKA) 4 by ACE (Fig. 5A, EC PGs).
  • This peptide exhibited significant affinity for secreted HUVEC PGs, although the average affinity was somewhat weaker than that exhibited by the peptide for heparin (PG K ⁇ 300 nM, heparin KdS 50 nM). Similar affinities were obtained for cell layer associated PGs (data not shown).
  • HUVEC PGs were subjected to various chemical and enzymatic degradations. Samples were then tested for their ability to bind to (ARKKAAKA PGs in which HS GAGs were chemically degraded by nitrous acid or enzymatically degraded by heparatinase I, were able to maintain comparable affinity for the peptide as was displayed by the total PG sample (Fig 5A, EC PGs/NA and Fig. 6).
  • PGs in which CS GAG chains were digested with chondroitinase ABC were also able to maintain comparable affinity for the peptide. Release of GAG chains from cores by borohydride reduction resulted in a 3-4 fold diminished affinity (Fig. 6).
  • heparin-binding sites are believed to form ⁇ -helices upon heparin-binding, and molecular modeling illustrates that basic amino acids in the binding sites align to one side of the helix to form a region of high positive charge through which heparin-binding occurs (Cardin, A. D. and H. J. R. Weintraub,
  • Additional consensus sequence peptides were designed to determine other aspects of peptide structure important to heparin-binding. Including gly cine in place of alanine in the hydropathic positions weakened heparin-binding, and peptides in which arginine was included in all basic positions displayed higher affinity for heparin than did those containing arginines and lysines. The latter is consistent with work showing a higher affinity interaction of arginine-heparin and arginine-HS than lysine-heparin or lysine-HS (Fromm, J. R. , et al., Arch Biochem Biophys, 232 (2):279-287, 1995).
  • heparin-binding characteristics of the peptides developed here may rely on amino acid type and arrangement in addition to ionic interactions. Inclusion of prolines within or between consensus sequence motifs weakened affinity for heparin, possibly as a result of alterations in peptide secondary conformation; this issue was investigated in our CD experiments. Finally, changing the spacing between consensus motifs weakened affinity for heparin; however, sequence orientation did not appear to influence binding ability as long as the motifs were contiguous and in one orientation. Molecular modeling of consensus sequences in native heparin-binding proteins predicts their presence within ⁇ -helical regions (Cardin, A. D. and H. J. R. Weintraub, Arteriosclerosis, 9:21-32, 1989).
  • GAG-directed conformational changes on polypeptides such as poly(L)-lysine and poly(L)-arginine have been identified (Gelman, R. A. , et al., Biopolymers, 12:541-558, 1973; Gelman, R. A. , and J. Blackwell, Arch Biochem Biophys, 159:427-433, 1973; Gelman, R. A., and J. Blackwell, Biopolymers, 13: 139-156, 1974).
  • Aqueous solutions of these polypeptides at neutral pH were shown by CD to adopt charged coil conformations, and to display ⁇ - helical conformations in the presence of heparin.
  • heparin-binding peptides designed here incorporate concatamers of heparin binding consensus sequences, which should rarely, if ever, appear in native proteins. Nonetheless, the proposed characteristics of heparin-binding motifs in proteins, as set forth by Cardin and Weintraub based on their theoretical analysis of putative heparin-binding domains of native proteins (Cardin, A. D. and H. J. R. Weintraub, Arteriosclerosis, 9:21-32, 1989), hold true with our model peptides. Thus, our data suggest that peptides containing the Cardin and Weintraub heparin- binding consensus sequences may show a selective advantage in heparin-binding over certain other sequences which do not fit their criteria.
  • optimally active heparin-binding peptides should include multiple sequences of the types: (XBBXBX)n and (XBBBXXBX)n. Sequence number and peptide Mr are the most critical features; peptides should be of at least approximately 30 residues, which could be decreased to 15 if cysteine is included near either terminus to promote dimerization. Peptides should contain contiguous sequence arrays, without intervening residues between sequences. Alanine, which stabilizes ⁇ -helical conformation, should occupy the hydropathic residue positions, and arginine the basic positions.
  • the high affinity PG- or GAG-binding peptides developed here, or derivatives thereof, could prove useful as tools for the promotion of cell-substratum attachment of PG-expressing cells, in the targeting of drugs to PG-expressing cells and PG-rich extracellular matrices, or as antagonists of GAG-mediated actions, e.g., neutralization of the anticoagulant activity of heparin, as is presented in this patent application.
  • Heparin is administered to patients either as unfractionated heparin or as heparin fragments.
  • the fragments are prepared in several different ways, and result in a heterogeneous mixture that varies according to the methods of preparation.
  • the low molecular weight heparins currently approved for clinical use, the standard dosage in anti-Xa units where known, and their molecular weight distributions are:
  • Heparin inhibits the activity of thrombin and Factor Xa by binding to AT III and thus enhancing the ability of antithrombin III to bind to these enzymes.
  • the higher- MW heparins presumably act as a bridge between ATIII and thrombin, and the binding of both proteins to the same molecule of heparin appears to be important for thrombin inhibition by ATIII.
  • the low molecular weight heparins do not affect thrombin activity, but still render ATIII capable of inhibiting Factor Xa activity.
  • heparin Solutions of Lovenox, Orgaran, or unfractionated heparin (Sigma) were prepared in 0.32% sodium citrate or in normal human plasma to contain 0.5U/ml anti- FXa activity. Calibrations were made against the standards provided by the Stachrom Heparin (Diagnostica Stago) assay kit. The heparin/ ATIII complex was allowed to form at 37°C for 2 minutes, peptide was added, the mixture was incubated for an additional 1-5 minutes, and then Factor Xa was added, and finally the color reagent, for one minute, and the absorbance was read at 405nm.
  • Plasma was obtained from normal donors.
  • Thrombin concentration human alpha thrombin, Enzyme Research Laboratories, South Bend, IN
  • Heparin was added at 0.5 IU anti-thrombin activity /ml.
  • the clotting time for heparin alone was approximately 3 minutes.
  • the peptides were added in concentrations ranging from 1-200 ⁇ g/ml. After one minute, thrombin was added and the clotting time determined. The effects of the peptides were concentration dependent. The clotting time was restored to normal values in all samples at the dosages shown below in Table III.
  • Rats 300-400 gm were anesthetized with ketamine/acepromazine and were cannulated in the left jugular vein and right femoral vein. Blood samples were all 0.1 ml. Blood was drawn immediately before injection of Lovenox to establish baseline Factor Xa activity. Lovenox (43 IU anti-FXa activity/kg in 0.1 ml saline, based on suggested dosage for humans) was injected through the jugular catheter, followed immediately by 0.2 ml of saline. Blood (0.1 ml) was collected into sodium citrate from the femoral vein every 30 seconds for 3 min.
  • the peptide was injected at 3 min through the jugular catheter in 0.1 ml of phosphate-buffered saline, followed by a 0.2 ml saline flush. Peptides were administered at 2 mg except where noted otherwise. Blood collection was immediately resumed every 30 seconds until 10 minutes after the initial Lovenox injection, then at 15, 20, 25 and 30 min. The samples were centrifuged to obtain plasma and were assayed for residual Lovenox by assay of anti-FXa activity by the Stachrom Heparin test kit. A405 was measured after a 1 -minute incubation with the chromogenic Factor Xa substrate. The assay is described in further detail in the Figure Legend to Fig. 7. Results
  • the maximal concentration of Lovenox in the plasma was 0.5-1.0 U/ml anti- Factor Xa activity for all the animals tested.
  • the maximal plasma heparin concentration was found by 2-2.5 minutes after injection. About half the Lovenox was cleared from the circulation by 25-30 minutes after injection, in an approximately linear fashion for 15 minutes and more slowly thereafter.
  • Panel A shows a representative clearance curve.
  • the three peptides at dosages shown in panels B-D caused no removal of Lovenox above that due to direct clearance from the circulation alone (Panel A).
  • the peptide (ARKKPAKA) 3 (Panel D) appeared to delay clearance of the heparin from the circulation.
  • a series of peptides have been generated, wherein said peptides have the ability to reverse the anti-FXa levels of Lovenox in rats within a few minutes at Lovenox concentrations to be expected in patients.
  • the ability of these peptides to reverse the effects of Lovenox in vivo appears to be consistent with their ability to reverse the effects in vitro.
  • the peptides reverse both anti-thrombin and anti-Xa activity of unfractionated heparin in vitro, and anti-Xa activity, unfractionated heparin, Lovenox, and Orgaran in vitro.
  • YPARRYQWNRAKP murine serglycin, cysteine replaced with alanine
  • Plasma was obtained from normal donors. Thrombin concentration was standardized to produce a clotting time of 20-22 seconds. Heparin was added at 0.5U/ml. The clotting time for heparin alone was approximately 3 minutes. One minute after addition of heparin to the plasma, the peptides were added in concentrations ranging from 1-200 ug/ml. After one minute, thrombin was added and the clotting time determined. The clotting time was normalized at the peptide concentrations shown below.
  • YPTQRARYQWVRCNP 100 human serglycin

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des peptides de différentes séquences et tailles, et des techniques d'utilisation de ces peptides qui présentent une forte affinité pour les glycosaminoglycanes et les protéoglycanes, ces peptides réagissant fortement avec l'héparine, et d'autres glycosaminoglycanes, ou protéoglycanes (PG).
PCT/US2000/002853 1999-02-02 2000-02-02 Activites de modulation des peptides de l'heparine, et d'autres glycosaminoglycanes ou proteoglycanes WO2000045831A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CA002361612A CA2361612A1 (fr) 1999-02-02 2000-02-02 Activites de modulation des peptides de l'heparine, et d'autres glycosaminoglycanes ou proteoglycanes
EP00905964A EP1156816A4 (fr) 1999-02-02 2000-02-02 Activites de modulation des peptides de l'heparine, et d'autres glycosaminoglycanes ou proteoglycanes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11827699P 1999-02-02 1999-02-02
US60/118,276 1999-02-02

Publications (1)

Publication Number Publication Date
WO2000045831A1 true WO2000045831A1 (fr) 2000-08-10

Family

ID=22377591

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/002853 WO2000045831A1 (fr) 1999-02-02 2000-02-02 Activites de modulation des peptides de l'heparine, et d'autres glycosaminoglycanes ou proteoglycanes

Country Status (3)

Country Link
EP (1) EP1156816A4 (fr)
CA (1) CA2361612A1 (fr)
WO (1) WO2000045831A1 (fr)

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002060488A1 (fr) * 2001-01-30 2002-08-08 Universite Catholique De Louvain Composes antitumoraux
WO2004059278A2 (fr) * 2002-12-31 2004-07-15 Rimonyx Pharmaceuticals Ltd. Procedes permettant de rechercher par criblage des medicaments anti-inflammatoires et utilisation de ces procedes
US7049286B2 (en) 2001-08-30 2006-05-23 Diatos, S.A. Insulin conjugates and methods of use thereof
EP1797901A1 (fr) 2005-12-16 2007-06-20 Diatos Conjugués peptidiques pénétrant dans les cellules en tant que vecteurs pour l'administration d'acides nucléiques
EP1818395A1 (fr) 2006-02-08 2007-08-15 Diatos Compositions et méthodes pour le traitement de maladies lysosomales
US7259140B2 (en) 2003-03-28 2007-08-21 Thomas Jefferson University Heparin-binding peptides and uses thereof
WO2007113687A2 (fr) 2006-03-30 2007-10-11 Diatos S.A. Conjugués de camptothécine et de peptide et compositions pharmaceutiques les contenant
EP1853917A2 (fr) * 2005-03-04 2007-11-14 Northwestern University Epitopes angiogéniques de liaison a l'héparine, amphiphiles peptidiques, compositions auto-assemblées et procédés d'utilisation associés
US7452679B2 (en) 2003-12-05 2008-11-18 Northwestern University Branched peptide amphiphiles, related epitope compounds and self assembled structures thereof
US7491690B2 (en) 2001-11-14 2009-02-17 Northwestern University Self-assembly and mineralization of peptide-amphiphile nanofibers
US7534761B1 (en) 2002-08-21 2009-05-19 North Western University Charged peptide-amphiphile solutions and self-assembled peptide nanofiber networks formed therefrom
US7544661B2 (en) 2003-12-05 2009-06-09 Northwestern University Self-assembling peptide amphiphiles and related methods for growth factor delivery
US7544664B2 (en) 2003-08-14 2009-06-09 Diatos, S.A. Sequences facilitating penetration of a substance of interest
US7554021B2 (en) 2002-11-12 2009-06-30 Northwestern University Composition and method for self-assembly and mineralization of peptide amphiphiles
US7683025B2 (en) 2002-11-14 2010-03-23 Northwestern University Synthesis and self-assembly of ABC triblock bola peptide amphiphiles
US7745708B2 (en) 2002-02-15 2010-06-29 Northwestern University Self-assembly of peptide-amphiphile nanofibers under physiological conditions
US8003595B2 (en) 2000-03-01 2011-08-23 Cellectis Amino acid sequences facilitating penetration of a substance of interest into cells and/or cell nuclei
US8076295B2 (en) 2007-04-17 2011-12-13 Nanotope, Inc. Peptide amphiphiles having improved solubility and methods of using same
US8450271B2 (en) 2009-04-13 2013-05-28 Northwestern University Peptide-based scaffolds for cartilage regeneration and methods for their use
WO2014127046A1 (fr) * 2013-02-12 2014-08-21 Headsight, Inc. Système diagnostique pour dispositif de commande

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997047312A1 (fr) * 1996-06-11 1997-12-18 Commonwealth Biotechnologies, Inc. Peptides se liant a l'heparine
US5770563A (en) * 1991-12-06 1998-06-23 The United States Of America As Represented By The Department Of Health And Human Services Heparin- and sulfatide binding peptides from the type I repeats of human thrombospondin and conjugates thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5614494A (en) * 1993-11-12 1997-03-25 University Of Michigan, The Board Of Regents Acting For And On Behalf Of Peptides for heparin and low molecular weight heparin anticoagulation reversal

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5770563A (en) * 1991-12-06 1998-06-23 The United States Of America As Represented By The Department Of Health And Human Services Heparin- and sulfatide binding peptides from the type I repeats of human thrombospondin and conjugates thereof
WO1997047312A1 (fr) * 1996-06-11 1997-12-18 Commonwealth Biotechnologies, Inc. Peptides se liant a l'heparine
US5877153A (en) * 1996-06-11 1999-03-02 Commonwealth Biotechnologies Inc. Heparin-binding peptides

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CARDIN ET AL.: "Molecular modeling of protein-glycosaminoglycan interactions", ARTERIOSCLEROSIS,, vol. 9, no. 1, January 1989 (1989-01-01), pages 21 - 32, XP002928185 *
HODGKINSON ET AL.: "Glycosaminoglycan binding characteristics of the insulin-like growth factor binding proteins", J. MOL. ENDOCRINOLOGY,, vol. 13, no. 1, August 1994 (1994-08-01), pages 105 - 112, XP002928184 *
See also references of EP1156816A4 *

Cited By (32)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8003595B2 (en) 2000-03-01 2011-08-23 Cellectis Amino acid sequences facilitating penetration of a substance of interest into cells and/or cell nuclei
WO2002060488A1 (fr) * 2001-01-30 2002-08-08 Universite Catholique De Louvain Composes antitumoraux
US7112562B2 (en) 2001-08-30 2006-09-26 Diatos, S.A. Insulin conjugates and methods of use thereof
US7049286B2 (en) 2001-08-30 2006-05-23 Diatos, S.A. Insulin conjugates and methods of use thereof
US7491690B2 (en) 2001-11-14 2009-02-17 Northwestern University Self-assembly and mineralization of peptide-amphiphile nanofibers
US7838491B2 (en) 2001-11-14 2010-11-23 Northwestern University Self-assembly and mineralization of peptide-amphiphile nanofibers
US8063014B2 (en) 2002-02-15 2011-11-22 Northwestern University Self-assembly of peptide-amphiphile nanofibers under physiological conditions
US7745708B2 (en) 2002-02-15 2010-06-29 Northwestern University Self-assembly of peptide-amphiphile nanofibers under physiological conditions
US7534761B1 (en) 2002-08-21 2009-05-19 North Western University Charged peptide-amphiphile solutions and self-assembled peptide nanofiber networks formed therefrom
US7554021B2 (en) 2002-11-12 2009-06-30 Northwestern University Composition and method for self-assembly and mineralization of peptide amphiphiles
US8124583B2 (en) 2002-11-12 2012-02-28 Northwestern University Composition and method for self-assembly and mineralization of peptide-amphiphiles
US7683025B2 (en) 2002-11-14 2010-03-23 Northwestern University Synthesis and self-assembly of ABC triblock bola peptide amphiphiles
WO2004059278A2 (fr) * 2002-12-31 2004-07-15 Rimonyx Pharmaceuticals Ltd. Procedes permettant de rechercher par criblage des medicaments anti-inflammatoires et utilisation de ces procedes
WO2004059278A3 (fr) * 2002-12-31 2005-03-03 Rimonyx Pharmaceuticals Ltd Procedes permettant de rechercher par criblage des medicaments anti-inflammatoires et utilisation de ces procedes
US7259140B2 (en) 2003-03-28 2007-08-21 Thomas Jefferson University Heparin-binding peptides and uses thereof
US8293700B2 (en) 2003-08-14 2012-10-23 Cellectis Anti-bacterial composition especially for controlling gram-negative bacteria, comprising a peptide and an advantageously hydrophobic anti-bacterial agent
US7544664B2 (en) 2003-08-14 2009-06-09 Diatos, S.A. Sequences facilitating penetration of a substance of interest
US7452679B2 (en) 2003-12-05 2008-11-18 Northwestern University Branched peptide amphiphiles, related epitope compounds and self assembled structures thereof
US7544661B2 (en) 2003-12-05 2009-06-09 Northwestern University Self-assembling peptide amphiphiles and related methods for growth factor delivery
US8580923B2 (en) 2003-12-05 2013-11-12 Northwestern University Self-assembling peptide amphiphiles and related methods for growth factor delivery
US8138140B2 (en) 2003-12-05 2012-03-20 Northwestern University Self-assembling peptide amphiphiles and related methods for growth factor delivery
EP1853917A4 (fr) * 2005-03-04 2008-09-10 Univ Northwestern Epitopes angiogéniques de liaison a l'héparine, amphiphiles peptidiques, compositions auto-assemblées et procédés d'utilisation associés
JP2008531733A (ja) * 2005-03-04 2008-08-14 ノースウエスタン ユニバーシティ 血管新生性のヘパリン結合エピトープ、ペプチド両親媒性物質、自己集合組成物、および関連する使用法
US7851445B2 (en) * 2005-03-04 2010-12-14 Northwestern University Angiogenic heparin-binding epitopes, peptide amphiphiles, self-assembled compositions and related methods of use
EP1853917A2 (fr) * 2005-03-04 2007-11-14 Northwestern University Epitopes angiogéniques de liaison a l'héparine, amphiphiles peptidiques, compositions auto-assemblées et procédés d'utilisation associés
EP1797901A1 (fr) 2005-12-16 2007-06-20 Diatos Conjugués peptidiques pénétrant dans les cellules en tant que vecteurs pour l'administration d'acides nucléiques
EP1818395A1 (fr) 2006-02-08 2007-08-15 Diatos Compositions et méthodes pour le traitement de maladies lysosomales
WO2007113687A2 (fr) 2006-03-30 2007-10-11 Diatos S.A. Conjugués de camptothécine et de peptide et compositions pharmaceutiques les contenant
US8410045B2 (en) 2006-03-30 2013-04-02 Drais Pharmaceuticals, Inc. Camptothecin-peptide conjugates and pharmaceutical compositions containing the same
US8076295B2 (en) 2007-04-17 2011-12-13 Nanotope, Inc. Peptide amphiphiles having improved solubility and methods of using same
US8450271B2 (en) 2009-04-13 2013-05-28 Northwestern University Peptide-based scaffolds for cartilage regeneration and methods for their use
WO2014127046A1 (fr) * 2013-02-12 2014-08-21 Headsight, Inc. Système diagnostique pour dispositif de commande

Also Published As

Publication number Publication date
CA2361612A1 (fr) 2000-08-10
EP1156816A1 (fr) 2001-11-28
EP1156816A4 (fr) 2004-12-29

Similar Documents

Publication Publication Date Title
WO2000045831A1 (fr) Activites de modulation des peptides de l'heparine, et d'autres glycosaminoglycanes ou proteoglycanes
Jackson et al. Glycosaminoglycans: molecular properties, protein interactions, and role in physiological processes
US5654273A (en) Synducin mediated modulation of tissue repair
Ricard-Blum et al. Matricryptins derived from collagens and proteoglycans
Brown et al. Histidine-rich glycoprotein and platelet factor 4 mask heparan sulfate proteoglycans recognized by acidic and basic fibroblast growth factor
US6319897B1 (en) Peptides which inhibit complement activation
US7259140B2 (en) Heparin-binding peptides and uses thereof
US6855801B1 (en) Peptides modulating activities of heparin other glycosaminoglycans or proteoglycans
JP2005187480A (ja) 殺菌／透過性増加タンパク質産物の治療用途
JPH03500492A (ja) ４型コラーゲン活性を有するポリペプチド類
EP0278781B1 (fr) Peptides à activité de laminine
Soncin et al. A cell-surface proteoglycan mediates human adenocarcinoma HT-29 cell adhesion to human angiogenin.
EP0255206A2 (fr) Peptides inhibant la liaison du facteur von Willebrand
US8173595B2 (en) Methods and compositions for the inhibition of thrombus formation
Bittner et al. Modulation of collagen gel contraction by decorin
US5618917A (en) Methods and compositions for detecting and treating kidney diseases associated with adhesion of crystals to kidney cells
D'Amore Heparin-endothelial cell interactions
US6506880B2 (en) Synthetic peptides that enhance atherogenic lipoprotein uptake and lower plasma cholesterol
US6906037B2 (en) Therapeutic peptide-based constructs
Shenoy et al. Development of heparin antagonists with focused biological activity
US20060252692A1 (en) Inhibitors for use in hemostasis
TW200529870A (en) Therapeutic use of factor XI
Ross et al. Fibrillin containing elastic microfibrils support platelet adhesion under dynamic shear conditions
EP0511830A2 (fr) Heparine purifiée par affinité
US20090181057A1 (en) Methods and Compositions for the Inhibition of Thrombus Formation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref country code: CA

Ref document number: 2361612

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 2000905964

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2000905964

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2361612

Country of ref document: CA

WWW Wipo information: withdrawn in national office

Ref document number: 2000905964

Country of ref document: EP