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WO1993025576A2 - Peptides having platelet-derived growth factor (pdgf) activity - Google Patents

Peptides having platelet-derived growth factor (pdgf) activity Download PDF

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Publication number
WO1993025576A2
WO1993025576A2 PCT/US1993/005325 US9305325W WO9325576A2 WO 1993025576 A2 WO1993025576 A2 WO 1993025576A2 US 9305325 W US9305325 W US 9305325W WO 9325576 A2 WO9325576 A2 WO 9325576A2
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Prior art keywords
seq
peptide
pdgf
peptides
amino acid
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PCT/US1993/005325
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French (fr)
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WO1993025576A3 (en
Inventor
Amrit K. Judd
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Sri International
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Priority to JP6501593A priority Critical patent/JPH07508510A/en
Publication of WO1993025576A2 publication Critical patent/WO1993025576A2/en
Publication of WO1993025576A3 publication Critical patent/WO1993025576A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/49—Platelet-derived growth factor [PDGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention is in the field of biologically active peptides. These peptides may also be incorporated into other peptides or proteins to yield hybrid, or chimeric, multifunctional molecules. More particularly, it concerns peptides which substantially correspond in sequence to fragments of platelet-derived growth factor (“PDGF”) and their preparation and activity as mitogens and as chemotactic agents for fibroblasts, vascular smooth muscle cells and other cells. These activities lead to uses for these peptides as wound healing agents, as agents for treatment of vascular diseases in addition to uses in analytical methods for determining PDGF in samples.
  • PDGF platelet-derived growth factor
  • PDGF is a 32-kD protein heterodimer composed of
  • a and B polypeptide chains linked by disulfide bonds It is stored in the ex-granules of platelets and released when platelets are activated by blood clotting and contact with sites of injury. It stimulates specific target cells by binding to cell-surface receptors, thereby mediating a cascade of events that leads to DNA synthesis and cell proliferation.
  • PDGF is a strong mitogen for fibroblasts, smooth muscle cells, and glial cells. In addition, it is a potent chemotactic factor for neutrophils, onocytes, fibroblasts, and smooth muscle cells. Thus, PDGF plays an important role in the migration of inflammatory cells and connective tissue cells to sites of inflammation and injury and in the repair or restructuring of injured tissues.
  • PDGF appears to play a part in atherosclerosis.
  • a degenerative disease of the arteries this condition is characterized by deposition of fatty substances in, and the fibrous thickening of, intima, resulting in the narrowing of the vessel passages and ultimately their hardening and loss of elasticity.
  • the earliest lesion of atherosclerosis is a ubiquitous fatty streak commonly found in -children. This grossly flat, lipid-rich lesion consists of macrophages and some smooth muscle.
  • the fibrous plaque is representative of the various forms of advanced atherosclerosis and is made up of increased internal smooth muscle cells surrounded by connective tissue matrix and containing variable amounts of intracellular and extracellular lipid. In the lumen of the artery, this lesion is generally covered by a dense, fibrous cap of smooth muscle and connective tissue.
  • PDGF platelet- derived growth factor
  • Plaque PDGF plays an important role in wound healing.
  • Activated monocytes have been shown to release a PDGF-like activity and to express the C-sis gene and to secrete a PDGF-like activity as well.
  • Pierce, G.F. at al. , J. Exp. Med. , 167, 974 (1988), reported that PDGF and recombinant C-sis gene homodimeric proteins augment in vivo incisional wound healing in rats.
  • PDGF increases collagen formation, DNA content, and protein levels.
  • HLE human leukocyte elastase
  • HLE cleavage produced by HLE is located at or near the N- and C-terminal ends of either the A or B chains since HLE preferentially cleaves at Val-X bonds and, to a lesser extent, at Ala-X bonds.
  • cathepsin G cleaves peptide bonds adjacent to the carboxyl group of phenylalanine, leucine, tyrosine, isoleucine, and methionine residues.
  • the sensitivity of PDGF fibroblasts' chemotactic activity to HLE but not the cathepsin G suggests that the active site for chemotactic activity is located in a small region(s) of the PDGF molecule.
  • PDGF Reduction and alkylation of PDGF results in loss of its ' mitogenic activity but has no effect on its chemotactic activity, thus suggesting that sulfhydryl groups of cysteine do not affect chemotaxis but do affect mitogenesis. This also indicates that dimers (homo or hetero) or disulfide loop structures are required for mitogenesis. Because p28 v"s ⁇ s , the transforming protein of the simian sarcoma virus, which is 92% homologous to the A chain of PDGF, has specific mitogenic activity identical with that of PDGF, it is likely that the A chain carries the active site for the mitogenic activity of PDGF.
  • the B chain may contain the active site for chemotactic activity or the A chain may have the active site for both the mitogenic activity and the chemotactic activity.
  • the native material While in some cases it might be possible to obtain, make and/or use the native material to moderate body functions and treat injury or disease or to use this native material in assays, this is not really practical.
  • the native material exists in such small amounts that its isolation on a commercial scale is not feasible. Also, the native material, at 32 kD, is too large to synthesize in any sort of reasonable yield. What is needed is synthetic peptides which exhibit desired PDGF activity but which are significantly shorter and which correspond substantially to a region of the PDGF sequence.
  • peptide fragments of from about 6 to about 26 amino acids in length and having amino acid sequences which have substantial homology to sequences found in the A or B chain of PDGF as well as salts, dimers and derivatives of such fragments can exhibit substantial PDGF-mimicking biological activity.
  • the invention provides novel peptides corresponding to a sequence having substantial homology to one of the following PDGF A chain sequences:
  • this invention provides novel active peptides corresponding to or having substantial homology to one of the following PDGF B-chain sequences:
  • this invention provides novel active peptide dimers, peptide ACM derivatives and oxidized peptides corresponding to or having substantial homology to one of the PDGF A-chain sequences shown in Table 3.
  • this invention provides novel active peptide dimers, peptide ACM derivatives and oxidized peptides corresponding to or having substantial homology to one of the PDGF B-chain sequences shown in Table 4.
  • this invention provides hybrid (i.e., chimeric) multifunctional molecules incorporating the peptides of this invention into other peptides and proteins.
  • this invention provides the salts of these peptides, the amide and acyl derivatives of the carbonyl and amino end groups of these peptides and their salts.
  • any of the active peptides of this invention may be conjugated to peptide polymers and proteins.
  • These immobilizing peptides and proteins are high molecular weight (5 kD to 80 kD) materials such as bovine serum albumin (BSA) , ovalbumin (OVA) , poly(lysine) and the like.
  • the peptides can also be attached to lipophillic moieties such as 12 to 24 carbon atom long saturated and unsaturated hydrocarbon and fatty acid residues.
  • this invention concerns labeled versions of these peptides, their salts and derivatives and their application to label-dependent assay methods such as radioimmunoassays (RIA) , fluoroassays, and enzyme-linked immunoabsorbent assays (ELISA) for the determination or assaying of PDGF in laboratory and clinical samples.
  • label-dependent assay methods such as radioimmunoassays (RIA) , fluoroassays, and enzyme-linked immunoabsorbent assays (ELISA) for the determination or assaying of PDGF in laboratory and clinical samples.
  • RIA radioimmunoassays
  • fluoroassays fluoroassays
  • ELISA enzyme-linked immunoabsorbent assays
  • active peptides, salts, derivatives and hybrids of this invention and particularly those of the B-chain are used as mitogens, and as chemotactic agents and the invention provides pharmaceutical agents based on these materials and provides for their use in wound healing, in the alleviation of the effects of vascular disease or injury, and in the modulation of calcium uptake into cells.
  • Figs. 1, 5 and 6 each show results of the competitive binding of 3T3 cells of three peptides of this invention
  • Figs. 3 and 4 graph the effect of a material of this invention and PDGF on intracellular calcium influx
  • Fig. 2 is a graph depicting competitive binding of 3T3 cells by PDGF itself as a standard for comparison.
  • the acyl group usually preferred in this invention is acetyl.
  • Acyl groups are used to block the terminal amino group of a peptide.
  • Amide refers to an amino-containing group formed to block the terminal carboxyl group of a peptide.
  • the amide group has the structure
  • R ⁇ and R 2 are each hydrogen or a lower alkyl.
  • Constant substitution refers to a substitution in a peptide of an amino acid by another amino acid which is similar in chemical and hydrophobicity properties to the original.
  • ELISA refers to an enzyme-1inked immunosorbent assay which employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of antigen or antibody present in a sample.
  • a description of the ELISA technique is found in Chapter 22 of the 4th Edition of Basic and Clinical Immunology by D.P. Sites et al, published by Lange Medical Publications of Los Altos, California, in 1982, which is incorporated herein by reference.
  • EMIT refers to an enzyme-multiplied immunoassay technique which uses (1) an enzyme-labeled hapten, (2) specific antibody to the hapten, (3) pretreatment reagent, (4) buffered-enzyme substrate, and (5) standards to detect the amount of an unknown in a sample.
  • a description of the EMIT technique is found in Enzyme Immunoassav. edited by E.T. Maggio, published in 1980 by CRC ' Press, Inc., Boca Raton, Florida, particularly on pp. 141-150, 234-5, and 242-3. These materials are incorporated by reference.
  • Fluoroimmunoassay refers to an antibody-based assay in which the species to be measured binds to, displaces or competes for binding with a material labelled with a fluorescent species in an antibody-ligand complex.
  • the complex is separated and the presence or absence of fluorescent species gives a measure of the amount of measured species.
  • the complex has different fluorescent properties than the uncomplexed fluorescent species so that the formation of the complex can be detected without separation of the complex.
  • fluoroimmunoassay techniques is found in "A Review of Fluoroimmunoassay and Immunofluorometric Assay", D.S. Smith et al. (1981) Ann. Clin. Bioche . (1981) 18:253-274 which is incorporated herein by reference.
  • Label refers to a detectable group in a molecule.
  • common labels are radioactive species useful in radioimmunoassays, fluorescent species useful in fluoroimmunoassays, and enzymatic species useful in the ELISA and EMIT methods and the like.
  • “Lower alkyl” refers to a straight or branched chain saturated hydrocarbon group having from 1 to 4 carbon atoms such as, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.
  • Peptide or “polypeptide” refers to relatively low molecular weight compounds which yield two or more, such as up to about 30 units of amino acid on hydrolysis.
  • “Pharmaceutically acceptable salt” and “salt” refer to salts that retain the desired antigenic activity of the parent peptide.
  • “Pharmaceutically acceptable salt” refers to salts that are suitable for ingestion or parenteral administration or the like in that they do not impart any undesired toxicological effects.
  • salts and pharmaceutically acceptable salts include (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid,- tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid, and the like; (b) salts with monovalent and polyvalent metal cations such as sodium, potassium, zinc, calcium, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or with an organic cation formed from N,N' -dibenzylethylenediamine or ethylened
  • Radioimmunoassay or “RIA” refers to an antibody-based assay in which the species to be measured binds to, displaces or competes for binding with a radiolabeled material in an antibody-ligand complex. The complex is separated and the presence or absence of radioactivity gives a measure of the amount of measured species.
  • substantially corresponding refers to the property of two amino acid sequences being identical to one another or differing from one another by no more than three amino acid units. Sequences can differ by having a different amino acid at a given position or by having an extra amino acid or by missing an amino acid. Preferably, the sequences have at most two points of difference and more preferably have one difference or are identical. As used herein, the following abbreviations are used for the amino acids described:
  • this invention relates to synthetic peptides and their salts; peptide dimers, and derivatives which have PDGF properties and activity.
  • the synthetic peptides have about 6 to 26 amino acids substantially corresponding to a 6 to 26 amino acid sequence of a PDGF chain.
  • the peptide can correspond to portions of the A chain in which case they can have sequences substantially corresponding to the peptides shown in Table 1.
  • the peptides correspond to portions of the B chain in which case they can have sequences substantially corresponding to the peptides shown in Table 2.
  • the peptides can also be in the form of dimers and derivatives substantially corresponding to those A- chain materials shown in Table 3.
  • the peptides ⁇ an also be in the form of dimers and derivatives substantially corresponding to those B- chain materials shown in Table 4.
  • Any of these materials can be in the form of pharmaceutically acceptable salts. Labeled forms can be prepared, as well.
  • any of these materials can also be incorporated into hybrid, multifunctional molecules. This is done by incorporating these peptide sequences into other peptides or proteins such as hirudin, or the like. These materials can be prepared by direct synthesis as exemplified by Church, F.C. et al., J. Biol. Chem. (1991) 266:11975-11979. This article illustrates the formation and hybrid activity of a hirudin sequence-containing peptide.
  • the active peptides of this invention can advantageously be coupled or conjugated to other moieties to enhance cell attachment or to immobilize the peptides.
  • These complexing and conjugating techniques are generally well known in the art and can employ methods and reagents of the art. Immobilization is usually brought about by covalently attaching the peptide to a peptide polymer or protein of 5 kD to 80 kD.
  • BSA and OVA are two well known proteins for this use as are the regular lysine polymers which range in size from about 5 kD to about 30 kD. This immobilization locks the conformation of the peptide and leads to higher activity.
  • the peptide can be coupled to lypophillic moieties of 6 to 20 carbons or so. These can be fatty acid residues of 12 to 20 carbons such as palmitic, oleic and linoleic acid and the like and alkyl amino acids where the alkyl substituent is from about 6 to 12 carbons in length and typically saturated and linear, such as aminooctanoic acid.
  • lypophillic moieties 6 to 20 carbons or so.
  • These can be fatty acid residues of 12 to 20 carbons such as palmitic, oleic and linoleic acid and the like and alkyl amino acids where the alkyl substituent is from about 6 to 12 carbons in length and typically saturated and linear, such as aminooctanoic acid.
  • the preparation of amino acids carrying these lypophillic groups is shown in Toth, I. et al. , In. Proc. of the 11th Amer. Peptide Symposium, J.E. Rivier and G.R. Marshall
  • the peptides may be synthesized by any techniques that are known to those skilled in the peptide art, such as may be found, in Meienhofer, J. , Hormonal Proteins and Peptides. Vol. 2, p. 46, Academic Press, New York, (1973) (for solid phase peptide synthesis) and Schroder, E. et al. , The Peptides. Vol. l, Academic Press, New York, (1965) (for classical solution synthesis) .
  • These methods comprise sequential addition of amino acids or suitably protected amino acids to a growing peptide chain.
  • amino acids or suitably protected amino acids are protected by a suitable protecting group.
  • the protected or derivatized amino acid is contacted with the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions suitable for forming the amide linkage.
  • the protecting group is then removed from this newly added amino acid residue and the next amino acid is then added.
  • any remaining protecting groups are removed sequentially or concurrently to afford the final peptide.
  • A_lso as is well known, it is possible to add more than one amino acid at a time to a growing chain.
  • a preferred method of preparing compounds of the. present invention involves solid phase peptide synthesis.
  • the alpha-amino function of the amino acids is protected by an acid or base-sensitive group.
  • Suitable protecting groups are t- utyloxycarbonyl (Boc) , fluorenyl methoxy carbonyl (FMOC) , benzyloxycarbonyl (Z) , and the like. Side chain active sites are protected, as well, to prevent undesired reactions or couplings.
  • Particularly preferred side chain protecting groups are, for arginine: nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, Z, Boc, and adamantyloxy carbonyl; for lysine: dichloro benzyloxyl carbonyl, t-Boc; for aspartic acid and glutamic acid: o-benzyl, t-butyl; for tyrosine: benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, cyclohexyl, cyclopentyl, and acetyl; for serine and threonme: benzyl, t-butyl and tetrahydropyranyl; for histidine: benzyl, p- toluenesulfonyl and 2,4-dinitrophenyl; and for trypt ⁇ phan:
  • the carboxyl-terminal amino acid is attached to a suitable solid support.
  • Suitable supports are inert to the reagents and reaction conditions of the reactions, as well as insoluble in the media used.
  • Suitable solid supports include chloromethylpolystyrenedivinylbenzene polymers and the like, especially chloromethylpolystyrene-1% divinylbenzene polymer.
  • a particularly useful support is the benzhydrylaminopolystyrenedivinyl- benzene polymer described by Vivaille, P. et al. (1971) Helv.
  • the attachment to the chloro-methyl polystyrene-divinylbenzene type of resin is made by means of the reaction of the alpha N-protected amino acid, especially the Boc-amino acid, as its cesium, tetramethylammonium, 4,5-diazabicyclo[5.4.0]undec-5-ene, or similar salt in ethanol, acetonitrile, N,N-dimethylformamide (DMF) , and the like, especially the cesium salt in DMF, with the chloromethyl resins at an elevated temperature, for example between about 40°C and 60°C, preferably about 50°C, for from about 12 to 48 hours, preferably about 24 hours.
  • the alpha N-Boc-amino acid is attached to the benzhydrylamine resin by means of an N,N' -dicyclohexylcarbodiimide (DCC)/
  • DCC N,N' -dicyclohexylcarbodiimide
  • HBT 1-hydroxybenzotriazole
  • the removal of the alpha N-protecting groups may be performed in the presence of, for example, a solution of trifluoroacetic acid in methylene chloride, or other strong acid solution, preferably 50% trifluoroacetic acid in dichloromethane at about ambient temperature.
  • Base-labile protecting groups may be removed by treatment with a base such as piperidine in DMF.
  • Each protected amino acid is preferably introduced in approximately 2.5 molar excess and coupling may be carried out in dichloromethane and the like, especially in dichloromethane at about ambient temperature.
  • the coupling agent is normally DCC in dichloromethane but may be N,N' -diisopropylcarbodiimide or other carbodiimide either alone or in the presence of HOBT, N-hydroxysuccinimide, other N-hydroxyimides or oximes.
  • protected amino acid active esters e.g., p-nitrophenyl, pentafluorophenyl and the like
  • symmetrical ' anhydrides may be used.
  • the peptide is either carried through another deprotection and neutralization cycle followed by acylation, preferably acetylation with acetic anhydride to yield an N-acetyl (N-Ac) blocked amino end group, or it may be removed from the resin directly. If the carboxy
  • the peptide may be either synthesized on the benzhydrylamino-polystyrene resin, which gives the amide directly, or it may be removed from the resin by ammonolysis with, for example, ammonia/methanol or a monia/ethanol, at a temperature of from about 0° to about 50°C, preferably about 25°C for about 12 to about 48 hours, preferably about 18 hours. If a peptide with a free amino-terminal and a carboxyl-terminal is desired, the peptide may be directly removed from the resin by treatment with anhydrous liquid hydrogen fluoride in the presence of a radical scavenger such as anisole.
  • a radical scavenger such as anisole.
  • the amino or carboxyl-blocked (protected) peptides are similarly deprotected by treatment with anhydrous liquid hydrogen fluoride.
  • anhydrous liquid hydrogen fluoride In cases where base-labile protection of the alpha N function is used in conjunction with t-butyl-based side chain protection, the final resin removal and deprotection step may be performed with trifluoroacetic acid.
  • the latter treatment may be used for simultaneous cleavage from the resin and deprotection to yield free-C0_H end groups when a normal benzylester linkage has been used or to form a C0-NH remind (amide) end groups when a benzhydrylamino linkage has been used.
  • the resin cleavage and deprotection steps may be combined in a single step utilizing liquid HF/anisole as described above. The fully protected polypeptide can then be purified by chromatographic steps.
  • the peptides can be obtained as salts, by simple adjustment of the pH of the medium from which they are finally recovered with acids or bases corresponding to the desired counter ions.
  • Radiolabeled versions of the polypeptides can be produced in several manners. For one, commercially
  • 3 H-amino acids can be prepared by the magnesium oxide procedure of Schwyzer et al. (1959) Helv. Clin. Acta. 42 2622. Except for the precautions routinely associated with radiochemicals, these processes can follow the usual synthesis route.
  • the finished polypeptide or conjugate can be radiolabeled by tritium exchange.
  • Enzy e labels can be incorporated by using an enzymic carrier for forming conjugates o.r by attaching an enzymatically active group to the carrier or the peptide.
  • Parallel dimers are synthesized by using acetamido methyl group (ACM) on appropriate cysteine residues, thereby selectively forming disulfide bonds to put the peptide chains in registered dimeric form.
  • ACM acetamido methyl group
  • the ACM group is resistant to HF and can be removed with iodine in ethanol, with simultaneous oxidation to a disulfide bond.
  • the cyclic .disulfides can be formed by air oxidation of a dilute solution of the corresponding linear peptide in water. After HF cleavage and extraction of the peptide off of the resin, the resulting solution is adjusted to pH 8.1 by addition of concentrated NH 4 OH in water and then shaken slowly on a shaker for 5-10 days. At the end of this time or during it, an Ellman test can be performed to confirm the completion of the disulfide bond formation. The peptide solution is adjusted to pH 6.5 by addition of a weak acid such as acetic acid. It is then passed through an acetic acid-treated and water-washed resin column.
  • a weak acid such as acetic acid
  • the peptide can be extracted with aqueous acetic acid.
  • This extract can be concentrated such as by rotary evaporation at 35°C at 1.0 mm Hg vacuum and reevaporated from water to remove most of the acetic acid.
  • the resulting residue can be purified by preparative HPLC using a linear gradient of acetonitrile-water with 0.1% trifluoroacetic acid.
  • the purified product fractions can be combined, organics evaporated off, and the aqueous phase lyophilized.
  • peptides and peptide analogs of this invention can be tested to determine their most advantageous PDGF-mimicking activity.
  • a major advantage of the peptides of this invention is their ability to exhibit only one or only a portion of the several activities ascribed to native PDGF.
  • they are first tested for their ability to bind to PDGF receptors on the surface of Balb/C 3TC fibroblasts by a competitive inhibition radioimmunoassay (RIA) by the test of R.M. Senior et al., J. Cell Biol. 100:351 (1988) . All the peptides are then tested for their ability to stimulate mitogenesis and to induce chemotaxis.
  • RIA radioimmunoassay
  • Biological assays on active peptides can be carried out on cultured smooth muscle cells (SMC) .
  • Rat aorta cells can be obtained from ATCC (CRL1476) , or alternatively, isolated and cultured by the procedure described by J. Nakao et al., Atherosclerosis 43 : 143 (1982) and R. Ross, J. Cell Biol. 50:172 (1971) .
  • SMCs can be isolated from media explants of thoracic aortas from male Wistar rats 6 to 8 weeks old. In culture, the cells begin to grow and migrate out of the explants after 10 to 14 days. Cells are then subcultured at a 1 to 2 split ratio when they become confluent and are maintained in tissue culture media (minimal essential medium [MEM] ) containing 10% fetal bovine serum (FBS) .
  • tissue culture media minimal essential medium [MEM]
  • FBS fetal bovine serum
  • the cell monolayers are solubilized with 1 ml of 1% Triton X-100 for 20 in at room temperature.
  • the amount of radioactivity contained in the lysates is determined on a Packard Gamma Counting System and the results are expressed as mean counts per well.
  • Fibroblast growth factor (FGF) and epidermal growth factor (EGF) are used as controls because they do not compete with the binding of PDGF. J.S. Huang et al., J. Biol. Chem. 257:8130 (1982).
  • the mitogenic activity of peptides is determined according to the procedure described by G.R. Grotendorst et al., J. Cell Physiol. 113:261 (1982). Fibroblasts or smooth muscle cells form primary explant cultures are trypsinized and resuspended in Dulbecco's Modified Eagle's Medium (DMEM) with 10% calf serum prepared from platelet-free plasma. The cells are then pelleted at a density of 6 x 10 4 cells/well in Costar microtissue culture wells (6.4 mm diameter). After 24 h, various dilutions of PDGF or peptides are added to the wells and the cells are incubated for an additional 48 h.
  • DMEM Dulbecco's Modified Eagle's Medium
  • the cells are released from the wells by trypsinization and then counted using an electronic cell counter.
  • One unit of PDGF activity is defined as that amount of PDGF which stimulates 50% of the cells to divide in 48 h.
  • Assays are also performed to determine the incorporation of 3 H-thymidine.
  • Cells are added to 96- well microcultures plates at a concentration of 1 x 10 4 cells/well in 0.1 ml of Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum (FCS) . Dilutions of PDGF or peptide are added in triplicate to these wells in 0.1 ml of DMEM.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FCS fetal calf serum
  • the plates are incubated for 48 h at ' 37°C, with the cultures being labelled with 0.2 ⁇ Ci 3 H-thymidine during the last 18-24 h.
  • the cultures are then harvested onto glass fiber filters and counted. Results are expressed as the percent increase in 3 H-thymidine incorporation by cells incubated with PDGF or peptide as compared to the cells incubated with media alone.
  • Chemotaxis assays are performed using fibroblasts or smooth muscle cells in a 48-well chemotaxis chamber (NeuroProbe, Cabin John, NJ) according to manufacturer's instructions and as described by W. Falk et al., J. Immunol. Meth. 3_3:239 (1980).
  • Various dilutions of chemotactic solutions are added in triplicate to bottom wells of the chamber in 25 ⁇ l of Hank's Balanced Salt Solution (BSS) containing 2% bovine serum albumin plate and the gasket and top plate is secured on the filter with thumb screws.
  • BSS Hank's Balanced Salt Solution
  • Approximately 3 x 10 4 cells are added to wells in the top chamber in 50 ⁇ l of Hank's BSS containing 2% BSA. After incubation for 2 h at 37°C, the apparatus is disassembled and the nonmigrated cells removed from the top of the filter by gentle scraping. The filter is then fixed with methanol, mounted on a glass slide, and dried. The filter is then stained with Diff-Quick and the number of cells migrating through the filter are counted microscopically. Ten microscope fields are counted per well and the results are expressed as the mean number of migrated cells per microscope field. Alternatively, the amount of stain is measured by determining the optical density. By these uses of these tests, or other similar tests known in the art, one can determine the best activity of the particular material of this invention and thus its most advantageous use.
  • the peptides, salts and derivatives of the present invention find use as medically active agents and as analytical agents. In medical applications they can be administered locally or systemically to promote the healing of wounds, burns and the like. They also can be administered systemically to treat atherosclerosis or to intervene in calcium uptake by cells. For systemic administration one or a mixture of two or more of these peptides may be administered according to any convenient or effective methods for introducing foreign substances into the blood stream of mammals, such as by oral, rectal, nasal, buccal, vaginal, or parenteral routes.
  • the effective dosage level is, for example, 0.01 to 100 mg/kg, preferably about 0.05 to 50 mg/kg. Doses of this size may be administered on a regimen of 1 to 4 times per day.
  • compositions can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable nontoxic carriers.
  • such compositions may be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) administration particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly in semisolid forms such as creams and suppositories; for oral or buccal administration particularly in the form of tablets or capsules; or intranasally particularly in the form of powders, nasal drops or aerosols.
  • parenteral subcutaneous, intramuscular or intravenous
  • vaginal or rectal administration particularly in semisolid forms such as creams and suppositories
  • oral or buccal administration particularly in the form of tablets or capsules
  • the compounds may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. 1975,
  • Formulations for parenteral administration may contain as common excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
  • Formulations for vaginal or rectal administration e.g., suppositories, may contain as excipients, for example, polyalkylene glycols, petroleum jelly, cocoa butter, and the like.
  • Formulations for inhalation administration may be solid and contain as excipients, for example, lactose, or may be aqueous or oily solutions for administration in the form of nasal drops.
  • a pharmaceutically acceptable nontoxic composition can be formed by the incorporation of any of the normally employed excipients, oral dose extenders or carriers such as, for ' example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like.
  • Such compositions can take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained-release formulations, and the like.
  • Such compositions may contain 0.1-95% active ingredient, preferably 1-70%, with the remainder being carrier.
  • the materials may be formulated into lotions, salves and creams with carriers such as water, mineral oil, salve base and the like.
  • the peptides of this invention can be employed as the sole active agent in a pharmaceutical composition or can be used in combination with other active ingredients ' .
  • the peptides of this invention can also be employed as reagents and standards in analytical schemes to detect the presence or quantity of PDGF in samples. To this end they can be used in labeled form in immunoassays such as fluoroim- munoassays, radioi munoassays, ELISA assays, EMIT assays and the like.
  • Example 1 Linear peptides of the invention are synthesized by solid-phase techniques on a Beckman Model 990C automated peptide synthesizer using commercially available t-BOC amino acid polystyrene resin and t-BOC protected amino acids with the following side-chain protecting groups: O-benzyl esters for aspartic acid and glutamic acid; O-benzyl ethers for threonine and serine; tosyl for arginine; DNP for histidine, p-methoxybenzyl or acetamido methyl for cysteine; O-chlorobenzyloxycarbonyl for lysine; and 2,6-dichlorobenzyl for tyrosine.
  • O-benzyl esters for aspartic acid and glutamic acid
  • O-benzyl ethers for threonine and serine
  • tosyl for arginine
  • DNP for histidine, p-methoxybenzyl or
  • Step Reagent or Solvent (min) 1. CH 2 C1 2 x 3 1.5 2. 40% TFA/CH 2 C1 2 prewash 5 3. 40% TFA/CH 2 C1 2 30 4. 1. ,5 5. 80% Isopropanol/CH 2 Cl 2 x 3 1.,5 6. CH 2 C1 2 x 3 1.,5 7. 5% Diisopropylethylamine/CH 2 Cl 2 x 2 10 8. CH 2 C1 2 x 3 1.5 9. Coupling; 3-fold excess of t-BOC amino 120 in CH 2 C1 2 :DMF (9:1; v/v) ; DCC/CH 2 C1 2
  • the peptide is cleaved from the resin using anhydrous hydrogen fluoride in the presence of 10% anisole as scavenger, at 4°C for 1 hr.
  • 10% anisole as scavenger 0.5% dimethyl sulfide is used.
  • the DNP group of His is removed before HF cleavage by treatment with 20-fold excess of thiophenol.
  • the peptides are separated from the various organic side- products by extraction with ether and isolated from the resin by extraction with various concentrations of aqueous acetic acid depending on the solubilities of the peptides.
  • the solutions are diluted with water to about 5% acetic acid concentration and lyophilized.
  • the crude peptides are then purified on Sephadex LH-20. Final purification can be achieved on HPLC using 50 cm/20 mm prep, column packed with Vydac 15-20 micron C lg . Purity of the peptides is checked by analytical HPLC and amino acid analys ' is.
  • This process was used to prepare a peptide corresponding to the 44-51 region of the PDGF "A" chain--T-G-C-C-N-T-S-S (SEQ ID N0:2) .
  • This material was tested to determine its ability to bind to receptors on the 3T3 fibroblast cells. It bound competitively and inhibited the binding of PDGF. The results of the test are given in Fig. 1. The results of the PDGF binding are shown in Fig. 2. This suggests that this peptide can inhibit biological activity of PDGF since cell binding is the initiating event for the induction of biological activity. As such, this peptide can find application as an antiatherosclerotic and antirestenosis agent.
  • This material was also tested for its ability to induce chemotaxis and it showed chemotactic activity. The results of this test are shown in Table 5. This suggests that this peptide can also find application in wound healing.
  • Example 2 The process and testing of Example 1 was repeated this time to make and test a peptide corresponding to the 108-125 region of the PDGF A chain--G-R-P-R-E-S-G-K-K-R-K-R-K-R-L-K-P-T (SEQ ID NO: 1
  • This peptide was found to exhibit chemotactic activity as shown in Table 5. This peptide was also tested for its ability to induce intracellular calcium influx and found to have activity of inducing intracellular calcium uptake. The results of this test are shown in Fig. 3. Induction of intracellular calcium influx by PDGF is shown in Fig. 4. This suggests that this peptide can stimulate cell proliferation and cell growth and find application in wound healing.
  • Example 3 The process of Example 1 was repeated to produce peptide corresponding to the 101-125 region of the PDGA A-chain--D-Y-R-E-E-D-T-G-R-P-R-E-S-G-K-K-R-K-R- K-R-L-K-P-T (SEQ ID NO:10) .
  • This peptide showed cell binding and chemotactic activity. Results are shown in Fig. 5 and Table 5.
  • This extract was concentrated by 5 rotary evaporation at 35°C at 1.0 mm Hg vacuum and reevaporated from water to remove most of the acetic acid.
  • the resulting residue was purified by preparative HPLC using a linear gradient of acetonitrile-water with 0.1% trifluoroacetic acid.
  • the purified product 0 fractions were combined, organics are evaporated off, and the aqueous phase lyophilized.
  • This product was tested for 3T3 receptor-binding activity using a competitive binding assay and found to be very strongly active. The results of this test are shown in Fig. 6.
  • This peptide being a very strong receptor antagonist can inhibit biological activities of PDGF, e.g., mitogenesis and chemotaxis and thereby can find therapeutic use in atherosclerosis. It can also inhibit intracellular calcium uptake and if properly targeted to 0 calcium-dependent cancer cells could intervene in the course of this disease. This peptide when tested alone, induced chemotaxis. The results are shown in Table 5.
  • the peptide was also treated to alter the ends to give free amino and carboxy ends, and amidated carboxy 5 ends. These products were tested with the results given in Table 6.
  • (A) 48 60 is formed.
  • the monomer is formed as in Example 1, but using an ACM-modified cysteine.
  • the product is then treated with iodine in methanol which removes the ACMs
  • Lys Lys Pro lie Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu 1 5 10 15

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Abstract

Peptides and salts and derivatives thereof (e.g., cyclic materials and dimers) having sequences substantially corresponding to PDGF domains exhibit PDGF activity and are useful in therapy and analytical systems.

Description

PEPTIDES HAVING PLATELET-DERIVED GROWTH FACTOR
(PDGF) ACTIVITY
Field of the Invention This invention is in the field of biologically active peptides. These peptides may also be incorporated into other peptides or proteins to yield hybrid, or chimeric, multifunctional molecules. More particularly, it concerns peptides which substantially correspond in sequence to fragments of platelet-derived growth factor ("PDGF") and their preparation and activity as mitogens and as chemotactic agents for fibroblasts, vascular smooth muscle cells and other cells. These activities lead to uses for these peptides as wound healing agents, as agents for treatment of vascular diseases in addition to uses in analytical methods for determining PDGF in samples.
Background Materials PDGF is a 32-kD protein heterodimer composed of
A and B polypeptide chains linked by disulfide bonds. It is stored in the ex-granules of platelets and released when platelets are activated by blood clotting and contact with sites of injury. It stimulates specific target cells by binding to cell-surface receptors, thereby mediating a cascade of events that leads to DNA synthesis and cell proliferation. PDGF is a strong mitogen for fibroblasts, smooth muscle cells, and glial cells. In addition, it is a potent chemotactic factor for neutrophils, onocytes, fibroblasts, and smooth muscle cells. Thus, PDGF plays an important role in the migration of inflammatory cells and connective tissue cells to sites of inflammation and injury and in the repair or restructuring of injured tissues. More especially, PDGF appears to play a part in atherosclerosis. A degenerative disease of the arteries, this condition is characterized by deposition of fatty substances in, and the fibrous thickening of, intima, resulting in the narrowing of the vessel passages and ultimately their hardening and loss of elasticity. The earliest lesion of atherosclerosis is a ubiquitous fatty streak commonly found in -children. This grossly flat, lipid-rich lesion consists of macrophages and some smooth muscle. The fibrous plaque is representative of the various forms of advanced atherosclerosis and is made up of increased internal smooth muscle cells surrounded by connective tissue matrix and containing variable amounts of intracellular and extracellular lipid. In the lumen of the artery, this lesion is generally covered by a dense, fibrous cap of smooth muscle and connective tissue.
Important events in the development of lesions of atherosclerosis involve mainly the injury to endothelial cells -- the initiating event in atherogenesis -- and advance lesions of atherosclerosis. Injured endothelial cells produce several growth factors in culture, including a mitogen resembling platelet- derived growth factor (PDGF) . Platelets contain PDGF, which may be of particular importance in atherogenesis because it is both chemotactic and mitogenic and thus can induce both smooth muscle cell migration and proliferation. It has been demonstrated that if platelets are absent from sites of endothelial injury or if platelet interaction can be pharmacologically prevented, then the intimal proliferative lesions that usually accompany such injury will not occur. Smooth muscle cells are found in both fatty streaks and fibrous plaques. Their proliferation is the key event that determines how extensive fibrous plaques will become. They can form enormous amounts of connective tissue matrix and can accumulate lipid. They contain receptors for PDGF and can respond to a number of chemotactic factors. Thus, the event leading to the development of lesions of atherosclerosis can be represented as: Injury to endothelium i
Release of PDGF i
Migration of smooth muscle cells from the media to intima
I Smooth muscle cell proliferation i Plaque PDGF plays an important role in wound healing.
Although autologous cellular grafts and reconstructed skin are beneficial in certain instances, such costly specialized and time-consuming procedures are not applicable for all clinical situations. Enhancement of wound healing using pharmaceutical agents can be of great benefit.
The cellular events leading to the repopulation of cells and restoration of a wounded area are controlled in large part by specific peptide factors, which regulate the migration and proliferation of cells involved in the repair process. These factors have a variety of cellular sources, including many cell types that are an integral part of the cellular machinery of wound repair. Activated monocytes (macrophages) have been shown to release a PDGF-like activity and to express the C-sis gene and to secrete a PDGF-like activity as well. Pierce, G.F., at al. , J. Exp. Med. , 167, 974 (1988), reported that PDGF and recombinant C-sis gene homodimeric proteins augment in vivo incisional wound healing in rats. PDGF increases collagen formation, DNA content, and protein levels.
The following review articles describe the native PDGF material and its activity (Deuel, Thomas F., Current Topics in Cellular Regulation. 2ϋ:51 (1985); Deuel, Thomas F. et al., J. Clin. Invest. 74;669 (1984)) . It has been reported by Senior, R.M. et al. , J. Cell Biol. 100:351 (1985) that separate domains exist in PDGF for fibroblast chemotactic and mitogenic activity and for onocyte and fibroblast chemotactic activity. This raises the possibility of selectively modifying the biological activities of PDGF. Senior et al. demonstrated that human leukocyte elastase (HLE) abolishes the chemotactic activity of PDGF for fibroblasts but has no effect on the chemotactic activity of monocytes or its mitogenic activity for 3T3 cells or its capacity to bind to 3T3 cells. Cathepsin G has no effect on the chemotactic or mitogenic activities of PDGF, suggesting that the active site of PDGF for chemotaxis is different for different cell types and that different sites on PDGF are involved in its chemotactic activity and mitogenic activity for fibroblasts. Observations of these investigators also suggest that the cleavage produced by HLE is located at or near the N- and C-terminal ends of either the A or B chains since HLE preferentially cleaves at Val-X bonds and, to a lesser extent, at Ala-X bonds. On the other hand, cathepsin G cleaves peptide bonds adjacent to the carboxyl group of phenylalanine, leucine, tyrosine, isoleucine, and methionine residues. The sensitivity of PDGF fibroblasts' chemotactic activity to HLE but not the cathepsin G suggests that the active site for chemotactic activity is located in a small region(s) of the PDGF molecule. Reduction and alkylation of PDGF results in loss of its'mitogenic activity but has no effect on its chemotactic activity, thus suggesting that sulfhydryl groups of cysteine do not affect chemotaxis but do affect mitogenesis. This also indicates that dimers (homo or hetero) or disulfide loop structures are required for mitogenesis. Because p28v"sιs, the transforming protein of the simian sarcoma virus, which is 92% homologous to the A chain of PDGF, has specific mitogenic activity identical with that of PDGF, it is likely that the A chain carries the active site for the mitogenic activity of PDGF. The B chain may contain the active site for chemotactic activity or the A chain may have the active site for both the mitogenic activity and the chemotactic activity.
Previous studies involving the receptors of PDGF have been based on the premise that a single cell- surface receptor binds all three isoforms of PDGF (AA, BB, and AB) . However, Hart et al., Science 240:1529 (1988) recently reported that two populations of PDGF receptor exist and can be distinguished by their ligand- binding specificity. The B receptor binds only the BB dimer, whereas the A/B receptor binds AA, BB, and AB dimers. They also reported that human dermal fibroblasts appear to express seven times more B receptor than A/B receptor. Their observations indicate that B receptor is responsible for most PDGF receptor phosphorylation. Studies of Escobedo et al., Science 240:1532 (1988) and Nature 335:85 (1988) suggest that a common PDGF receptor is activated by homodimeric A and B forms of PDGF. They observed that both forms of PDGF bind to the transfected receptor, stimulate the receptor tyrosine kinase activity, and elicit a mitogenic response of Balb/c 3T3 cells. Their data indicate that the tyrosine kinase and mitogenic responses of the Balb/c 3T3 cells to AA and BB forms of PDGF can be attributed to a single type of receptor and show that the AA form, like the BB form, is a true mitogen.
Another recent study, Khachigian, L.M. et al., J. Biol. Chem. 267, No. 3, 1660 (1992) described an 18 unit peptide corresponding to the 194-211 region of the A chain of PDGF and its ability to bind cultured cells and interact with other important growth factors. All of this prior activity shows that PDGF is an important material which plays a part in many biological processes.
While in some cases it might be possible to obtain, make and/or use the native material to moderate body functions and treat injury or disease or to use this native material in assays, this is not really practical. The native material exists in such small amounts that its isolation on a commercial scale is not feasible. Also, the native material, at 32 kD, is too large to synthesize in any sort of reasonable yield. What is needed is synthetic peptides which exhibit desired PDGF activity but which are significantly shorter and which correspond substantially to a region of the PDGF sequence.
Statement of the Invention
In accord with this invention, it has now been found that peptide fragments of from about 6 to about 26 amino acids in length and having amino acid sequences which have substantial homology to sequences found in the A or B chain of PDGF as well as salts, dimers and derivatives of such fragments can exhibit substantial PDGF-mimicking biological activity.
Accordingly, in one aspect, the invention provides novel peptides corresponding to a sequence having substantial homology to one of the following PDGF A chain sequences:
33-53, 38-46, 44-51, 48-60, 53-63, 55-68, 61-71, 10l-i25, or 106-125. These sequences are shown in Table 1.
In another aspect this invention provides novel active peptides corresponding to or having substantial homology to one of the following PDGF B-chain sequences:
25-42, 30-47, 36-50, 40-50, 45-52, 76-91, 80-95, 84-97, 89-101, 94-109, 98-111, 104-116, 110-122, or 115-128. These sequences are shown in Table 2.
In another aspect this invention provides novel active peptide dimers, peptide ACM derivatives and oxidized peptides corresponding to or having substantial homology to one of the PDGF A-chain sequences shown in Table 3.
In yet another aspect this invention provides novel active peptide dimers, peptide ACM derivatives and oxidized peptides corresponding to or having substantial homology to one of the PDGF B-chain sequences shown in Table 4.
In another aspect, this invention provides hybrid (i.e., chimeric) multifunctional molecules incorporating the peptides of this invention into other peptides and proteins.
In other aspects this invention provides the salts of these peptides, the amide and acyl derivatives of the carbonyl and amino end groups of these peptides and their salts. In addition, any of the active peptides of this invention may be conjugated to peptide polymers and proteins. These immobilizing peptides and proteins are high molecular weight (5 kD to 80 kD) materials such as bovine serum albumin (BSA) , ovalbumin (OVA) , poly(lysine) and the like. The peptides can also be attached to lipophillic moieties such as 12 to 24 carbon atom long saturated and unsaturated hydrocarbon and fatty acid residues.
In an additional aspect this invention concerns labeled versions of these peptides, their salts and derivatives and their application to label-dependent assay methods such as radioimmunoassays (RIA) , fluoroassays, and enzyme-linked immunoabsorbent assays (ELISA) for the determination or assaying of PDGF in laboratory and clinical samples. In addition, non- labeled materials can be used as standards and the like in assay methods.
In further aspects, active peptides, salts, derivatives and hybrids of this invention and particularly those of the B-chain are used as mitogens, and as chemotactic agents and the invention provides pharmaceutical agents based on these materials and provides for their use in wound healing, in the alleviation of the effects of vascular disease or injury, and in the modulation of calcium uptake into cells.
Detailed Description of the Invention
Brief Description of the Drawings This invention will be described with reference being made to the accompanying drawings wherein Figs. 1, 5 and 6 each show results of the competitive binding of 3T3 cells of three peptides of this invention; Figs. 3 and 4 graph the effect of a material of this invention and PDGF on intracellular calcium influx; and Fig. 2 is a graph depicting competitive binding of 3T3 cells by PDGF itself as a standard for comparison. Definitions
In this specification and claims, reference will be made to certain terms which are defined as follows. As used herein:
"Acyl" refers to an alkyl-containing carbonyl group, e.g., R-C(=0)-, wherein R is an alkyl group having from 1 to 8 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, hexyl, octyl and the like. The acyl group usually preferred in this invention is acetyl.
Acyl groups are used to block the terminal amino group of a peptide.
"Amide" refers to an amino-containing group formed to block the terminal carboxyl group of a peptide. The amide group has the structure
-C(=0) -N-R^, where R^ and R2 are each hydrogen or a lower alkyl.
"Conservative substitution" refers to a substitution in a peptide of an amino acid by another amino acid which is similar in chemical and hydrophobicity properties to the original.
"ELISA" refers to an enzyme-1inked immunosorbent assay which employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of antigen or antibody present in a sample. A description of the ELISA technique is found in Chapter 22 of the 4th Edition of Basic and Clinical Immunology by D.P. Sites et al, published by Lange Medical Publications of Los Altos, California, in 1982, which is incorporated herein by reference.
"EMIT" refers to an enzyme-multiplied immunoassay technique which uses (1) an enzyme-labeled hapten, (2) specific antibody to the hapten, (3) pretreatment reagent, (4) buffered-enzyme substrate, and (5) standards to detect the amount of an unknown in a sample. A description of the EMIT technique is found in Enzyme Immunoassav. edited by E.T. Maggio, published in 1980 by CRC' Press, Inc., Boca Raton, Florida, particularly on pp. 141-150, 234-5, and 242-3. These materials are incorporated by reference.
"Fluoroimmunoassay" refers to an antibody-based assay in which the species to be measured binds to, displaces or competes for binding with a material labelled with a fluorescent species in an antibody-ligand complex. In some embodiments of this assay, the complex is separated and the presence or absence of fluorescent species gives a measure of the amount of measured species. In other embodiments, the complex has different fluorescent properties than the uncomplexed fluorescent species so that the formation of the complex can be detected without separation of the complex. A description of fluoroimmunoassay techniques is found in "A Review of Fluoroimmunoassay and Immunofluorometric Assay", D.S. Smith et al. (1981) Ann. Clin. Bioche . (1981) 18:253-274 which is incorporated herein by reference.
"Label" refers to a detectable group in a molecule. Among the common labels are radioactive species useful in radioimmunoassays, fluorescent species useful in fluoroimmunoassays, and enzymatic species useful in the ELISA and EMIT methods and the like.
"Lower alkyl" refers to a straight or branched chain saturated hydrocarbon group having from 1 to 4 carbon atoms such as, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.
"Peptide" or "polypeptide" refers to relatively low molecular weight compounds which yield two or more, such as up to about 30 units of amino acid on hydrolysis. "Pharmaceutically acceptable salt" and "salt" refer to salts that retain the desired antigenic activity of the parent peptide. "Pharmaceutically acceptable salt" refers to salts that are suitable for ingestion or parenteral administration or the like in that they do not impart any undesired toxicological effects. Examples of such salts and pharmaceutically acceptable salts include (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid,- tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid, and the like; (b) salts with monovalent and polyvalent metal cations such as sodium, potassium, zinc, calcium, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or with an organic cation formed from N,N' -dibenzylethylenediamine or ethylenediamine; and (c) combinations of (a) and (b) , e.g., a zinc tannate salt and the like.
"Radioimmunoassay" or "RIA" refers to an antibody-based assay in which the species to be measured binds to, displaces or competes for binding with a radiolabeled material in an antibody-ligand complex. The complex is separated and the presence or absence of radioactivity gives a measure of the amount of measured species.
"Substantially corresponding" or "substantial homology" refers to the property of two amino acid sequences being identical to one another or differing from one another by no more than three amino acid units. Sequences can differ by having a different amino acid at a given position or by having an extra amino acid or by missing an amino acid. Preferably, the sequences have at most two points of difference and more preferably have one difference or are identical. As used herein, the following abbreviations are used for the amino acids described:
Alanine A
Arginine R
Asparagine N Aspartic Acid D
Cysteine C
Glutamine Q
Glutamic Acid E
Glycine G
Histidine H
Isoleucine I
Leucine L
Lysine K
Methionine M
Phenylalanine F
Proline P
Serine S
Threonine T
Tryptophan W Tyrosine Y
Valine V
These represent L-amino acids with the exception of the achiral amino acid glycine. All peptide sequences mentioned herein are written according to the generally accepted convention whereby the N-terminal (or amino-terminal) amino acid is on the left and C-terminal (carboxyl-terminal) amino acid is on the right.
Description of Preferred Embodiments
In one aspect, this invention relates to synthetic peptides and their salts; peptide dimers, and derivatives which have PDGF properties and activity. The synthetic peptides have about 6 to 26 amino acids substantially corresponding to a 6 to 26 amino acid sequence of a PDGF chain.
The peptide can correspond to portions of the A chain in which case they can have sequences substantially corresponding to the peptides shown in Table 1.
The peptides correspond to portions of the B chain in which case they can have sequences substantially corresponding to the peptides shown in Table 2. The peptides can also be in the form of dimers and derivatives substantially corresponding to those A- chain materials shown in Table 3.
The peptides ςan also be in the form of dimers and derivatives substantially corresponding to those B- chain materials shown in Table 4.
Any of these materials can be in the form of pharmaceutically acceptable salts. Labeled forms can be prepared, as well.
Any of these materials can also be incorporated into hybrid, multifunctional molecules. This is done by incorporating these peptide sequences into other peptides or proteins such as hirudin, or the like. These materials can be prepared by direct synthesis as exemplified by Church, F.C. et al., J. Biol. Chem. (1991) 266:11975-11979. This article illustrates the formation and hybrid activity of a hirudin sequence-containing peptide.
Table 1
38 46
V E V K R C- T G C (SEQ ID NO:l)
44 51
T G C C N T S S (SEQ ID NO:2)
48 60
N T S S V K C Q P S R V H (SEQ ID NO:3)
53 63
K C Q P S R V H H R S (SEQ ID N0:4)
55 68
Q P S R V H H R S V K V A K (SEQ ID NO:5)
61 71
H R S V K V A K V E Y (SEQ ID NO:6)
66 78
V A K V E Y V R K K P K L (SEQ ID NO:7)
106 125 D T G R P R E S G K K R K R K R L K P T (SEQ ID NO:8)
33 53
I W P P C V E V K R C T G C C N T S S V K (SEQ ID NO:9)
101 125
D Y R E E D T G R P R E S G K K R K R K R L K P T
(SEQ ID NO:10)
106 125
D T G R P R E S G K K R K R K R L K P T (SEQ ID NO:11)
108 125
G R P R E S G K K R K R K R L K P T (SEQ ID NO:12) Table 2
25 42
I S R R L I.D R T N A N F L V W P P (SEQ ID NO:13)
30 47
I D R T N A N F L V W P P C V E V Q (SEQ ID NO:14)
36 50
N F L V W P P C V E V Q R C S (SEQ ID NO:15)
40 50
W P P C V E V Q R C S (SEQ ID NO:16)
45 52
E V Q R C S G C (SEQ ID NO:17)
76 91
E I V A K K P I F K K A T V T L (SEQ ID NO:18)
80 95
K K P I F K K A T V T L E D H L (SEQ ID NO:19)
84 97 F K K A T V T L E D H L A C (SEQ ID NO:20)
89 101 V T L E D H L A C K C E T (SEQ ID NO: 21)
94 109
H L A C K C E T V A A A R P V T (SEQ ID NO: 22)
98 111
K C E T V A A A R P V T R S (SEQ ID NO: 23)
104 116
A A R P V T R S P G G S Q (SEQ ID NO:24)
110 122
R S P G G S Q E Q R A K T (SEQ ID NO: 25)
115 128
S Q E Q R A K T P Q T R V T (SEQ ID NO:26) Table 3
38 - 46
V E V K R C T G C (SEQ ID NO: 27)
I I
Acm Acm
38 46
V E V K R C T G C (SEQ ID NO:28) I I
38 46
V E V K R C T G C (SEQ ID NO: 29)
I I
V E V K R C T G C (SEQ ID NO: 29)
44 51
T G C C N T S S (SEQ ID NO:30)
AcmAcm
44 51
T G C C N T S S (SEQ ID NO:31)
44 51
T G C C N T S S (SEQ ID NO: 32)
I I
T G C C N T S S (SEQ ID NO: 32)
48 60
N T S S V K C Q P S R V H (SEQ ID NO: 33)
I
Acm
48 60
(N T S S V K C Q P S R V H) 2 (SEQ ID NO: 34)
Table 4
36 ■ 50
N F L V W P P C V E V Q R C S (SEQ ID NO:35)
N F L V W P P C V E V Q R C S (SEQ ID NO:35)
40 50
W P P C V E V Q R C S (SEQ ID NO:36)
W P P C V E V Q R C S (SEQ ID NO:36)
45 52
E V Q R C S G C (SEQ ID NO:37)
E V Q R C I S G CI (SEQ ID NO:37)
94 109
H L A C K C E T V A A A R P V T (SEQ ID NO:38)
H L A C I K CI E T V A A A R P V T (SEQ ID NO:38)
98 111
( C E T V A A A R P V T R S)2 (SEQ ID NO:39)
Acm peptide analogs of 35 and 36 Oxidized analogs of 35, 36, 37 and 38
Conjugates The active peptides of this invention can advantageously be coupled or conjugated to other moieties to enhance cell attachment or to immobilize the peptides. These complexing and conjugating techniques are generally well known in the art and can employ methods and reagents of the art. Immobilization is usually brought about by covalently attaching the peptide to a peptide polymer or protein of 5 kD to 80 kD. BSA and OVA are two well known proteins for this use as are the regular lysine polymers which range in size from about 5 kD to about 30 kD. This immobilization locks the conformation of the peptide and leads to higher activity.
The peptide can be coupled to lypophillic moieties of 6 to 20 carbons or so. These can be fatty acid residues of 12 to 20 carbons such as palmitic, oleic and linoleic acid and the like and alkyl amino acids where the alkyl substituent is from about 6 to 12 carbons in length and typically saturated and linear, such as aminooctanoic acid. The preparation of amino acids carrying these lypophillic groups is shown in Toth, I. et al. , In. Proc. of the 11th Amer. Peptide Symposium, J.E. Rivier and G.R. Marshall (Eds.) ESCOM, Leiden 1990, pp. 1078-1079. All of these adducts are cumulatively described as "conjugates."
General Preparative Techniques
Chemical Synthesis of the Peptide Sequence The peptides may be synthesized by any techniques that are known to those skilled in the peptide art, such as may be found, in Meienhofer, J. , Hormonal Proteins and Peptides. Vol. 2, p. 46, Academic Press, New York, (1973) (for solid phase peptide synthesis) and Schroder, E. et al. , The Peptides. Vol. l, Academic Press, New York, (1965) (for classical solution synthesis) .
These methods comprise sequential addition of amino acids or suitably protected amino acids to a growing peptide chain. Generally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid is contacted with the next amino acid in the sequence having the complimentary (amino or carboxyl) group suitably protected, under conditions suitable for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue and the next amino acid is then added. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support) are removed sequentially or concurrently to afford the final peptide. A_lso, as is well known, it is possible to add more than one amino acid at a time to a growing chain. A preferred method of preparing compounds of the. present invention involves solid phase peptide synthesis. In this method the alpha-amino function of the amino acids is protected by an acid or base-sensitive group. Suitable protecting groups are t- utyloxycarbonyl (Boc) , fluorenyl methoxy carbonyl (FMOC) , benzyloxycarbonyl (Z) , and the like. Side chain active sites are protected, as well, to prevent undesired reactions or couplings. Particularly preferred side chain protecting groups are, for arginine: nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, Z, Boc, and adamantyloxy carbonyl; for lysine: dichloro benzyloxyl carbonyl, t-Boc; for aspartic acid and glutamic acid: o-benzyl, t-butyl; for tyrosine: benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, cyclohexyl, cyclopentyl, and acetyl; for serine and threonme: benzyl, t-butyl and tetrahydropyranyl; for histidine: benzyl, p- toluenesulfonyl and 2,4-dinitrophenyl; and for tryptόphan: formyl.
The carboxyl-terminal amino acid is attached to a suitable solid support. Suitable supports are inert to the reagents and reaction conditions of the reactions, as well as insoluble in the media used. Suitable solid supports include chloromethylpolystyrenedivinylbenzene polymers and the like, especially chloromethylpolystyrene-1% divinylbenzene polymer. . For the special case where the carboxy-terminal amino acid of the peptide becomes an amide [-C(=0) -NH„] , a particularly useful support is the benzhydrylaminopolystyrenedivinyl- benzene polymer described by Vivaille, P. et al. (1971) Helv. Chim.'Acta. !54:2772. The attachment to the chloro-methyl polystyrene-divinylbenzene type of resin is made by means of the reaction of the alpha N-protected amino acid, especially the Boc-amino acid, as its cesium, tetramethylammonium, 4,5-diazabicyclo[5.4.0]undec-5-ene, or similar salt in ethanol, acetonitrile, N,N-dimethylformamide (DMF) , and the like, especially the cesium salt in DMF, with the chloromethyl resins at an elevated temperature, for example between about 40°C and 60°C, preferably about 50°C, for from about 12 to 48 hours, preferably about 24 hours. The alpha N-Boc-amino acid is attached to the benzhydrylamine resin by means of an N,N' -dicyclohexylcarbodiimide (DCC)/
1-hydroxybenzotriazole (HBT) mediated coupling for from about 2 to about 24 hours, preferably about 12 hours at a temperature of between about 10 C and 50 C, preferably 25°C in a solvent such as dichloromethane or DMF, preferably dichloromethane.
The removal of the alpha N-protecting groups may be performed in the presence of, for example, a solution of trifluoroacetic acid in methylene chloride, or other strong acid solution, preferably 50% trifluoroacetic acid in dichloromethane at about ambient temperature. Base-labile protecting groups may be removed by treatment with a base such as piperidine in DMF. Each protected amino acid is preferably introduced in approximately 2.5 molar excess and coupling may be carried out in dichloromethane and the like, especially in dichloromethane at about ambient temperature. The coupling agent is normally DCC in dichloromethane but may be N,N' -diisopropylcarbodiimide or other carbodiimide either alone or in the presence of HOBT, N-hydroxysuccinimide, other N-hydroxyimides or oximes. Alternatively, protected amino acid active esters (e.g., p-nitrophenyl, pentafluorophenyl and the like) or symmetrical' anhydrides may be used. At the end of the solid phase synthesis, the peptide is either carried through another deprotection and neutralization cycle followed by acylation, preferably acetylation with acetic anhydride to yield an N-acetyl (N-Ac) blocked amino end group, or it may be removed from the resin directly. If the carboxy
[-C(=0)-0H] terminal is to be blocked as the amide, the peptide may be either synthesized on the benzhydrylamino-polystyrene resin, which gives the amide directly, or it may be removed from the resin by ammonolysis with, for example, ammonia/methanol or a monia/ethanol, at a temperature of from about 0° to about 50°C, preferably about 25°C for about 12 to about 48 hours, preferably about 18 hours. If a peptide with a free amino-terminal and a carboxyl-terminal is desired, the peptide may be directly removed from the resin by treatment with anhydrous liquid hydrogen fluoride in the presence of a radical scavenger such as anisole. The amino or carboxyl-blocked (protected) peptides, either on the resin or removed from the resin by ammonolysis, are similarly deprotected by treatment with anhydrous liquid hydrogen fluoride. In cases where base-labile protection of the alpha N function is used in conjunction with t-butyl-based side chain protection, the final resin removal and deprotection step may be performed with trifluoroacetic acid.
Other means of removal of the (side chain) protecting groups from the polypeptide are treatment with hydrogen fluoride/pyridine complex, treatment with tris(trifluoroacetyl)boron and trifluoroacetic acid, by reduction with hydrogen and palladium on carbon or polyvinylpyrrolidone, or by reduction with sodium in liquid ammonia or with liquid hydrogen fluoride plus anisole at a temperature between about -10 and +10 C, preferably about 0 C, for between about 15 minutes and 1 hour, preferably about 30 minutes. The latter treatment (HF/anisole) may be used for simultaneous cleavage from the resin and deprotection to yield free-C0_H end groups when a normal benzylester linkage has been used or to form a C0-NH„ (amide) end groups when a benzhydrylamino linkage has been used. For the amide terminal peptides on the benzhydrylamine resins, the resin cleavage and deprotection steps may be combined in a single step utilizing liquid HF/anisole as described above. The fully protected polypeptide can then be purified by chromatographic steps.
Salt Formation
The peptides can be obtained as salts, by simple adjustment of the pH of the medium from which they are finally recovered with acids or bases corresponding to the desired counter ions.
Preparing Labeled Versions of the Peptides Radiolabeled versions of the polypeptides can be produced in several manners. For one, commercially
14 available carbon-labeled ammo acids can be employed in the synthesis of the polypeptides. Similarly 3H-amino acids can be prepared by the magnesium oxide procedure of Schwyzer et al. (1959) Helv. Clin. Acta. 42 2622. Except for the precautions routinely associated with radiochemicals, these processes can follow the usual synthesis route. Alternatively, the finished polypeptide or conjugate can be radiolabeled by tritium exchange. Enzy e labels can be incorporated by using an enzymic carrier for forming conjugates o.r by attaching an enzymatically active group to the carrier or the peptide.
Synthesis of the Dimers
Parallel dimers are synthesized by using acetamido methyl group (ACM) on appropriate cysteine residues, thereby selectively forming disulfide bonds to put the peptide chains in registered dimeric form. The ACM group is resistant to HF and can be removed with iodine in ethanol, with simultaneous oxidation to a disulfide bond.
Synthesis of Cyclic Derivatives The cyclic .disulfides can be formed by air oxidation of a dilute solution of the corresponding linear peptide in water. After HF cleavage and extraction of the peptide off of the resin, the resulting solution is adjusted to pH 8.1 by addition of concentrated NH4OH in water and then shaken slowly on a shaker for 5-10 days. At the end of this time or during it, an Ellman test can be performed to confirm the completion of the disulfide bond formation. The peptide solution is adjusted to pH 6.5 by addition of a weak acid such as acetic acid. It is then passed through an acetic acid-treated and water-washed resin column. After washing the column with water, the peptide can be extracted with aqueous acetic acid. This extract can be concentrated such as by rotary evaporation at 35°C at 1.0 mm Hg vacuum and reevaporated from water to remove most of the acetic acid. The resulting residue can be purified by preparative HPLC using a linear gradient of acetonitrile-water with 0.1% trifluoroacetic acid. The purified product fractions can be combined, organics evaporated off, and the aqueous phase lyophilized. Assays for Biological Activity
All the peptides and peptide analogs of this invention can be tested to determine their most advantageous PDGF-mimicking activity. As already noted, a major advantage of the peptides of this invention is their ability to exhibit only one or only a portion of the several activities ascribed to native PDGF. In a preferred testing approach, they are first tested for their ability to bind to PDGF receptors on the surface of Balb/C 3TC fibroblasts by a competitive inhibition radioimmunoassay (RIA) by the test of R.M. Senior et al., J. Cell Biol. 100:351 (1988) . All the peptides are then tested for their ability to stimulate mitogenesis and to induce chemotaxis. Biological assays on active peptides can be carried out on cultured smooth muscle cells (SMC) . Rat aorta cells can be obtained from ATCC (CRL1476) , or alternatively, isolated and cultured by the procedure described by J. Nakao et al., Atherosclerosis 43 : 143 (1982) and R. Ross, J. Cell Biol. 50:172 (1971) . SMCs can be isolated from media explants of thoracic aortas from male Wistar rats 6 to 8 weeks old. In culture, the cells begin to grow and migrate out of the explants after 10 to 14 days. Cells are then subcultured at a 1 to 2 split ratio when they become confluent and are maintained in tissue culture media (minimal essential medium [MEM] ) containing 10% fetal bovine serum (FBS) .
Cell Binding Assay Fibroblasts or smooth muscle cells are grown in
2 -well tissue culture plates in a 1:1 mixture of Dulbecco's modified Vogt's medium and Ham's F12 medium. When the cells reach confluence (3-4 days) , the plates are washed with cold phosphate-buffered saline (PBS) and then filled with 1 ml of binding serum (PBS + 1% human serum albumin) containing various concentrations of synthetic peptide or PDGF standards (in triplicate) . After 3 h incubation at 4°C, approximately 5 ng of 125i- labelled PDGF is added to each well, and the plates are incubated for an additional 3 h at 4°C. After three washes with binding medium, the cell monolayers are solubilized with 1 ml of 1% Triton X-100 for 20 in at room temperature. The amount of radioactivity contained in the lysates is determined on a Packard Gamma Counting System and the results are expressed as mean counts per well. Fibroblast growth factor (FGF) and epidermal growth factor (EGF) are used as controls because they do not compete with the binding of PDGF. J.S. Huang et al., J. Biol. Chem. 257:8130 (1982).
Assay for Mitogenic Activity The mitogenic activity of peptides is determined according to the procedure described by G.R. Grotendorst et al., J. Cell Physiol. 113:261 (1982). Fibroblasts or smooth muscle cells form primary explant cultures are trypsinized and resuspended in Dulbecco's Modified Eagle's Medium (DMEM) with 10% calf serum prepared from platelet-free plasma. The cells are then pelleted at a density of 6 x 104 cells/well in Costar microtissue culture wells (6.4 mm diameter). After 24 h, various dilutions of PDGF or peptides are added to the wells and the cells are incubated for an additional 48 h. The cells are released from the wells by trypsinization and then counted using an electronic cell counter. One unit of PDGF activity is defined as that amount of PDGF which stimulates 50% of the cells to divide in 48 h. Assays are also performed to determine the incorporation of 3H-thymidine. Cells are added to 96- well microcultures plates at a concentration of 1 x 104 cells/well in 0.1 ml of Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal calf serum (FCS) . Dilutions of PDGF or peptide are added in triplicate to these wells in 0.1 ml of DMEM. The plates are incubated for 48 h at'37°C, with the cultures being labelled with 0.2 μCi 3H-thymidine during the last 18-24 h. The cultures are then harvested onto glass fiber filters and counted. Results are expressed as the percent increase in 3H-thymidine incorporation by cells incubated with PDGF or peptide as compared to the cells incubated with media alone.
Assay for Chemotactic Activity Chemotaxis assays are performed using fibroblasts or smooth muscle cells in a 48-well chemotaxis chamber (NeuroProbe, Cabin John, NJ) according to manufacturer's instructions and as described by W. Falk et al., J. Immunol. Meth. 3_3:239 (1980). Various dilutions of chemotactic solutions are added in triplicate to bottom wells of the chamber in 25 μl of Hank's Balanced Salt Solution (BSS) containing 2% bovine serum albumin plate and the gasket and top plate is secured on the filter with thumb screws. Approximately 3 x 104 cells are added to wells in the top chamber in 50 μl of Hank's BSS containing 2% BSA. After incubation for 2 h at 37°C, the apparatus is disassembled and the nonmigrated cells removed from the top of the filter by gentle scraping. The filter is then fixed with methanol, mounted on a glass slide, and dried. The filter is then stained with Diff-Quick and the number of cells migrating through the filter are counted microscopically. Ten microscope fields are counted per well and the results are expressed as the mean number of migrated cells per microscope field. Alternatively, the amount of stain is measured by determining the optical density. By these uses of these tests, or other similar tests known in the art, one can determine the best activity of the particular material of this invention and thus its most advantageous use.
Utility and Administration
The peptides, salts and derivatives of the present invention find use as medically active agents and as analytical agents. In medical applications they can be administered locally or systemically to promote the healing of wounds, burns and the like. They also can be administered systemically to treat atherosclerosis or to intervene in calcium uptake by cells. For systemic administration one or a mixture of two or more of these peptides may be administered according to any convenient or effective methods for introducing foreign substances into the blood stream of mammals, such as by oral, rectal, nasal, buccal, vaginal, or parenteral routes. The effective dosage level is, for example, 0.01 to 100 mg/kg, preferably about 0.05 to 50 mg/kg. Doses of this size may be administered on a regimen of 1 to 4 times per day.
Peptides here provided can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable nontoxic carriers. As mentioned above, such compositions may be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) administration particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly in semisolid forms such as creams and suppositories; for oral or buccal administration particularly in the form of tablets or capsules; or intranasally particularly in the form of powders, nasal drops or aerosols. The compounds may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. 1975, incorporated by reference. Formulations for parenteral administration may contain as common excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Formulations for vaginal or rectal administration, e.g., suppositories, may contain as excipients, for example, polyalkylene glycols, petroleum jelly, cocoa butter, and the like. Formulations for inhalation administration may be solid and contain as excipients, for example, lactose, or may be aqueous or oily solutions for administration in the form of nasal drops. For buccal administration typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinated starch, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition can be formed by the incorporation of any of the normally employed excipients, oral dose extenders or carriers such as, for' example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. Such compositions can take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Such compositions may contain 0.1-95% active ingredient, preferably 1-70%, with the remainder being carrier.
For local administration, the materials may be formulated into lotions, salves and creams with carriers such as water, mineral oil, salve base and the like. The peptides of this invention can be employed as the sole active agent in a pharmaceutical composition or can be used in combination with other active ingredients'. In addition to these medical uses, the peptides of this invention can also be employed as reagents and standards in analytical schemes to detect the presence or quantity of PDGF in samples. To this end they can be used in labeled form in immunoassays such as fluoroim- munoassays, radioi munoassays, ELISA assays, EMIT assays and the like.
Examples The following examples serve to illustrate the invention. They should not be construed as narrowing it, or limiting its scope.
Example 1 Linear peptides of the invention are synthesized by solid-phase techniques on a Beckman Model 990C automated peptide synthesizer using commercially available t-BOC amino acid polystyrene resin and t-BOC protected amino acids with the following side-chain protecting groups: O-benzyl esters for aspartic acid and glutamic acid; O-benzyl ethers for threonine and serine; tosyl for arginine; DNP for histidine, p-methoxybenzyl or acetamido methyl for cysteine; O-chlorobenzyloxycarbonyl for lysine; and 2,6-dichlorobenzyl for tyrosine. All couplings are performed using a 3-molar excess of t-BOC amino acid and dicyclohexylcarbodiimide (DCC) over the number of milliequivalents of amino acid on the resin. In the cases of Asn and Gin, a 3-molar excess of t-BOC- amino acid, DCC, and hydroxybenzotriazole (HOBT) is used. All couplings are monitored by the ninhydrin test. 40% TFA-CH2C12 containing 10% anisole and 0.1% indole as scavenger is used for BOC deprotection. The details of the synthetic cycle are as follows:
' SCHEDULE OF EVENTS FOR ASSEMBLING A PEPTIDE ON A RESIN
Time
Step Reagent or Solvent (min) 1. CH2C12 x 3 1.5 2. 40% TFA/CH2C12 prewash 5 3. 40% TFA/CH2C12 30 4. 1. ,5 5. 80% Isopropanol/CH2Cl2 x 3 1.,5 6. CH2C12 x 3 1.,5 7. 5% Diisopropylethylamine/CH2Cl2 x 2 10 8. CH2C12 x 3 1.5 9. Coupling; 3-fold excess of t-BOC amino 120 in CH2C12:DMF (9:1; v/v) ; DCC/CH2C12
10. CH2C12 x 3 1.5
11. 80% Isopropanol/CH2Cl2 x 3 1.5
After completion of the synthesis, the peptide is cleaved from the resin using anhydrous hydrogen fluoride in the presence of 10% anisole as scavenger, at 4°C for 1 hr. In the case of methionine, cysteine, and tryptophan, 0.5% dimethyl sulfide is used.
The DNP group of His is removed before HF cleavage by treatment with 20-fold excess of thiophenol. The peptides are separated from the various organic side- products by extraction with ether and isolated from the resin by extraction with various concentrations of aqueous acetic acid depending on the solubilities of the peptides. The solutions are diluted with water to about 5% acetic acid concentration and lyophilized. The crude peptides are then purified on Sephadex LH-20. Final purification can be achieved on HPLC using 50 cm/20 mm prep, column packed with Vydac 15-20 micron Clg. Purity of the peptides is checked by analytical HPLC and amino acid analys'is. This process was used to prepare a peptide corresponding to the 44-51 region of the PDGF "A" chain--T-G-C-C-N-T-S-S (SEQ ID N0:2) . This material was tested to determine its ability to bind to receptors on the 3T3 fibroblast cells. It bound competitively and inhibited the binding of PDGF. The results of the test are given in Fig. 1. The results of the PDGF binding are shown in Fig. 2. This suggests that this peptide can inhibit biological activity of PDGF since cell binding is the initiating event for the induction of biological activity. As such, this peptide can find application as an antiatherosclerotic and antirestenosis agent. This material was also tested for its ability to induce chemotaxis and it showed chemotactic activity. The results of this test are shown in Table 5. This suggests that this peptide can also find application in wound healing.
Table 5 CHEMOTACTIC ACTIVITY OF PEPTIDES
Figure imgf000034_0001
aPeptides were considered positive when their OD was sig¬ nificantly higher than that of the media.
Table 6
COMPETITIVE INHIBITION FOR RECEPTOR BINDING
ON 3T3 CELLS BY ANALOGS OF PDGF B (45-52) CYCLIC PEPTIDE
Figure imgf000035_0002
Figure imgf000035_0001
Table 7
COMPETITIVE INHIBITION FOR RECEPTOR BINDING ON 3T3 CELLS BY PDGF PEPTIDES AND THEIR BSA CONJUGATES
Cone. Max.
Figure imgf000036_0001
Peptide concentration per milligram of conjugate. Example 2 The process and testing of Example 1 was repeated this time to make and test a peptide corresponding to the 108-125 region of the PDGF A chain--G-R-P-R-E-S-G-K-K-R-K-R-K-R-L-K-P-T (SEQ ID
NO:12) . This peptide was found to exhibit chemotactic activity as shown in Table 5. This peptide was also tested for its ability to induce intracellular calcium influx and found to have activity of inducing intracellular calcium uptake. The results of this test are shown in Fig. 3. Induction of intracellular calcium influx by PDGF is shown in Fig. 4. This suggests that this peptide can stimulate cell proliferation and cell growth and find application in wound healing.
Example 3 The process of Example 1 was repeated to produce peptide corresponding to the 101-125 region of the PDGA A-chain--D-Y-R-E-E-D-T-G-R-P-R-E-S-G-K-K-R-K-R- K-R-L-K-P-T (SEQ ID NO:10) . This peptide showed cell binding and chemotactic activity. Results are shown in Fig. 5 and Table 5.
Example 4
I 1
The cyclic disulfide E-V-Q-R-C-S-G-C (SEQ ID
NO:17) was formed by air oxidation of a dilute solution of B-chain 45-52 peptide in water. After HF cleavage of the peptide off of the synthesis resin and extraction, the resulting solution is adjusted to pH 8.1 by addition of 50% concentrated NH4OH in water and then shaken slowly on a shaker for 7 days. At the end of this time, an Ellman test was performed to confirm the completion of the disulfide bond formation. The peptide solution was adjusted to pH 6.5 by addition of a few drops of acetic acid. It was then passed through an acetic acid-treated and water-washed Biorex 70 resin column. After washing the column with water, the peptide was extracted into 50% aqueous acetic acid. This extract was concentrated by 5 rotary evaporation at 35°C at 1.0 mm Hg vacuum and reevaporated from water to remove most of the acetic acid. The resulting residue was purified by preparative HPLC using a linear gradient of acetonitrile-water with 0.1% trifluoroacetic acid. The purified product 0 fractions were combined, organics are evaporated off, and the aqueous phase lyophilized. This product was tested for 3T3 receptor-binding activity using a competitive binding assay and found to be very strongly active. The results of this test are shown in Fig. 6. 5 This peptide being a very strong receptor antagonist can inhibit biological activities of PDGF, e.g., mitogenesis and chemotaxis and thereby can find therapeutic use in atherosclerosis. It can also inhibit intracellular calcium uptake and if properly targeted to 0 calcium-dependent cancer cells could intervene in the course of this disease. This peptide when tested alone, induced chemotaxis. The results are shown in Table 5.
The peptide was also treated to alter the ends to give free amino and carboxy ends, and amidated carboxy 5 ends. These products were tested with the results given in Table 6.
Example 5
The peptide dimer
30 (N T S S V K C Q P S R V H)2 (SEQ ID NO:3)
(A) 48 60 is formed. The monomer is formed as in Example 1, but using an ACM-modified cysteine. The product is then treated with iodine in methanol which removes the ACMs
_R and gives rise to the desired cysteine-linked dimer. The dimer is recovered and purified.
Example 6 Conjugation of Peptides to Bovine Serum Albumin (BSA) Peptides were conjugated to BSA by the method of Atassi, M.Z. et al. Biochimia. Biophysica Acta. 670:300-302 (1981). BSA (100 mg) was dissolved in 0.025 M borate buffer (20 mL) , pH 9.3. To this a solution of succinic anhydride (540 mg) in dioxane (10 mL) was added in small aliquots over a period of 30 min and the reaction mixture was stirred magnetically while maintaining the pH at 9.3 by the addition of 3 M NaOH. Following the last addition of succinic anhydride the acylation reaction was allowed to continue for 3 hr. The solution was then dialyzed against four changes of 0.01 M triethylamme (Et3N) , freeze-dried and finally dried in a desiccator over P205.
Succinylated BSA (14 mg) was suspended in 1 mL of anhydrous DMF and the suspension stirred magnetically for 4 hr while shielding from direct light. p-Nitro- phenol (6.5 mg) was added. The mixture was stirred for 15 min after which 5 mg of dicyclohexylcarbodiimide was added. The reaction mixture was allowed to stir at room temperature for 3 hr. To this was added 10 mg of peptide and 100 μL of Et3N. The reaction mixture was stirred at room temperature for 24 hr while protected from direct light. After this it was diluted with 3 mL of water and dialyzed extensively against distilled water and lyophilized. The process was used to form several conjugates which were found to be active. The results of these tests are given in Table 7. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: SRI INTERNATIONAL
(ii) TITLE OF INVENTION: PEPTIDES CORRESPONDING TO ACTIVE DOMAINS OF PLATELET-DERIVED GROWTH FACTOR (PDGF)
(iii) NUMBER OF SEQUENCES: 39
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SRI INTERNATIONAL
ATTN: INTELLECTUAL PROPERTY COUNSEL
(B) STREET: 333 Ravenswood Avenue
(C) CITY: Menlo Park
(D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 94025
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/894,497
(B) FILING DATE: 05-JUN-1992
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: CLARK, JANET P.
(B) REGISTRATION NUMBER: 34,799
(C) REFERENCE/DOCKET NUMBER: PCT-2679
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 859-2446
(B) TELEFAX: (415) 859-3880
(C) TELEX: 334486
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NOil:
Val Glu Val Lys Arg Cys Thr Gly Cys 1 5
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Thr Gly Cys Cys Asn Thr Ser Ser
1 5
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:3:
Asn Thr Ser Ser Val Lys Cys Gin Pro Ser Arg Val His
1 5 10
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Lys Cys Gin Pro Ser Arg Val His His Arg Ser 1 5 10
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Gin Pro Ser Arg Val His His Arg Ser Val Lys Val Ala Lys 1 5 10
(2) INFORMATION FOR SEQ ID NO:6: —
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
His Arg Ser Val Lys Val Ala Lys Val Glu Tyr 1 5 10
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Val Ala Lys Val Glu Tyr Val Arg Lys Lys Pro Lys Leu 1 5 10
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Asp Thr Gly Arg Pro Arg Glu Ser Gly Lys Lys Arg Lys Arg Lys Arg 1 5 10 15
Leu Lys Pro Thr 20
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:9: lie Trp Pro Pro Cys Val Glu Val Lys Arg Cys Thr Gly Cys Cys Asn 1 5 10 15
Thr Ser Ser Val Lys 20
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:10:
Asp Tyr Arg Glu Glu Asp Thr Gly Arg Pro Arg Glu Ser Gly Lys Lys 1 5 10 15
Arg Lys Arg Lys Arg Leu Lys Pro Thr 20 25
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Asp Thr Gly Arg Pro Arg Glu Ser Gly Lys Lys Arg Lys Arg Lys Arg 1 5 10 15
Leu Lys Pro Thr 20
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Gly Arg Pro Arg Glu Ser Gly Lys Lys Arg Lys Arg Lys Arg Leu Lys 1 5 10 15
Pro Thr
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: lie Ser Arg Arg Leu lie Asp Arg Thr Asn Ala Asn Phe Leu Val Trp 1 5 10 15
Pro Pro
(2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: lie Asp Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu 1 5 10 15
Val Gin
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gin Arg Cys Ser 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Trp Pro Pro Cys Val Glu Val Gin Arg Cys Ser 1 5 10
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Glu Val Gin Arg Cys Ser Gly Cys 1 5
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Glu lie -Val Ala Lys Lys Pro lie Phe Lys Lys Ala Thr Val Thr Leu 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Lys Lys Pro lie Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu Ala Cys 1 5 10
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Val Thr Leu Glu Asp His Leu Ala Cys Lys Cys Glu Thr 1 5 10
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
His Leu Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser 1 5 10
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Ala Ala Arg Pro Val Thr Arg Ser Pro Gly Gly Ser Gin 1 5 10
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: 1inear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Arg Ser Pro Gly Gly Ser Gin Glu Gin Arg Ala Lys Thr 1 5 10
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Ser Gin Glu Gin Arg Ala Lys Thr Pro Gin Thr Arg Val Thr 1 5 10 (2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "This position is Acm-modified. "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 9
(D) OTHER INFORMATION: /note= "This position is Acm-modified. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Val Glu Val Lys Arg Cys Thr Gly Cys 1 5
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: both
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Val Glu Val Lys Arg Cys Thr Gly Cys 1 5
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "This position has disulfide bond to corresponding position on identical sequence."
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 9
(D) OTHER INFORMATION: /note= "This position has disulfide bond to corresponding position of identical sequence. " (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Val Glu Val Lys Arg Cys Thr Gly Cys 1 5
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION: /note= "This position is modified with Acm. "
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= "This position is modified with Acm. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Thr Gly Cys Cys Asn Thr Ser Ser 1 5
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: both
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:31:
Thr Gly Cys Cys Asn Thr Ser Ser 1 5
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 3..4
(D) OTHER INFORMATION: /note= "This position has disulfide bond to corresponding position of identical sequence. " (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Thr Gly Cys Cys Asn Thr Ser Ser
1 5
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(i ) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(D) OTHER INFORMATION: /note= "This position modified with Acm."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Asn Thr Ser Ser Val Lys Cys Gin Pro Ser Arg Val His 1 5 10
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Asn Thr Ser Ser Val Lys Cys Gin Pro Ser Arg Val His 1 5 10
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 8
(D) OTHER INFORMATION: /note= "This position has disulfide bond to corresponding position of identical sequence. "
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 14
(D) OTHER INFORMATION: /note= "This position has disulfide bond to corresponding position of identical sequence." (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gin Arg Cys Ser 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= "This position has disulfide bond to corresponding position on identical sequence. "
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 10
(D) OTHER INFORMATION: /note= "This position has disulfide bond to corresponding position on identical sequence. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Trp Pro Pro Cys Val Glu Val Gin Arg Cys Ser 1 5 10
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 5
(D) OTHER INFORMATION: /note= "This position has disulfide bond to corresponding position on identical sequence. "
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 8
(D) OTHER INFORMATION: /note= "This position has disulfide bond to corresponding position of identical sequence. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Glu Val Gin Arg Cys Ser Gly Cys 1 5 (2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids (B)' TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 4
(D) OTHER INFORMATION: /note= "This position has disulfide bond to corresponding position on identical sequence. "
(ix) FEATURE:
(A) NAME/KEY: Disulfide-bond
(B) LOCATION: 6
(D) OTHER INFORMATION: /note= "This position has disulfide bond to corresponding position on identical sequence. "
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
His Leu Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr 1 5 10 15
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser Lys Cys 1 5 10 15
Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Ser 20 25

Claims

Claims:
1. A PDGF-activity-displaying peptide or peptide salt having a peptide sequence substantially corresponding to a PDGF A-chain sequence selected from the group consisting of
38 46
V E V K R C T G C (SEQ ID N0:1)
44 51 T G C C N T S S (SEQ ID NO:2)
48 60
N T S S V K C Q P S R V H (SEQ ID NO:3)
53 63
K C Q P S R V H H R S (SEQ ID NO:4)
55 68
Q P S R V H H R S V K V A K (SEQ ID NO:5)
61 71
H R S V K V A K V E Y (SEQ ID NO:6)
66 78
V A K V E Y V R K K P K L (SEQ ID NO:7)
106 125
D T G R P R E S G K K R K R K R L K P T (SEQ ID NO:8)
33 53
I W P P C V E V K R C T G C C N T S S V K (SEQ ID NO:9)
101 125
D Y R E E D T G R P R E S G K K R K R K R L K P T
(SEQ ID NO: 10)
106 125
D T G R P R E S G K K R K R K R L K P T (SEQ ID NO:11)
108 125
G R P R E S G K K R K R K R L K P T (SEQ ID NO:12)"
2. The peptide of claim 1.
3. The peptide salt of claim 1.
4. The peptide or peptide salt of claim 1 in conjugation with a moiety selected from the group consisting of protein, peptide polymer and a lipophilic group.
5. A PDGF-activity-displaying peptide, peptide salt or peptide conjugate having a peptide sequence substantially corresponding to a PDGF B-chain sequence selected from the group consisting of
25 42
I S R R L I D R T N A N F L V W P P (SEQ ID NO:13)
30 47
I D R T N A N F L V W P P C V E V Q (SEQ ID NO:14)
36 50
N F L V W P P C V E V Q R C S (SEQ ID NO:15)
40 50
W P P C V E V Q R C S (SEQ ID NO:16)
45 52
E V Q R C S G C (SEQ ID NO:17)
76 91
E I V A K K P I F K K A T V T L (SEQ ID NO:18)
80 95
K K P I F K K A T V T L E D H L (SEQ ID NO: 19)
84 97
F K K A T V T L E D H L A C (SEQ ID NO:20)
89 101
V T L E D H L C K C E T (SEQ ID NO:21)
94 109 H L A C K C E T V A A A R P V T (SEQ ID NO:22)
98 111
K C E T V A A A R P V T R S (SEQ ID NO:23)
104 116
A A R P V T R S P G G S Q (SEQ ID NO:24)
110 122
R S P G G S Q E Q R A K T (SEQ ID NO:25)
115 128
S Q E Q R A K T P Q T R V T (SEQ ID NO:26)
6. The peptide of claim 5.
7. The peptide salt of claim 5.
8. The peptide or peptide salt of claim 5 in conjugation with a moiety selected from the group consisting of protein, peptide polymer and a lipophilic group.
9. A PDGF-activity-displaying peptide or peptide dimer or derivative, conjugate or salt having a peptide sequence substantially corresponding to a PDGF A- chain sequence selected from the group consisting of
38 46
V E V K R C T G C (SEQ ID NO:27)
Figure imgf000054_0001
38 46 V E V K R C T G C (SEQ ID NO:28)
38 46
V E V K R C T G C (SEQ ID NO:29)
V E V K R C T G C (SEQ ID NO:29) 44 51
T G C C N T S S (SEQ ID NO: 30)
I I
AcmAcm
44 51
T G C C N T S S (SEQ ID NO:31)
44 51
T G C C N T S S (SEQ ID NO:32)
T G C I CI N T S S (SEQ ID NO:32)
48 60
N T S S V K C Q P S R V H (SEQ ID NO:33)
I Acm
48 60
(N T S S V K C Q P S R V H)2 (SEQ ID NO:34)
10. The peptide of claim 9.
11. The peptide salt of claim 9.
12. The peptide or peptide salt of claim 9 in conjugation with a moiety selected from the group consisting of protein, peptide polymer and a lipophilic grou .
13. A PDGF-activity-displaying peptide or peptide dimer or derivative or salt or conjugate having a peptide sequence substantially corresponding to a PDGF B- chain sequence selected from the group consisting of
36 50
N F L V W P P C V E V Q R C S (SEQ ID NO:35)
N F L V W P P C I V E V Q R CI S (SEQ ID NO:35)
40 50 W P P C V E V Q R C S (SEQ ID NO:36)
W P P C I V E V Q R CI S (SEQ ID NO:36)
45 52
E V Q R C S G C (SEQ ID NO:37) E V Q R C I S G CI (SEQ ID NO:37)
94 109
H L A C K C E T V A A A R P V T (SEQ ID NO: 38) i I
H L A C K C E T V A A A R P V T (SEQ ID NO: 38)
98 111
(K C E T V A A A R P V T R S)2 (SEQ ID NO: 39) .
14. The peptide of claim 13.
15. The peptide salt of claim 13.
16. The peptide or peptide salt of claim 13 in conjugation with a moiety selected from the group consisting of protein, peptide polymer and a lipophilic grou .
17. An ACM derivative of the peptide of claim 14.
18. An oxidized analog of a peptide sequence substantially corresponding to a PDGF B-chain sequence selected from the group consisting of
45 52
E V Q R C S G C (SEQ ID NO:37)
I I
E V Q R C S G C (SEQ ID NO:37)
94 . 109
H L A C K C E T V A A A R P V T (SEQ ID NO:38)
H L A C K C E T V A A A R P V T (SEQ ID NO:38)
19. A multifunctional hybrid molecule comprising a peptide or peptide salt of claim 1, claim 5, claim 9 or claim 13 in hybrid combination with another peptide or protein.
20. A method for achieving a PDGF-related effect in a patient comprising administering to said patient a material selected from the group consisting of a peptide of claim 1, 5, 9 and 13, a peptide salt of claim 1, 5, 9, and 13 and a peptide conjugate of claim 4, 8, 12 and 16 a peptide derivative of claim 17 and an oxidized analog of claim 18.
PCT/US1993/005325 1992-06-05 1993-06-03 Peptides having platelet-derived growth factor (pdgf) activity WO1993025576A2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP6501593A JPH07508510A (en) 1992-06-05 1993-06-03 Platelet-derived growth factor (PDGF) activity

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US89449792A 1992-06-05 1992-06-05
US07/894,497 1992-06-05

Publications (2)

Publication Number Publication Date
WO1993025576A2 true WO1993025576A2 (en) 1993-12-23
WO1993025576A3 WO1993025576A3 (en) 1994-04-14

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Country Status (3)

Country Link
JP (1) JPH07508510A (en)
CA (1) CA2137367A1 (en)
WO (1) WO1993025576A2 (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995011259A1 (en) * 1993-10-22 1995-04-27 Ellerman Pharmaceuticals Limited Platelet-derived growth factor analogues
US5952304A (en) * 1993-10-22 1999-09-14 Trigen Limited Platelet-derived growth factor analogues
EP2419117A4 (en) * 2009-04-13 2014-10-22 Elc Man Llc Methionine sulfoxide peptide, compositions and methods of use
WO2014207534A3 (en) * 2013-06-25 2015-04-09 Sépia Pesquisa E Desenvolvimento Bradykinin receptor modulators and use thereof
PL425038A1 (en) * 2018-03-26 2019-10-07 Uniwersytet Gdański New peptide derivatives of platelet-derived growth factor (PDGF), method for obtaining, pharmaceutical composition and application
WO2024236323A1 (en) * 2023-05-17 2024-11-21 Ucl Business Ltd Therapeutic pdgf-a molecules

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE131532T1 (en) * 1986-08-13 1995-12-15 Zymogenetics Inc EXPRESSION OF BIOLOGICALLY ACTIVE PDGF ANALOGUES IN EUKARYOTIC CELLS
WO1992011364A1 (en) * 1990-12-21 1992-07-09 Creative Biomolecules, Inc. Biosynthetic pdgf antagonists

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995011259A1 (en) * 1993-10-22 1995-04-27 Ellerman Pharmaceuticals Limited Platelet-derived growth factor analogues
US5952304A (en) * 1993-10-22 1999-09-14 Trigen Limited Platelet-derived growth factor analogues
US6350731B1 (en) 1993-10-22 2002-02-26 Trigen Limited Platelet-derived growth factor analogues
EP2419117A4 (en) * 2009-04-13 2014-10-22 Elc Man Llc Methionine sulfoxide peptide, compositions and methods of use
WO2014207534A3 (en) * 2013-06-25 2015-04-09 Sépia Pesquisa E Desenvolvimento Bradykinin receptor modulators and use thereof
EP3013851A4 (en) * 2013-06-25 2016-12-28 Sépia Pesquisa E Desenvolvimento Bradykinin receptor modulators and use thereof
US9920096B2 (en) 2013-06-25 2018-03-20 Sepia Pesquisa E Desenvolvimento Bradykinin receptor modulators and use thereof
PL425038A1 (en) * 2018-03-26 2019-10-07 Uniwersytet Gdański New peptide derivatives of platelet-derived growth factor (PDGF), method for obtaining, pharmaceutical composition and application
WO2024236323A1 (en) * 2023-05-17 2024-11-21 Ucl Business Ltd Therapeutic pdgf-a molecules

Also Published As

Publication number Publication date
CA2137367A1 (en) 1993-12-23
JPH07508510A (en) 1995-09-21
WO1993025576A3 (en) 1994-04-14

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