WO1992005796A1

WO1992005796A1 - Peptide inhibitors of neutrophil activation proteins

Info

Publication number: WO1992005796A1
Application number: PCT/US1991/007560
Authority: WO
Inventors: Paul H. Johnson; William C. Castor; Elizabeth M. Smith
Original assignee: Sri International; The Regents Of The University Of Michigan
Priority date: 1990-10-09
Filing date: 1991-10-09
Publication date: 1992-04-16
Also published as: AU8939691A

Abstract

Peptides having neutrophil activation protein inhibitory activity are disclosed. These peptides correspond to the first from 10 to 19 amino acids at the NH2 end of CTAP-III.

Description

PEPTIDE INHIBITORS OF NEUTROPHIL ACTIVATION PROTEINS

Description

Technical Field The present invention relates to peptides that inhibit neutrophil activation protein (NAP) and to the pharmaceutical use of these peptides to attenuate neutrophil-dependent myocardial ischemic injury.

Background

Activation of neutrophils with release of lysosomal enzymes and production of oxygen free radicals is an important part of the host defense mechanism against microbial infection. Activated neutrophils have, however, been implicated in the pathogenesis of several disease processes, including emphysema, adult respiratory distress syndrome, and myocardial infarction. In the case of regional myocardial ischemia and reperfusion experienced in infarction, neutrophils may adhere to the vessel walls at sites of inflammation and release toxic products capable of damaging the adjacent endothelium as well as myocytes within the reperfused region. It is well recognized that neutrophils can release a variety of mediators capable of promoting tissue injury, including proteolytic enzymes, platelet activating factor, arachidonic acid metabolites and active species of oxygen (e.g., superoxide anion, hydroxy1 radical, and hypochlorous anion) . The generation of these cytotoxic metabolites of oxygen within the microenvironment formed between the adherent activased neutrophils and altered endothelial cells leads to an increase in vascular permeability and myocyte damage which escalates upon reperfusion as neutrophils are directed and attracted to the reperfused region under the influence of the local accumulation of chemoattractants.

Others have suggested alleviating neutrophil- mediated tissue damage by depleting circulating neutrophils using anti-neutrophil antibody or extracorporeal filtration. Alternatively, others have suggested "mopping up" the mediators that promote tissue injury. For example, by treating with superoxide dismutase (SOD) to remove oxygen radicals produced following neutrophil activation. In both cases, the source of the damaging factors, namely activation of neutrophils, has not been avoided. Rather, these approaches are a step removed from the cause and attempt to alleviate the damage caused by the source of the mediators of injury.

The present invention employs peptides which have sequences which mirror the first 10 to 19 amino acids of Human connective tissue activating peptide III.

Human connective tissue-activating peptides (CTAPs) are a group of naturally occurring polypeptides that are capable of activating connective tissue cells. These peptides are present in platelets and leukocytes and stimulate mitogenesis, glycosaminoglycan and hyaluronic acid synthesis, prostaglandin E- and cyclic AMP formation, plasminogen activator secretion, fibroblast chemotaxis, glucose transport and glycolysis. CTAPs are being investigated as pharmaceuticals for regenerating connective tissue (e.g., would healing). Castor, C.W., et al. , PNAS (USA) (1983) 80:765-769 reports the amino acid sequence of one CTAP, known as CTAP-III, and the biological characteristics of CTAP- III. United States Patent 4,897,348 issued Jan. 30, 1990 to Johnson et al. discloses that analogs of CTAP- III in which the 21 position ethionine is replaced with leucine or a like acyclic side chain hydrophobic amino acid have CTAP-III activity with enhanced stability.

This patent also discloses genetic engineering techniques for producing CTAP-III and its 21-position analogs and is incorporated herein by reference.

Connective tissue activating peptide III (CTAP- III) is a human platelet granule-derived growth factor found in 1000 times the quantity of other growth factors presently known to be in platelets. CTAP-III stimulates synthesis of DNA, hyaluronic acid (HA) , sulfated glycosaminoglycan (GAG) chains, proteoglycan monomer and proteoglycan core protein in human synovial fibroblast cultures (1-6) . These references are listed below under "References." CTAP-III also stimulates glucose transport, formation of prostaglandin E₂, HA synthetase activity and plasminogen activator activity (7-10) . CTAP-III isolated by immunoaffinity chromatography showed significant molecular size heterogeneity by SDS PAGE when visualized by silver stains and/or Western blotting (11) . Further, CTAP-III purified by immunoaffinity methods appeared heterogeneous by analytical isoelectric focusing (IEF) (12) . This heterogeneity was detectable immediately after extraction from platelet α-granules freshly obtained from individual donors as well as in pooled outdated blood bank platelets. Such microheterogeneity was thought likely to have biologic significance since fractions of CTAP-III isolated by preparative IEF had varying specific activities in stimulating DNA and glycosaminoglycan synthesis in human synovial cell cultures (13) . Evidence for microhetero¬ geneity due to proteolytic processing of CTAP-III from human platelets was recently reported (11) . Two NH₂- terminal cleavage products were identified: CTAP-III (des 1-13) and CTAP-III (des 1-15). CTAP-III (des 1-13) had a pi of 8.6 and was a stable proteolytic cleavage product that retained the capacity to stimulate [ C]GAG synthesis in human synovial cell cultures. CTAP-III (des 1-15) was an elastase and chymotrypsin cleavage product identical to NAP-2, an entity thought to have neutrophil activating properties (14) . The data reported now demonstrate that the molecular size and charge heterogeneity of CTAP-III is due, in part, to deamidation, nonenzymatic glycosylation and proteolytic cleavage; only proteolytic cleavage clearly influences biologic activities measured in vitro.

References

The following references are cited herein by their reference numbers:

1. Castor, C.W. et al., connective tissue activation. XI. Stimulation of glycosaminoglycan and

DNA formation by a platelet factor. Arthritis Rheum. (1977) 20:859-68.

2. Castor, C.W. et al., Connective tissue activation XIV. Composition and actions of a human platelet autocoid mediator. Arthritis Rheum. (1979) 22_:260-72.

3. Walz, D.A. et al., Connective Tissue Activation: Structural Studies on a Human Platelet Mitogen [Abstract]. Clin. Res. (1979) 27:649. 4. Castor, C.W. et al., structural and biological characteristics of connective tissue activating peptide (CTAP-III) , a major human platelet- derived growth factor. Proc. Natl. Acad. Sci. USA (1983) 80:765-9. 5. Castor, C.W. et al., connective tissue activation XXV. Regulation of proteoglycan synthesis in human synovial cells. Arthritis Rheum. (1983) 26:522-7.

6. Castor, C.W. et al., connective tissue activation XXI. Regulation of glycosaminoglycan metabolism by lymphocyte (CTAP-I) and platelet (CTAP- III) growth factors. In Vitro (1981) 12:777-85.

7. Castor, C.W. et al., connective tissue activation XXIX: Stimulation of glucose transport by connective tissue activating peptide-III. Biochemistry (1985) 24.:1762-7.

8. Castor, C.W. et al., connective tissue activation XX. Stimulation of prostaglandin secretion by mediators from lymphocytes (CTAP-I) and platelets (CTAP- III). Arthritis Rheum. (1981) 24.504-9.

9. Sisson J.C. et al., connective tissue activation XVIII. Stimulation of hyaluronic acid synthetase activity. J. Lab Clin. Med. (1980) 96:189- 97. 10. Ragsdale, C.G. et al., connective tissue activating peptide-III. Induction of synthesis and secretion of plasminogen activator by synovial fibroblasts. Arthritis Rheum. (1984) 27:663-7.

11. Castor, C.W. et al., connective tissue activation XXXIII: Biologically active cleavage products of CTAP-III from human platelets. Bioche . Biophvs. Res. Co mun. (1989) 163:1071-8.

12. Green, M.S. et al., connective tissue activation XXX: Isoelectric point microheterogeneity of CTAP-III, a human platelet derived growth factor. Proc. Soc. EXP. Biol. Med. (1986) 181:555-9.

13. Castor, C.W. et al., connective tissue activation: evidence for molecular heterogeneity of a platelet derived growth factor (CTAP-III) [Abstract]. Clin. Res. (1983) 3_1:784. 14. Walz, A. et al. , a novel cleavage product of β-thromboglobulin formed in cultures of stimulated mononuclear cells activates human neutrophils. Biochem. Biophvs. Res. Commun. (1989) 159:969-75. 15. Castor, C.W. , connective tissue activating peptide III (CTAP-III) and its isoforms. In: Barnes D. , ed. Methods in Enzymology, Peptide Growth Factors, Part C. Orlando, FL: Academic Press (in press) .

16. Castor, C.W. et al., connective tissue activating peptide III (CTAP-III) : Characteristics of a biosynthetic form expressed in E. coli [Abstract]. Arthritis Rheum. (1986) 29:S43.

17. Schneider, C. et al. , a one-step purification of membrane proteins using a high efficiency im unomatrix. J. Biol. Chem. (1982) 257:10766-9.

18. Oyama, V.I. et al., measurement of cell growth in tissue culture with a phenol reagent (Folin- Ciocalteau) . Proc. Soc. Exp. Biol. Med. (1956) 91:305- 7. 19. Waddell, W.J. et al. , a simple ultraviolet spectrophotometric method for the determination of protein. J. Lab. Clin. Med. (1956) 48:311-4.

20. Castor, C.W. et al., Connective Tissue Activating Peptides. In: Sabato g., ed. Methods in Enzvmoloσv. Orlando, Florida: Academic Press, (1988) 731-48.

21. Anderson, B.L. et al., a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 90,000. Anal. Biochem. (1983) 132:365- 75.

22. Morrissey, J.H., silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Analvt. Biochem. (1981) 117:307-10. 23. Heukeshoven, J. et al., simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis (1985) :103-12. 24. Matsudiara, P., sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J. Biol. Chem. (1987) 262:10035- 8.

25. Prestidge, R.L. et al., preparative flatbed electrofocusing in granulated gels with natural pH gradients generated from simple buffers. Anal. Biochem. (1979) 97:95-102.

26. Seetharama, A. et al., reversibility of the ketoamine linkages of aldoses with proteins. 27. Cole, L.A. et al. , the structures of the serine-linked sugar chains on human chorionic gonadotropin. Biochem. Biophvs. Res. Com . (1985) 126:333-9.

28. Hewick, R.M. et al., a gas-liquid solid phase peptide protein sequenator. J. Biol. Chem. (1981)

256:7990-7.

29. Castor, C.W. et al. , connective tissue activation XXI. Regulation of glycosaminoglycan metabolism by lymphocyte (CTAP-I) and platelet (CTAP- III) growth factors. In Vitro (1981) 17:777-85.

30. Johnson, P.H. et al., connective tissue activating peptide-III (CTAP-III) : cloning the synthetic gene and characterization of the protein expressed in E coli [Abstract]. Fed. Proc. (1986) 45:1790. 31. Waleh, N.S., et al. , structural and functional organization of the colicin El operon. Proc. Natl. Acad. Sci. USA (1985) 82:8389-93.

32. Wenger, R.H. et al., cloning of cDNA coding for connective tissue activating peptide III from a human platelet-derived λgtll expression library. Blood (1989) 73:1498-1503.

33. Begg, G.S. et al., complete covalent structure of human β-thromboglobulin. Biochemistry (1978) 17:1739-44.

34. Walz, A.R. et al., effects of the neutrophil-activating peptide NAP-2, platelet basic protein, connective tissue activating peptide III, and platelet factor 4 on human neutrophils. J. Exp. Med. (1989) 170:1745-50.

35. Katz, I.R. et al., protease-induced immunoregulatory activity of platelet factor 4. Proc. Natl. Acad. Sci. USA (1986) 83:3491-5.

36. Matsushima, K. et al.. Molecular cloning of a human monocyte-derived neutrophil chemotactic factor

(MDNCF) and the induction of MDNCF mRNA by Interleukin 1 and tumor necrosis factor. J. Ex . Med. (1988) 167:1883- 93.

37. Sugano, S. et al., transformation by Rous sarcoma virus induces a novel gene with homology to a mitogenic platelet protein. Cell (1987) 49:321-8.

38. Bedard, P.A. et al., constitutive expression of a gene encoding a polypeptide homologous to biologically active human platelet protein in Rous sarcoma virus-transformed fibroblasts. Proc. Natl. Acad. Sci. USA (1987) 84:6715-9.

39. Anisowicz, A. et al., constitutive overexpression of a growth-related gene in transformed Chinese hamster and human cells. Proc. Natl. Acad. Sci. USA (1987) 84:7188-92.

Disclosure of the Invention

One aspect of the invention is a peptide of from 10 to 19 amino acids in length corresponding to the first 10 to 19 amino acids present in the N-terminal region of CTAP-III. This peptide has the ability to bind to and inhibit neutrophil activating protein (NAP) .

Another aspect of the invention are individual peptides containing 10 to 19 of the first 10 to 19 amino acids of the N-terminal region of CTAP-III.

Another aspect of the invention is a pharmaceutical composition comprising a peptide of the invention in an injectable carrier. A further aspect of the invention is a method for treating an individual suffering from a myocardial infarction by attenuating neutrophil-dependent myocardial tissue ischemic injury comprising administering to said individual a sufficient amount of a peptide of the invention to attenuate neutrophil invasion of myocardial tissue during reperfusion.

A still further aspect of the invention is a method for treating an individual suffering from a cerebral vascular disease by attenuating neutrophil- dependent cerebral tissue ischemic injury comprising administering to said individual a sufficient amount of a peptide of the invention to attenuate neutrophil invasion of cerebral tissue during reperfusion.

Brief Description of the Drawings

In this application reference will be made to the accompanying drawings in which Figure 1 is a graph showing the results of preparative IEF over a range of pH 3-10 separated CTAP-III into four major peaks, each possessing the capacity to stimulate [ 3H]DNA synthesis.

Figure 2 is a silver stained SDS-PAGE gel which shows two CTAP-III preparations. Lane 1 contains a typical CTAP-III preparation eluted from a heparin affinity column with 0.3M NaCl. Band A shows the CTAP- III sequence on microsequencing, band B contains variable mixtures of small isoforms (see text) . Lane 2 contains molecular weight markers, Lane 3 shows CTAP-III forms which on microsequencing of Immobilon blots revealed the structural alterations (CTAP-III [Asp-1] , CTAP-III [des 1-14]) as labeled in the figure.

Figure 3 is a pair of graphs. The top panel plots the mitogenic activity (mean ± S.E.M.) of the CTAP- III (des 1-14) isoform and CTAP-III at different concentrations; the lower panel records the GAG stimulating activity of the two forms. The mitogenic activity of the des 1-14 isoform is not significantly different from CTAP-III. The apparent reduction of GAG stimulating activity of the des 1-14 isoform was significant (P<O.01).

Figure 4 is a pair of graphs in which i munoaffinity purified CTAP-III is compared to an aliquot cleaved to the des 1-15 form with porcine elastase. The heparin affinity column used to separate elastase from the isoforms yielded two affinity forms of CTAP-III (des l-15)/NAP-2, (0.3M and 0.5M). Biologic activity (mean ± SEM) is plotted versus increasing concentrations of the peptides. Both affinity forms had mitogenic activity similar to the parental form (P<0.15). The 0.3M form showed a significam; increase in GAG stimulating activity (P<0.01); the 0.5M form appeared similar to the parental CTAP-III in activity.

Figure 5 is a pair of graphs which show the biologic activities (mean ± S.E.M.) of rCTAP-III-Leu-21 and its elastase cleavage products, des 1-15, 0.3M and 0.5M heparin affinity forms plotted as a function of concentration. The upper panel shows that rCTAP-III- Leu-21 had no mitogenic activity for human synovial cells; the lower panel shows that the intact recombinant molecule had very little GAG stimulating activity. The GAG stimulating capacity of the des 1-15 forms was signi icantly enhanced compared to the intact molecule (P<0.001). In the mitogenic assay both des 1-15 forms were significantly more active than the parent molecule (for the 0.3M isoform, P<0.02; for the 0.5M isoform, P<0.001)..

Figure 6 is a schematic representation of CTAP- III. The single letter code identifies the amino acid residues in this schematic representation of CTAP-III; cleavage sites giving rise to the isoforms studied in this report are indicated.

Modes for Carrying out the Invention

The Peptides and their Preparation

The peptides of this invention are from 10 to 19 amino acids in length. In sequence, they follow the first 10 to 19 amino acids present at the NH~ and of CTAP-III. Figure 6 shows the sequence of CTAP-III. They are used in substantially pure form that is typically containing not more than about 50% by weight of other proteins. Some of these materials occur in impure form in platelets and can be prepared by isolation therefrom as shown below. They also can be prepared using synthetic routes.

The polypeptides 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 complementary (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 polypeptide. Also, 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-butyloxycarbonyl (Boc) , fluorenyl methyloxy carbonyl (FMCC) , 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 Asp and Glu: o-benzyl, t-butyl; for tyrosine: benzyl, o-bromobenzyloxycarbony1, 2,6-dichlorobenzyl, isopropyl, cyclohexyl, cyclo pentyl, and acetyl; for serine and threonine: benzyl, t-butyl and tetrahydro pyranyl; for histidine: benzyl, p-toluenesulfonyl and 2,4-dinitrophenyl; and for Trp: 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 benzhydrylamino-polystyrene-divinylbenzene 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 methylene chloride 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 HBT, 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 polypeptide 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 ammonia/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-C0₂H end groups when a normal benzylester linkage has been used or to form a CO-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.

Biological Activity

Once the peptides of the present invention have been synthesized they may be analyzed to determine their ability to inactivate CTAP-III/NAP. The preparation of recombinant CTAP-III and analogs are disclosed in U.S. Patent No. 4,897,348 to Johnson et al. , issued 30 January 1990, the contents of which are incorporated herein by reference. Once CTAP-III has been isolated or generated recombinantly the removal of N-terminal the present 10 to 19 amino acids results in activation of the CTAP-III into a Neutrophil Activating Protein (or "NAP"). (See, e.g., U.S. Serial No. 595,443, Attorney Docket No. 2575-0098, filed on even date herewith, the contents of which are incorporated herein by reference; and see Examples infra.) NAP activity may be inactivated by the addition of the peptides of the present invention. Although not wishing to be bound by this theory, it is believed that the addition of a peptide corresponding to the first 10 to 19 amino acids of the CTAP-III results in a tight, noncovalent association between the peptide and NAP. It is this association that is believed to inhibit the ability of the binding domain on NAP to activate neutrophils.

Peptides of various lengths corresponding to the N-terminus of CTAP-III may be prepared and assayed to determine their ability to inhibit NAP activity. This may be done, e.g. , by comparing the specific activity of NAP in the presence of the peptides of the present invention to the specific activity of NAP alone. In this manner, peptides of various lengths can be compared to determine the optimal length at which the peptides exhibit maximal inhibitory activity. In addition, the stability of the various length peptides in vitro may be compared so as to optimize lengths having the greatest stability.

Utility and Administration

In the practice of the medical and veterinary methods of this invention an effective amount of a peptide of this invention or a pharmaceutical composition containing the same is administered to the subject in need of such treatment. These peptide compositions may be administered by any of a variety of routes depending upon the specific end use, including particularly parenterally (including subcutaneous, intramuscular, and intravenous administration) .

The materials can be administered to mammals such as humans, monkeys, dogs, rodents, and the like. In such uses, the compositions generally include a pharmaceutical diluent such as injectable saline, mineral oil or the like. The compound or composition may also be administered by means of slow-release, depot, or implant formulations, as is well known in the art. The polypeptides described herein are usually administered in amounts of 0.001 to 1000 micrograms per kg of body weight, particularly in amounts of 1-500 micrograms per kg of body weight, although higher or lower amounts may be used.

This invention will be further explained by the following Example and experimental description which is intended to illustrate the practice of the invention but not limit its scope.

Example 1 Preparation of Synthetic Peptide

The synthetic peptide is synthesized by solid-phase techniques on a Beck an Model 990C peptide synthesizer using commercially available t-Boc amino acid polystyrene resin and t-Boc protected amino acids with the following side-chain protecting groups: 0-benzyl esters for Asp and Glu; 0-benzyl ethers for Thr and Ser; dnp for His; and orthochlorobenzyloxycarbonyl for Lys. All couplings are performed using a 3-molar excess of t-Boc amino acid and dicyclohexyl carbodiimide (DCC) over the number of milli-equivalents of amino acid on the resin. In the case of Asn and Gin, a 3-molar excess of the t-Boc amino acid, DCC, and N-hydroxybenzotriazole (HOBT) are used. All couplings are monitored by the ninhydrin test (Kaiser, et al. Anal Biochem 34:595 (1970).) Forty percent TFA-CH.Cl- is used for Boc deprotection. 2-Mercaptoethanol (0.1%) is added to the TFA/CH_C1_ when methionine is present. (Methionine is used with an unprotected sulfhydryl side chain.) The details of the synthetic cycle are given in Schedule 1. 0

Schedule 1

SCHEDULE OF EVENTS FOR ASSEMBLING THE PEPTIDE ON RESIN

15 Step Reagent or Solvent Time(min)

20

25

After completion of the synthesis, the peptide is cleaved from the resin using a low-high HF procedure (see Tam et al, (1982) Tett. Lett.. 23. 2939, and J. Am. Chem.

30 Soc.. (1983) 105. 6442) , and S_N2 deprotection reaction in HF-reaction Apparatus Type II (Peninsula Labs) . The peptide resin is first treated with an

HF:dimethylsulfide:p_-cresol mixture (25:65:10) at 4°C for 1 hr. HF and dimethyl-sulfide are evaporated completely.

35 Next, more HF is distilled into the reaction vessel to make the ratio of HF-p_-cresol 9:1. The mixture is stirred at 4°C for 45 min. After that, HF is evaporated completely, first at 4 C and then at room temperature, under vacuum. At low HF concentration. Asp and Glu side-chain benzyl ester groups are cleaved, with minimum acylation side reactions. The high-HF step removes more acid-stable groups such as Arg (Tos) and cleaves the peptide from the resin completely. The DNP group of His is removed before HF cleavage by treatment with 1000-fold excess of 2-mercaptoethanol.

The peptide is separated from the various side products by extraction with ether and isolated from the resin by extraction with 5% (or higher, depending on the solubility of the peptide) acetic acid and subsequent lyophilization. The crude peptide is subjected to gel filtration on Sephadex LH-20. Final purification is achieved on HPLC using 50 cm/20 mm preparative column packed with Vydac 15-20 micron C _. The purity of the peptide is checked by analytical HPLC and amino acid analysis.

After three lyophilizations from water, pure peptide is obtained as the acetate salt.

Experi enta1

Methods

Preparation of CTAP-III: Outdated platelets were obtained from local blood banks and the Southeast Michigan Red Cross Blood Services. Quality control studies on eight separate platelet packs five days after bleeding showed 0.84 x 10 platelets per pack and 3.2 x 10⁸ ± 2.9 x 10 leukocytes per pack; that is, there are approximately 1-2 leukocytes/1000 platelets. Platelet- rich plasma was centrifuged at 4°C and the pellet preparation was stored at -20°C.

CTAP-III was isolated from platelet pellets by extraction into acid/ethanol and precipitation with cold acetone (1,2,4) . In practice, 50g of pelleted platelets were added to 500 ml of acid ethanol (5 ml 1.25 N HC1/95 ml ethanol) ; this was stirred slowly at 4°C for 16 hr, centrifuged (15,000g, 10 min) , and the supernatant fluid was added to 1500 ml cold (4°C) acetone. After 1 hr at 4°C the supernatant fluid was removed by aspiration and centrifugation; the precipitated protein was dissolved in 30-50 ml of 0.5M acetic acid and dialyzed overnight against 0.2M acetate buffer, pH 4.0, containing protease inhibitors (0.001M Benzamadine and 0.005M EACA) . The dialyzed preparation was applied to a Sephacryl S-200 HR column (5x72 cm) and eluted with the same buffer and inhibitors in 15 ml fractions at a flow rate of 4.0 ml/min. Fractions containing CTAP-III were identified by immunodiffusion with anti-CTAP-III antisera and pooled. Glycosaminoglycan polysulfate (Arteparon, Luitpold Werke, Jupiter, Fla.), a proteinase inhibitor, was then added at a concentration of 100 μg/ml. This resulted in prompt flocculation of several protein species including essentially all of the CTAP-III. The turbid preparation was allowed to settle for 2 hr at 4°C and then centrifuged at 12,000g; the resultant pellet was dispersed in PBS, pH 7.5, allowed to stand overnight at 4°C and then centrifuged at 12,000g and the CTAP-III rich supernatant fluid was recovered for further processing. The isolation process was continued by heparin affinity and/or immunoaffinity chromatography. Heparin affinity columns were made by coupling heparin (Sigma crude unbleached heparin, Sigma Chemical Co., St. Louis, MO) to Affigel 15 (Bio-Rad Laboratories, Richmond, CA) as directed by the manufacturer (15) . Partially purified CTAP-III was applied to a heparin affinity column in phosphate-buffered saline, pH 7.0; CTAP-III was eluted with 0.3M sodium chloride and dialyzed against PBS. Monospecific immunoaffinity isolated anti-CTAP-III IgG was prepared by passing polyclonal rabbit anti-CTAP-III antisera over an antigen column of highly purified CTAP- III/BTG or rCTAP-III-Leu-21 coupled to activated CH- Sepharose 4B (16) . Bound monospecific anti-CTAP-III IgG was eluted at low pH, neutralized and cross-linked to protein A Sepharose with dimethylpimelimidate using a modification of the method of Schneider (17) . CTAP-III was applied to the immunoaffinity column, the column was washed with PBS and CTAP-III was eluted with 0.1M acetic acid (pH 2.8). To reduce growth factor binding to plastic and glass, either polyethylene glycol or highly purified human albumin was added before lyophilization in amounts sufficient to make their concentrations 0.01% in final preparations.

Protein measurement and antisera development: Protein was measured by a colorimetric method (18) and/or UV .absorption (19) . Antisera to both platelet-derived and recombinant CTAP-III-Leu-21 were raised in rabbits. Eight- to 10-week old male New Zealand white rabbits were immunized with 50 μg of CTAP-III or rCTAP-III in 0.15M NaCl in Freund's complete adjuvant. Booster injections were given at 6 and 12 weeks with antigens in incomplete Freund's adjuvant. Animals were bled at 4 weeks and then at biweekly intervals after initial immunization. Measurement of CTAP-III by radial immunodiffusion (RID) utilized filtered, heat inactivated rabbit anti-human CTAP-III (20) .

Analytic polyacrylamide gel electrophoresis: Highly purified CTAP-III and its isoforms were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 8M urea/8% total acrylamide and by analytical IEF in ampholyte gradients pH 3-10 (15,21). Proteins separated by SDS-PAGE were detected by silver stain and on IEF by both silver and Coomassie Brilliant Blue R-250 staining (15,22,23). Proteins were prepared for sequencing by blotting onto Immobilon-P using a semi-dry blotter (Polyblot, American Bionetics, Inc., Hayward, CA 94545) and identified by Coomassie Brilliant Blue R-250 or immunostaining with antisera to recombinant CTAP-III (15,20,24). Western blots of proteins following electrophoretic separation and immobilization in a nitrocellulose membrane were accomplished as described (15) . Membrane-bound antigens were probed with antisera to CTAP-III or rCTAP-III (1:500) and the complexes detected with a Bio-Rad Immunoblot (GAR-HRP) assay kit (BioRad Laboratories, Richmond, CA) .

Preparative isoelectric focusing of CTAP-III: Platelet preparations were fractionated using the Pharmacia Flat Bed Apparatus FBE 3000 and the Electrophoresis Constant Power Supply ECPS 3000/150.

Sephadex G-75M was washed with 2 x deionized water and dried. A stable pH gradient was achieved using a system of amphoteric and nonamphoteric buffers according to the method of Prestidge and Hearn (25) . The gel bed was then cut into 26 separate segments and the pH of each was measured. The protein focused in individual segments was eluted with PBS pH 7.0, concentrated, dialyzed against PBS, and stored frozen until assay.

Carbohydrate analysis: Carbohydrate analyses were performed by methods previously described in detail

(26,27). Three different acid hydrolyses were used for each sample aliquot. Peptides were incubated 1 hr at 80°C in 0.1N sulfuric acid to release sialic acid residues for assay. For analyses of neutral sugars, samples were hydrolyzed in 4N trifluoroacetic acid at 100°C for 2 hr. Analysis of amino sugars required hydrolyses in 6N HCl at 100°C for 3 hr. Sialic acid was measured by the thiobarbituric acid method; neutral sugars were converted to the corresponding glycamines by reductive amination with 0.2M sodium cyanoborohydride in 1M ammonium sulfate (100°C, 90 min) . Amino sugars and neutral sugar glycamines were separated and quantitated as described for amino acids, by cation exchange on a Kratos automated amino acid analyzer using post-column o- phthalaldehyde derivatization and fluorometric detection. Amino acid analyses: Amino acid compositions were determined as previously described (27) . One aliquot of CTAP-III was reduced with sodium borohydride prior to acid hydrolysis in order to preserve glucosyl-lysine linkages (26). Briefly, CTAP-III, with and without borohydride reduction, was subjected to 6N HCl hydrolysis (3 hr 100°C) , dried, and applied to a Kratos automated amino acid analyzer where the residues were separated by cation exchange, reacted with hypochlorite and an o- phthalaldehyde reagent and then detected with a Kratos FS950 fluorometer. Effluent peaks were compared with standards using a Hewlett-Packard 3390A integrator.

Amino acid sequence determination: Stained protein bands on Immobilon-P were cut out and destained in 100% methanol and subjected to NH₂~terminal sequencing using an Applied Biosyste s 470A Gas Phase Protein Sequencer interfaced with an Applied Biosystems 120A Analyzer (28) . Alternatively, aqueous protein solutions were applied to a glass fiber filter pretreated with 2.0 mg of Biobrene and sequenced as described above. The authentic carboxyterminal peptide of CTAP-III (residues 78-85) was isolated by HPLC C₁₈ chromatography; its sequence was subsequently determined (data not shown) . The presence of the authentic CTAP-III carboxyterminus in the isoforms was confirmed by comparing the HPLC profile of their tryptic digests with the native CTAP-III HPLC digest profile.

Cell culture methods: Normal human fibroblastic cells (synovium and cartilage) were developed from explants obtained at amputation or arthrotomy; fibroblasts from dermis were obtained following reduction ma moplasty as described earlier (1,2). Cells were grown as monolayer cultures in T-75 flasks in CMRL 1066 (Gibco, Grand Island, NY) medium supplemented with 5% human serum and 15% fetal calf serum (FCS) , sodium bicarbonate, L- glutamine, 0.02M Hepes buffer, penicillin, streptomycin and gentamicin. Trypsin dispersal was performed to facilitate cell propagation, study, and preparation for cold storage. Isotope incorporation studies: Measurement of

[ C]glucosamine incorporation into [ C]GAG, primarily [ 14C]HA, was accomplished by plating cells m 96-well microtiter plates at a density of 10⁴ cells/well in 200 μl semisynthetic medium (Leibovitz medium, L-15, Hazelton Biologies, Inc., Lenexa, Kansas), containing 1% FCS, L- glutamine, penicillin, gentamicin, streptomycin, sodium carbonate, and 0.02M Hepes buffer, pH 7.6 (15,29). Identification of [ C]HA was accomplished by incubating labeled media with hyaluronidase (ex.Streptomyces) in an appropriate buffer and then subjecting digested and undigested samples to the CPC-fixation-wash procedure (6,15).

To measure [ 3H]thymι.dm. e i.ncorporati.on into fibroblast DNA, cells were plated, 10 cells/microtiter well in 100 μl of ESM medium supplemented with 3% FCS, antibiotics, L-glutamine and Hepes buffer and incubated in a humidified chamber at 35-37°C (15,20). After the preliminary 20 hr incubation period, test samples or vehicles were added (5-15 μl/well) and incubation continued for 24 hr at which time [ H]-methylthymidine (1.5 μCi/15 μl ESM/well) was added and incubation resumed for the final 24 hr. Medium was aspirated, discarded, and the cell sheets were washed twice, each separately, with phosphate-buffered saline (pH 7.0), 5% trichloroacetic acid and absolute methanol. After air drying (at 35°C) , the cells were lysed for 1 hr at 37^βC with 50 μl of 0.3N sodium hydroxide. Fifty μl of cell lysate was pipetted into a counting vial containing Ecolume cocktail and radioactivity was measured in a scintillation counter.

Preparation of rCTAP-III: Recombinant CTAP-III- Leu-21, an analogue containing a leucine substitution for methionine at position 21, was produced from a synthetic gene using an E. coli expression system, pNP6, based on the genetic regulatory elements of the colicin El operon (30,31). Six liter shake-flask cultures were grown to an optical density (650 nm) of 0.4 and gene expression was induced by addition of mitomycin C to the culture at a final concentration of 0.5 μg/ml for 4 hr. Cells were collected by centrifugation and suspended in 25 mM Tris, 10 mM EDTA, 50 mM glucose, pH 8.0, containing 1M guanidinium hydrochloride. Cells were lysed by sonication and the insoluble material was removed by centrifugation. The resulting soluble material containing rCTAP-III-Leu-21 was dialyzed against 0.1N acetic acid, lyophilized and dissolved in 70% formic acid and incubated with a 50-200 molar excess of cyanogen bromide at room temperature for 12 hr. This treatment removed a short leader sequence connected to CTAP-III- Leu-21 by a single methionine residue and resulted in generating the native amino-terminal end. The protein reaction mixture was dialyzed against 0.1N acetic acid and partially purified by gel filtration chromatography using sephacryl-200 (Pharmacia) . Fractions containing rCTAP-III-Leu-21 were identified by polyacrylamide gel electrophoresis, pooled and lyophilized. The protein was dissolved in 50 mM Tris buffer, pH 8.5 containing 6M guanidinium hydrochloride at a concentration of 0.2 mg per ml. Protein folding was initiated by the addition of a redox agent to a final concentration of 2 mM oxidized glutathione and 1 mM reduced glutathionine and dialyzed against 100 volumes of buffer without GndHCl for 12 hr at room temperature. Under these conditions, rCTAP-III- Leu-21 refolded to the native conformation as verified by analysis using analytical reverse-phase HPLC and purified platelet-derived CTAP-III as a control. Finally, the rCTAP-III-Leu-21 was purified to greater than 95% purity by heparin affinity chromatography using sodium chloride gradient elution.

Results

Isoelectric point (pi) microheterogeneity: Highly purified, biologically active CTAP-III was separated by preparative IEF. Fractions were examined for total protein content, CTAP-III content by RID, purity by SDS- PAGE and biologic activity was measured in human synovial cell cultures. Mitogenic activity of the IEP (isoelectric point) variants of CTAP-III is shown in Figure 1; previously we showed a similar biologic activity profile by measuring the incorporation of

[¹ C]glucosamine into [¹⁴C]HA (12) . An analytical IEF gel (inset, Figure 1) suggests that the preparative fractions are mixtures of charge isomers. Data summarized in Figure l suggest that the specific biologic activity of CTAP-III varies with the IEP of the different fractions. Three additional preparative IEF experiments generated similar data. The relative magnitude of two CTAP-III anabolic activities, measured as enhanced GAG and DNA synthesis, were concordant in the different fractions. Glycosylation of CTAP-III: To explain some of the IEP heterogeneity we examined immunoaffinity isolated CTAP-III for covalently linked carbohydrate before and following acid hydrolysis. A mixture of CTAP-III variants not separated by IEF was studied. Unhydrolyzed CTAP-III (4.76 nmole based on amino acid composition) contained 0.6 nmole glucose; acid hydrolyzed CTAP-III had 10.0 nmole glucose/4.76 nmole CTAP-III. Therefore, this mixture of CTAP-III IEP variants contained 1.97 nmole of covalently linked glucose/nmole CTAP-III. Only glucose was detected; no galactose or amino sugars were found. Seven additional biologically active samples, including four IEP species of CTAP-III (from two separate preparative IEF studies) , were examined by the same procedures (see Table 1) . Glucose and lysine content of the four separate CTAP-III charge-isomers is shown. The amino acid composition of CTAP-III charge-isomers was examined prior to and following treatment with sodium borohydride to stabilize sugar-lysyl residue Schiff base adducts. Lysyl residues not determined by virtue of borohydride treatment are an estimate of sugar-lysyl adducts and are labeled "% lysine glycosylated." In the two IEF preparations with isoelectric points from 6.89 to 9.32, bound glucose ranged from 1.97 to 2.81 nmole glucose/nmole CTAP-III. In two of the eight preparations there was no evidence for glycosylation. Glycosylation of CTAP-III, then, occurs commonly, varies with the preparation and was not clearly related to pi or biologic activity. Glycosylation was not required for biologic activity; one nonglycosylated preparation (PI861_g5-I M) stimulated [¹⁴C]HA synthesis by over 900%.

Amino-terminal Deamidation, (CTAP-III [Asp-1]): CTAP-III (Asp-l) was detected by microsequencing the band from an Immobilon blot of an analytical IEF gel at the pi 7.0 locus, as well as in the SDS-PAGE gel shown in Figure 2. Our earlier studies showed asparagine to be the amino terminal residue of CTAP-III (3,4); further, the gene for human CTAP-III was recently shown to code for an amino terminal asparagine residue (32) . In this context it was interesting that several recent CTAP-III preparations from outdated platelet packs which had been stored frozen showed 100% aspartic acid at the NH₂-terminus. Other preparations revealed 40% Asp/60% Asn. Data illustrating a measure of biologic activity attending partial or complete deamidation of the amino terminal asparagine of CTAP-III are shown in Table 2. It is clear that neither partial nor complete NH₂-terminal deamidation abolishes the capacity of CTAP-III to stimulate [¹⁴C]GAG synthesis by synovial cella; in fact the range of activities seen here is similar to that found in preparations without evidence of deamidation. In [³H] thymidine assays, NH₂- terminal deamidated CTAP-III (P-1890 and P-1892) stimulated DNA synthesis by 146% and 86% in synovial cell cultures. Amino terminal sequence cleavage isoforms of CTAP- III, isolation, molecular characteristics and biologic activities: CTAP-III (des 1-13) was identified in purified preparations of CTAP-III recovered from prolonged cold storage (11) . SDS-PAGE gels showed a single silver stained band which immunostained with anti- rCTAP-III in a Western blot. The apparent molecular weight of the isoform was 6200 Da, the pi was 8.6 and NH₂-terminal sequencing showed:

NH₂-Leu-Tyr-Ala-Glu-Leu-Arg-Cys-Met-Cys-Ile-Lys-Thr.

Carboxyterminal analysis of CTAP-III (des 1-13) was the same as that for intact CTAP-III. CTAP-III (des 1-13) stimulated synthesis of [ 14C]HA in human synovial cell cultures with a specific activity similar to that attributed to the parent molecule; interestingly, no mitogenic activity was detected.

CTAP-III (des 1-14) was identified for the first time during the present study as an isoform which failed to bind to a heparin column. After isolation from an immunoaffinity column, an aliquot of this material was separated by SDS-PAGE. A Western blot of CTAP-III antigen using antik-rCTAP-III antibody then showed the major reactive species to have a molecular weight of 6500-7000 Da; a minor fraction had a molecular weight of 9300 Da (Figure 2) . Amino terminal sequencing of the bands from an Immobilon blot showed that the larger protein, representing 25% of the total, was CTAP-III (Asp-1) . The 6500 Da fragment was sequenced through 10 cycles which showed:

NH₂-Tyr-Ala-Glu-Leu-Arg-Cys-Met-Cys-Ile-Lys-COOH

This analysis corresponds to CTAP-III (des 1-14) ; subsequent studies have detected CTAP-III (des 1-14) in material which had bound to heparin, hence it is clear that this isoform may occur with or without a modified heparin binding domain. Biologic activity measurements of CTAP-III (des 1-14) and its accompanying larger form showed that the isoform stimulated DNA synthesis with a specific activity similar to that shown by a typical preparation of the parent molecule (Figure 3) . The des 1-14 isoform appeared slightly less active than the parent molecule in stimulating GAG synthesis. The significance of this finding is uncertain in view of the presence of 25% CTAP-III (Asp-1) in the preparation. In addition, CTAP-III (des 1-14) stimulated by 50-60 percent the incorporation of [ 35S0₄] into [35S]GAG formed in human chondrocyte cultures. The 75%/25% mixture of CTAP- III (des 1-14) /CTAP-III (Asp-l) contributes some degree of ambiguity to the activity studies reported here. For CTAP-III (Asp-1) to be responsible for the observed activity, its specific activity would have to be much greater than the parent (control) molecule. As noted earlier, CTAP-III (Asp-1) appears to have "normal" specific biologic activity; therefore it seems unlikely that this minor component could account for all of the biologic activity shown in Figure 3. Consequently, we attribute the major portion of the biologic activity of this preparation to CTAP-III (des 1-14) .

CTAP-III (des 1-15) was detected in platelet- derived CTAP-III preparations as a small isoform (11) . NH₂~terminal sequencing of the electrophoretically- separated band blotted onto Immobilon showed:

NH₂-Ala-Glu-Leu-Arg-Cys-Met-Cys-Ile-Lys-Thr.

We noted earlier that incubation of the parent molecule with porcine elastase or chymotrypsin resulted in cleavage to the CTAP-III (des 1-15) isoform (11) .

In the present study, we incubated 28.5 μg porcine elastase with 2.5 mg of CTAP-III for 17 hr at 21°C in order to generate this isoform for biologic activity measurements. Following enzymatic cleavage, the digestion mixture was eluted from a heparin affinity column with increasing concentrations of NaCl applied in a stepwise manner. The elastase was removed in the unbound fraction and CTAP-III (des 1-15) was eluted at both 0.3M and 0.5M NaCl. Both salt fractions were sequenced and shown to be CTAP-III (des 1-15) . An HPLC comparison of the tryptic digest of native CTAP-III with CTAP-III (des 1-15) . An HPLC comparison of the tryptic digest of native CTAP-III with CTAP-III (des 1-15) (both the 0.3 and 0.5M eluates) as well as CTAP-III (des 1-14) revealed that the carboxyterminal peptide (residues 78- 85) was identical (intact) in each (data not shown) . The anabolic biologic activity of CTAP-III (des 1-15)/NAP-2 is illustrated in Figure 4. It is clear that the 0.3M salt fraction of CTAP-III (des 1-15) stimulates glycosaminoglycan synthesis with a specific activity greater than that of the parent molecule; the 0.5M salt fraction, however, was not significantly more active than CTAP-III. The mean specific activity of the des 1-15 isoforms with respect to DNA synthesis, while generally greater than the parent molecule was not significantly increased.

CTAP-III (des 1-10) was detected when microsequencing a small isoform band blotted onto Immobilon. This band contained approximately 20 picomoles of CTAP-III (des 1-14) , 20 picomoles of CTAP- III (des 1-15) and 10 picomoles of CTAP-III (des 1-10) . The (des 1-10) variant should probably be considered a naturally-occurring form in platelet extracts. It has not yet been possible to test the biologic activity of this form as a single entity.

Biologic activity studies of rCTAP-III-Leu-21 and rCTAP-III-Leu-21 (des 1-15) : Reco binant CTAP-III-Leu- 21 was subjected to the elastase cleavage procedure described above for platelet-derived CTAP-III; digestion products were applied to a heparin affinity column and fractions were eluted with 0.3M and 0.5M NaCl. The cleaved rCTAP-III fractions had a MW of approximately 7,000 Da by SDS-PAGE and microsequencing confirmed the des 1-15 structure. The marked increase in biologic activity following cleavage to the des 1-15 form is demonstrated in Figure 5. Both isoforms of rCTAP-III- Leu-21 (des 1-15) stimulated [¹ C]GAG synthesis far more than the parent molecule; the specific activity was increased by 200-700 percent. The recombinant cleavage

3 isoforms stimulated [ H]DNA synthesis markedly compared to the parental molecule which actually appeared slightly inhibitory.

Discussion The present application provides evidence for several naturally-occurring isoforms of CTAP-III not previously known, including CTAP-III (des 1-14) , and des 1-10. Further, these studies show that CTAP-III (des 1-

14) and CTAP-III (des 1-15) retain or enhance the anabolic biologic properties of uncleaved CTAP-III.

Recombinant CTAP-III-Leu-21 clearly acquired increased specific activity with respect to stimulating DNA and GAG synthesis after cleavage to the des 1-15 form. The structural relationships of the CTAP-III cleavage isoforms to CTAP-III and β-TG are illustrated in Figure 6 (4,33). The carboxyterminus of these CTAP-III isoforms described in detail was found to be intact. The "activation" of rCTAP-III-Leu-21 by removal of the amino terminal 15 residues raises the possibility that platelet-derived CTAP-III owes much or all of its biological activity to trace amounts of small isoforms coisolated with intact CTAP-III. Current evidence from comparing platelet-derived CTAP-III and CTAP-III (des 1-

15) does not support the idea that all biologic activity resides in t he cleaved form. The most degraded preparation of platelet-derived CTAP-III contained a maximum of 20% (determined at sequencing) as small isoforms. If one postulates that all of the biologic activity derives from a CTAP-III (des 1-15) component representing one fifth of the total protein, then the specific activity of the isolated des 1-15 form would be expected to be five times that of an 80%/20% mixture of CTAP-III and CTAP-III (des 1-15) . Measurements show, however, that increases in specific activity fall short of that prediction; thus it is premature to ascribe all of the biologic activity to the cleaved forms.

Coisolation of CTAP-III and its isoforms cannot be avoided during organic extraction and conventional molecular sieve chromatography. Further, most of the isoforms bind to heparin affinity columns and all bind to immunoaffinity columns. The fact that all of the isoforms discussed in this report react with polyvalent antisera raised against CTAP-III has one unfortunate consequence: published studies of plasma CTAP-III/β-TG antigen levels measured by RIA or ELISA have much less specificity than once thought.

CTAP-III and CTAP-III (des 1-15) /NAP-2 have recently been separated by reverse-phase HPLC with a gradient of acetonitrile in 0.1% trifluoroacetic acid

(34) . HPLC offers an attractive approach to separating CTAP-III from its isoforms if it is accomplished without modifying the biologic activities of the proteins. This might allow separate testing of the platelet-derived CTAP-III and its isoforms and permit the "activation by cleavage experiment'* described above for rCTAP-III-Leu- 21.

Im unoaffinity isolated CTAP-III exhibits significant microheterogeneity as determined by SDS-PAGE and IEF analytical methods. Table 3 shows that the calculated IEPs of known and hypothetical members of the CTAP-III family range from about 7 to 9.3; measured values agree reasonably well with the predicted IEPs. These data account for a substantial portion of the observed IEP heterogsneity and identify some of the cationic forms possessing increased specific biologic activity. Nonenzymatic glycosylation of lysine in CTAP- III was considered as a cause for IEP heterogeneity. Table 3 shows that pi values computed for hypothetical glycosylated CTAP-III isoforms appear to support this hypothesis. Experimentally we found that six of eight CTAP-III preparations contained modest amounts of covalently-bound glucose. It is not known whether glycosylation occurs in vivo or in vitro. It is also clear that glycosylation is not required for biologic activity, and that the degree of glycosylation of CTAP- III does not explain variations in biologic activity.

At least seven mammalian proteins with extensive homology to CTAP-III (see Tables 4 and 5) are now known. CTAP-III and/or its isoforms are potent stimulators of DNA and GAG synthesis, glucose transport, glycolysis, PGE₂ formation and plasminogen activator secretion. Further, it is reported to be a chemoattractant for neutrophils after cleavage to the des 1-15/NAP-2 form (34) . There has been little clear evidence of biologic activity for PBP and β-TG. Another platelet α-granule protein, PF-4, has extensive homology and different actions; it is noted for its heparin neutralizing properties, chemotactic activity, and has been shown to be an immunoregulator which reverses immunosuppression in mice (35) . It is pertinent that the immunoregulatory activity of PF-4 is protease-induced immediately after platelet aggregation.

A monocyte-derived protein (MDNCF/NAP-1/I1-8) has extensive homology with CTAP-III and is a potent neutrophil chemotactic agent (36) . Chick fibroblasts, activated by serum or Rous sarcoma virus (RSV) are induced to form the 9E3 protein which is homologous to CTAP-III (37,38). RSV-transformed cells form copious amounts of hyaluronic acid, as do human fibroblasts stimulated by CTAP-III. A tumorigenic hamster cell line produces increased amounts of mRNA coding for a protein (CHEF-GRO) with homology to CTAP-III (39) . Recombinant gamma-interferon treated human fibroblasts, monocytes and endothelial cells exhibit induction of a gene which codes for a protein, gamma-IP-10, that also shows homology to CTAP-III and PF-4 (40) . Melanoma growth stimulatory activity (MGSA) isolated from Hs294T melanoma cells shows striking homology to CTAP-III and is a potent mitogen which exists in two molecular weight forms (41) .

These 10 to 19-des CTAP-III amino acid sequence homologies reflect a family of materials which may play roles in inflammation, wound healing and growth (37) , and may be derived from a common ancestral gene. This plethora of structurally-related molecules, viewed in the context of the complexity of the inflammatory response, argues the importance of relating specific molecular forms to specific biologic activities. The data reported here indicate that the biologic activities of CTAP-III are generated mainly by selective NH₂-terminal proteolytic processing.

Table 1

Glucose Content of CTAP-III Charge-isomers

* — % Expected lysine not found, presumably due to glycosylation.

** = Not performed.

*** = Isolated by immunoaffinity chromatography.

Table 2

Effect of Amino Terminal Deamidation on [ C]glucosamine Incorporation

* The platelets from which P-1908 were prepared were collected recently; the other platelets had been stored for an extended period.

Table 3

Isoelectric Points of CTAP-III and Its Isoforms

Residues Ref

1-85 (2)

1-85

5-85 (42

14-85

15-85

16-85

1-85^**

1-85^*** CTAP-III glycosylated at 71 and 76 6.99

^# Isoelectric points were calculated using the IBI Pustell Sequ

Programs, version 2.02.

Numbering of residues is from (4) .

Random glycosylation of a single lysine, here residue 71, was calculation of pi, glycine was substituted in the amino acid seq

*** Random multiple glycosylation sites, here at positions 71 an assumed. No differences were observed when alternative lysine r

32, 42, 56, 60, 72 or 77) were assumed to be glycosylated.

Claims

1. A peptide of from 10 to 19 amino acids in length corresponding to the first 10 to 19 amino acids present in the N-terminal region of CTAP-III.

2. The peptide of claim 1 in substantially pure form in isolation from other peptides.

3. The peptide of claim 1 being 10 amino acids in length and corresponding to the first 10 amino acids present in the N-terminal region of CTAP-III.

4. The peptide of claim 1 being 11 amino acids in length and corresponding to the first 11 amino acids present in the N-terminal region of CTAP-III.

5. The peptide of claim 1 being 12 amino acids in length and corresponding to the first 12 amino acids present in the N-terminal region of CTAP-III.

6. The peptide of claim 1 being 13 amino acids in length and corresponding to the first 13 amino acids present in the N-terminal region of CTAP-III.

7. The peptide of claim 1 being 14 amino acids in length and corresponding to the first 14 amino acids present in the N-terminal region of CTAP-III.

8. The peptide of claim 1 being 15 amino acids in length and corresponding to the first 15 amino acids present in the N-terminal region of CTAP-III.

9. The peptide of claim 1 being 16 amino acids in length and corresponding to the first 16 amino acids present in the N-terminal region of CTAP-III.

10. The peptide of claim 1 being 17 amino acids in length and corresponding to the first 17 amino acids present in the N-terminal region of CTAP-III.

11. The peptide of claim 1 being 18 amino acids in length and corresponding to the first 18 amino acids present in the N-terminal region of CTAP-III.

12. The peptide of claim 1 being 19 amino acids in length and corresponding to the first 19 amino acids present in the N-terminal region of CTAP-III.

13. A pharmaceutical composition comprising a peptide of claim 1 in an injectable carrier.

14. A method for treating an individual suffering from a myocardial infarction by attenuating neutrophil-dependent myocardial tissue ischemic injury comprising administering to said individual a sufficient amount of the peptide of claim 1 to attenuate neutrophil invasion of myocardial tissue during reperfusion.

15. A method for treating an individual suffering from a cerebral vascular disease by attenuating neutrophil-dependent cerebral tissue ischemic injury comprising administering to said individual a sufficient amount of the peptide of claim 1 to attenuate neutrophil invasion of cerebral tissue during reperfusion.