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WO1993025576A2 - Peptides ayant un facteur de croissance derive des plaquettes (pdgf) - Google Patents

Peptides ayant un facteur de croissance derive des plaquettes (pdgf) 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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WO1993025576A3 (fr
Inventor
Amrit K. Judd
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Sri International
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Priority to JP6501593A priority Critical patent/JPH07508510A/ja
Publication of WO1993025576A2 publication Critical patent/WO1993025576A2/fr
Publication of WO1993025576A3 publication Critical patent/WO1993025576A3/fr

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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 et sels, ainsi que leurs dérivés (p.ex. matériaux cycliques et dimères) ayant des séquences qui correspondent sensiblement aux domaines PDGF et présentant une activité PDGF. Ils sont utiles en thérapie et dans des systèmes analytiques.
PCT/US1993/005325 1992-06-05 1993-06-03 Peptides ayant un facteur de croissance derive des plaquettes (pdgf) WO1993025576A2 (fr)

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JP6501593A JPH07508510A (ja) 1992-06-05 1993-06-03 血小板由来増殖因子（ｐｄｇｆ）活性

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995011259A1 (fr) * 1993-10-22 1995-04-27 Ellerman Pharmaceuticals Limited Analogues du facteur de croissance derive de plaquettes
US5952304A (en) * 1993-10-22 1999-09-14 Trigen Limited Platelet-derived growth factor analogues
EP2419117A2 (fr) * 2009-04-13 2012-02-22 ELC Management LLC Peptide de méthionine sulfoxyde, compositions et procédés d'utilisation
WO2014207534A3 (fr) * 2013-06-25 2015-04-09 Sépia Pesquisa E Desenvolvimento Modulateurs des récepteurs de la bradykinine et leur utilisation
PL425038A1 (pl) * 2018-03-26 2019-10-07 Uniwersytet Gdański Nowe peptydowe pochodne płytkopochodnego czynnika wzrostu (PDGF), sposób ich otrzymywania, kompozycja farmaceutyczna oraz zastosowanie
WO2024236323A1 (fr) * 2023-05-17 2024-11-21 Ucl Business Ltd Molécules thérapeutiques de pdgf-a

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0259632A1 (fr) * 1986-08-13 1988-03-16 Zymogenetics, Inc. Expression d'analogues de PDGF biologiquement actifs dans des cellules eucaryotiques
WO1992011364A1 (fr) * 1990-12-21 1992-07-09 Creative Biomolecules, Inc. Antagonistes biosynthetiques de facteur de croissance plaquettaire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0259632A1 (fr) * 1986-08-13 1988-03-16 Zymogenetics, Inc. Expression d'analogues de PDGF biologiquement actifs dans des cellules eucaryotiques
WO1992011364A1 (fr) * 1990-12-21 1992-07-09 Creative Biomolecules, Inc. Antagonistes biosynthetiques de facteur de croissance plaquettaire

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF BIOLOGICAL CHEMISTRY., vol.267, no.11, 15 April 1992, BALTIMORE US pages 7478 - 7482 L.M.KHACHIGIAN ET AL 'Synthetic peptides representing the alternatively spliced exon of the PDGF A-chain modulate mitogenesis stimulated by normal human serum and several growth factors.' *
JOURNAL OF BIOLOGICAL CHEMISTRY., vol.267, no.3, 25 January 1992, BALTIMORE US pages 1660 - 1666 L.M.KHACHIGIAN ET AL 'A tyronisated peptide representing the alternatively spliced exon of the PDGF A-chain binds specifically to cultured cells and interferes with binding of several growth factors.' cited in the application *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995011259A1 (fr) * 1993-10-22 1995-04-27 Ellerman Pharmaceuticals Limited Analogues du facteur de croissance derive de plaquettes
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
EP2419117A2 (fr) * 2009-04-13 2012-02-22 ELC Management LLC Peptide de méthionine sulfoxyde, compositions et procédés d'utilisation
EP2419117A4 (fr) * 2009-04-13 2014-10-22 Elc Man Llc Peptide de méthionine sulfoxyde, compositions et procédés d'utilisation
WO2014207534A3 (fr) * 2013-06-25 2015-04-09 Sépia Pesquisa E Desenvolvimento Modulateurs des récepteurs de la bradykinine et leur utilisation
EP3013851A4 (fr) * 2013-06-25 2016-12-28 Sépia Pesquisa E Desenvolvimento Modulateurs des récepteurs de la bradykinine et leur utilisation
US9920096B2 (en) 2013-06-25 2018-03-20 Sepia Pesquisa E Desenvolvimento Bradykinin receptor modulators and use thereof
PL425038A1 (pl) * 2018-03-26 2019-10-07 Uniwersytet Gdański Nowe peptydowe pochodne płytkopochodnego czynnika wzrostu (PDGF), sposób ich otrzymywania, kompozycja farmaceutyczna oraz zastosowanie
WO2024236323A1 (fr) * 2023-05-17 2024-11-21 Ucl Business Ltd Molécules thérapeutiques de pdgf-a

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