The invention relates to neutralization of heparin with peptides. Background of the Invention

Applications of advancing medical technology, such as cardiopulmonary bypass, are associated with a variety of complications. Even in short term use, blood oxygenators produce sufficient activation of clotting pathways to require the use of heparin to inhibit blood coagulation.

An invariable complication of such surgical procedures is a hemorrhagic state manifested by a prolonged bleeding time, causes of which include failure to adequately neutralize heparin and the continuous stimulation of platelets, as manifested by a fall in platelet count, stimulation of thromboxane synthesis and release of platelet granule constituents (See Colman, J. Aneεthesiology , 66:595, 1987). Reversal of heparin is required to restore normal coagulation status and reduce post-operative blood loss.

Protamine is an arginine-rich polypeptide (32 amino acids from salmon) commonly used at the conclusion of cardiovascular surgical procedures to neutralize the anticlotting effects of heparin. The use of protamine, however, has been linked to several post-surgical complications, some of which are postoperative systemic hypotension, allergic reactions, catastrophic pulmonary vasoconstriction, acute pulmonary hypertension, complement activation, noncardiogenic pulmonary edema, decreased cardiac output (later event) , and thrombocytopenia/leukopenia.

The underlying biochemical basis for these physical complications is poorly understood, but allergic reactions to protamine, have been well documented. Since

protamine, usually isolated from fish, can be recognized as a foreign protein by the human immune system, patients with prior protamine exposure are at particular risk during subsequent exposures (Just Viera, Amer. Surgeon 50:151, 1984). Additionally, studies suggest that a non- immunological pathway via complement activation may be responsible for many of the acute reactions observed during protamine reversal of heparin anticoagulation. To avoid the use of protamine, a number of approaches have been proposed. Construction of bypass circuits with materials that do not activate the coagulation cascade have been suggested, as well as the use of non-heparin anticoagulate preparations. Neutralizing agents for heparin other than protamine are also currently being sought (Horrow, in Effective

Hemostasis in Cardiac Surgery, Ellison et al., eds, in press, 1988) . All of these alternatives, presently in various stages of research, have yet to reveal a suitable substitute for protamine that has gained widespread acceptance.

Platelet factor 4 (PF4) is a well characterized heparin-binding protein which is secreted from the α-granules of platelets upon aggregation (Niewiarowski et al., Nature 222:1269-1270, 1969; Levine et al., J. Biol . Che . 251:324-328, 1976). Human PF4 contains 70 amino acid residues and has a molecular weight of 7,800 daltons (Deuel et al, Proc . Natl . Acad . Sci . USA 74:2256-2258, 1977; alz et al, Throm . Res . 11:893-898, 1977). Under physiological conditions, PF4 exists as a tetramer complexed with a high-molecular-weight carrier that is also secreted by the platelets (Barber et al., Biochim . Biophys . Acta 286:312-329. 1972; Moore et al. , Biochim . Biophys . Acta 379:370-384, 1975).

Platelet factor 4 (PF4) has been proposed to play a significant role in blood coagulation due to its

binding to heparin and its ability to neutralize the physiological activity of heparin (Niewiarowski et al., supra , Levine et al., supra) . In plasma, in in vitro studies, PF4 has been demonstrated to reverse the effect of heparin on clot formation (Michalski,

Brit . J. Haematol 38:561-571, 1978). In vivo platelet concentrate has been shown to have heparin neutralizing activity when administered to humans after bypass surgery, and the effect was attributed to PF4 (Walker, Br. Heart J. 52:12, 1984).

Current models suggest that a number of factors are responsible for the efficient neutralization of heparin by PF4. For example, there is evidence that the α-helical carboxy terminus, which contains two pairs of lysines (61, 62 and 65, 66) interspersed among pairs of aliphatic residues, is essential for heparin binding. However, peptides from the C-terminal of PF4, such as PF4 58-70 and PF4 47-70 bind heparin very weakly (Rucinski et al., Thromb . Haemost . 63:493-498, 1990). In addition, monomeric low-affinity platelet factor 4 (LAPF4) , which is 50% homologous to PF4 and contains an α-helical C- terminus, also binds heparin poorly (Mayo, Biochem . 30:925-934, 1990). Thus, it has been proposed that the native conformation of the PF4, determined by two disulfide bridges 10-cys 36 and cys 12-cys 52) , as well as tetramer formation, are important for heparin binding (Loscalzo et al., Arch . Biochem . Biophys . 240:446-455, 1985; St. Charles et al., J. Biol . Chem . 264:2092-2099, 1989; Rucinski et al., supra ; Mayo, supra) . summary of the invention

The invention features a peptide which is capable of neutralizing the anticoagulant effects of heparin, and which contains the sequence, Y X__ X ₂ Y X ₃ X X ₅ Y X ₅ X ₇ Y, wherein, (a) each Y, independently, is selected from the group consisting of leucine, isoleucine, valine,

phenylalanine, tyrosine and tryptophan, and (b) each X, independently, is selected from the group consisting of alanine, arginine, asparagine, cysteine, gluta ine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine.

In preferred embodiments, at least 2 of the Y's are leucine, and more preferably, all of the Y ^, s are leucine. Preferably, two or more of the X's are lysine; more preferably X ₃ and X ₄ are lysine, or X ₆ and X ₇ are lysine; and most preferably X ₃ , X ₄ , X ₆ , and X ₇ are lysine. Also preferably, the peptide is between 8 and 60 amino acid residues in length, more preferably between 8 and 40 amino acid residues, and most preferably between 8 and 20 amino acid residues.

Neutralization of the anticoagulant effects of heparin by the peptides of the invention may be established by any standard blood coagulation assay, e.g., an in vitro Factor Xa assay as described herein. In preferred embodiments, the peptides of the invention bind heparin with an K _d less than 1.09 x 10 ^"5 , more preferably they bind heparin with a K _d less than 1.0 x 10 ^{" 6} , and most preferably they bind heparin with a K _d of 5.1 x 10 ^"5 or less. In a related aspect, the peptide of the invention is capable of self-associating to form a multimer of two or more peptides at reasonably low peptide concentrations, e.g., 0.02 to 0.6 mg/mL, under physiological conditions. Multimer formation may be tested by any standard procedure including circular dichroism spectroscopy, and size exclusion chromatography as described herein. Preferably, it is capable of forming a tetramer but may also form a hexamer or octamer. Most preferably, the peptide is RP316 (SEQ ID NO.: 1) or RP382 (SEQ ID NO. : 2) .

In another aspect, the invention features a method for neutralizing the anticoagulant effects of heparin in a mammal (preferably a human) having an undesirably high level of circulating heparin following administration of heparin in connection with a medical procedure by administering to the mammal a heparin neutralizing amount of the peptide of the invention.

Preferably, the peptide is administered in an amount which is sufficient to neutralize the anticoagulant effects of heparin administered at more than 50 units/kg of body weight of said mammal. Usually, the peptide is administered at 0.1 to 100 mg/kg of body weight of the mammal, and preferably, is administered at 10 to 30 mg/kg of body weight of the mammal. The method of the invention may be used to neutralize heparin for any medical procedure including heart surgery, cardiopulmonary bypass, organ transplantation, kidney dialysis, catheterization (e.g., cardiac catheterization, or for angioplasty) , and for the prevention of blood clot formation.

The peptides of the invention are small, stable molecules which can be readily obtained in large quantities of substantially pure material and can be used to restore normal blood coagulation status in mammals treated with heparin. These peptides have certain advantages over protamine with respect to toxicity and efficaciousness.

Other features and advantages of the invention will be apparent from the following description and from the claims.

Detailed Description Drawings

The drawings will first be briefly described.

FIG. 1 is a representation of the circular dichroism spectrum of RP316 in the mean residue ellipticity mode.

FIG. 2 is a graph of the mean residue ellipticity of RP316 at 222 nm as a function of total peptide concentration.

FIG. 3A is a chromatogram of gel filtration standards having the following molecular weights: peak 3, 67 kD; peak 4, 44 kD; peak 5, 17 kD; peak 8, 1.35 kD.

FIG. 3B is a gel filtration chromatogram of RP316. FIG. 3C is a graph of the log of the molecular weight of RP316 as a function of retention time on a Superose 12 column. FIG. 4 is a graph illustrating RP316 (D) , rPF4(»), and the C-terminal peptide PF4 58-70 (*) binding to heparin as a function of protein or peptide concentration.

FIG. 5 is a graph illustrating the effect of PF4 (D) , RP316 (♦), and PF4 59-70 (■) on Factor Xa activity.

FIG. 6 is a graph illustrating the effect of RP316 (♦), PF4 59-70 (■) , and PF4 (D) on Factor Xa activity in two experiments.

FIG. 7 is a graph illustrating the effect of PF4, RP316, and RP382 on plasma coagulation time.

There now follows a description of the syntheses of heparin-binding peptides, the analysis of the tertiary structure of these peptides, and the assays which demonstrate the heparin-binding and neutralizing characteristics of the synthesized peptides.

PF4 and recombinant PF4 (rPF4) were prepared as described previously (Cooke et al., Circulation, 85:1102- 1109, 1992).

Peptide Design and Synthesis Peptides derived from the amino acid sequence of the carboxy-terminus of PF4 were designed to create peptides with the potential for self-associating into multimers of α-helices.

Peptides PF4 58-70, RP316, and RP382 were prepared by solid phase peptide synthesis using an Applied

Biosystems Inc. 430 automated synthesizer according to standard methods using Boc/benzyl protected amino acids and anhydrous hydrogen fluoride cleavage/deprotection (e.g., see Stewart et al.. Solid Phase Peptide Synthesis, Pierce chemical Com. Rockford, II., 1984).

The peptides were purified to greater than 98% homogeneity by one pass over a 2 x 25 cm reverse phase HPLC column (Vydac) using a water/acetonitrile (0.1% trifluoroacetic acid) gradient. Peptides were identified and quantitated using a Waters Picotag (Milford, MA) amino acid analysis system and Electrospray Mass spectrometry was performed at M-Scan Inc. (West Chester, PA) , according to standard methods.

The calculated mass for RP316 is 1,939.4 daltons; the actual mass obtained was 1,939 daltons. The calculated mass for RP382 is 1,923.4 daltons; the actual mass obtained was 1,923.1 daltons. Circular Dichroism Spectrum of RP316

Spectroscopy was performed by Analytical Biotechnology Services, Boston MA according to standard methods. Samples of 0.022 mg/mL, 0.067 mg/mL, 0.20 mg/mL, 0.60 mg/mL, in phosphate buffered saline were run at 25° C using 2.0, 1.0, 0.5, and 0.2 cm path length cells respectively.

Size Exclusion Chromatography

Size exclusion chromatography of RP316 was carried out using a Superose 12 column (Pharmacia) . The column was pre-equilibrated with a buffer containing 50 mM phosphate, 0.5 M NaCl, pH 7.0. 150 ug RP316 were dissolved in 100 uL of the same buffer (1.5 mg/mL), injected onto the column, and eluted at a flow rate of 0.3 mL/min in the same buffer. The apparent molecular weight was determined by interpolation from the standard curve generated using gel filtration standards run on the same day. Heparin Binding Assay

0.5 itiL of [ ³ H]-heparin stock solution (New England Nuclear, average MW ca. 12,000) in binding buffer (10 mM Tris-HCl, pH 8.0, 0.2% bovine serum albumin, 150 mM NaCl) was mixed with increasing amounts of protein or peptide, and incubated at room temperature for 45 minutes. The samples were then filtered slowly through presoaked nitrocellulose filters (0.1 micron, 2.5 cm, Millipore VCWP 02500) and allowed to dry at 40°C for 40 minutes.

The activity was measured by liquid scintillation and the

K _d calculated as the concentration of protein at half saturation.

Human Plasma Clotting Assay 0.1 ml of Actin FS activated PTT Reagent (Baxter), 0.1 mL of freshly reconstituted human plasma (Baxter) containing 2 units/mL heparin were mixed with increasing amounts of PF4 or peptide in 5.0 mL glass test tubes. The resulting solution was incubated at 37°C for 3 minutes. Then 0.1 mL of 0.02 M CaCl ₂ was added and the time required for visible clot formation was recorded. Normal clotting time was determined using a sample without heparin. Factor Xa Assay

Heparin neutralization was measured in a Factor Xa assay as previously described (Denton et al. Biochem. J. 209:455-460, 1983). Titrations were performed in 96 well microtiter plates. To 180 uL of buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl) were added the following solutions in order: 30 uL 3 units/mL heparin (Sigma) , 30 uL 6 units/mL antithrombin III (Boehringer Mannheim) 30 uL 6 mM chromozym X (Boehringer Mannheim) , varying amounts of either PF4, RP316, or RP382, 30 uL 0.2 units/mL factor Xa (Boehringer Mannheim) . The optical density at 405 nm was measured after 6-10 minutes. Complete heparin neutralization was determined using a sample without antithrombin III.

RESULTS Two of the synthesized peptides, RP316 and RP382, have the following amino acid sequences:

RP316 AcLAALKKILKKLLESLGGC-NH ₂ (SEQ ID NO.: 1) RP382 AcLAALKKLLKKLAESLGGC-NH ₂ (SEQ ID NO. : 2)

These peptides were purified and then examined for their structural properties and their ability to neutralize heparin.

Referring to FIG. 1, the circular dichroism spectrum of RP316 is displayed in the mean ellipticity mode. These data clearly indicate that RP316 demonstrates the characteristic absorption minima (206 nm and 220 nm) and maximum (195 nm) of an cn-helical structure.

The helicity of RP316 was further examined at varying peptide concentrations, and an increase in the signal observed at 220 nm as a function of increasing peptide concentration was observed (FIG. 2) . It is well known that concentration dependent phenomena are not usually characteristic of unimolecular processes, and

thus, these data provide evidence that RP316 is present in multi eric form.

The multimeric nature of RP316 was then examined by size exclusion chromatography (FIG. 3) . A plot of the log of the molecular weight as a function of retention time (FIG. 3C) shows that RP316, with a retention time of 51.0 minutes, migrates with an apparent mass of 9,000 kD. The expected mass for a tetramer of RP316 is 7,800. Within error, the difference between these two values is not thought to be significant. Thus, these data provide additional evidence that RP316 is present in solution primarily in tetrameric form. Similar characteristics were observed when the structural properties of RP382 were examined. Having established that RP316 and RP382 are α-helical peptides which appear to form tetramers, in vitro experiments were conducted to compare the effects of these peptides and intact PF4.

Referring to FIG. 4, it was demonstrated that RP316 binds heparin with a K _d of about 5.1 x 10~ ⁷ . This is approximately a 6-fold lower affinity than the affinity of PF4 (K _d =8.5 x 10 ^"8 ) for heparin, but 20-fold higher than the affinity of the C-terminal peptide PF4 58-70 (K _d =l.l x 10 ^"5 ) . The abilities of RP316 and RP382 to prevent heparin-inhibition of the coagulation enzyme Factor Xa were compared to PF4, the PF4 59-70, and protamine (FIGS. 5 and 6). In the Factor Xa assay, 0.5 units of heparin inhibit enzymatic activity almost 90%. While PF4 59-70 failed to show any activity in this assay, on a weight/volume basis, RP316 and RP382 were as effective as PF4 and protamine at restoring Factor Xa activity.

Neutralization of heparin in human plasma was also examined (FIG. 7) . The activated partial thromboplastin time (APTT) of normal human plasma increases from its

base level of approximately 60 seconds to 400 seconds by the addition of 0.67 unit/mL of heparin. When added during the incubation, RP316 and RP382 have approximately 50% of the heparin neutralization activity of PF4 on a weight/volume basis.

These data demonstrate the successful design of small peptides which are capable of binding to heparin with sufficient affinity to neutralize the anticoagulant affects of this molecule. These peptides appear to self- associate into multimers which are most likely tetramers of α-helical peptides arranged in a four-helix bundle.

Other heparin binding, four-helix bundle peptides can be designed based on the sequences of the peptides described herein. Generally, peptides which are capable of forming four-helix bundles are amphiphilic and contain alternating pairs of lipophilic and hydrophilic amino acid residues (for example, see Ho et al. J. Am . Chem . Soc. 109:6751-6758, 1987, hereby incorporated by reference) . Thus, peptides based on the sequence of PF4 or on the heparin binding domains of other proteins can be modified to include residues that promote formation of four-helix bundles.

USE

The peptides of the invention may be easily prepared in large amounts which are substantially free of contaminants using the methods described herein. These peptides can be administered intravenously following heparin treatment, for example, using a slow drip intravenous infusion over the course of 10 minutes. The peptides of the invention may be employed following heparin treatment in any surgical procedure in which heparin is employed, or therapeutically when heparin is used to avoid clotting. A typical effective dose of these peptides is approximately 10-30 mg per kg of body

weight, based on an effective PF4 dose of 6.35 mg per kg of body weight. However, the actual dosage may vary with the dosage of heparin,

Other Embodiments Other embodiments are within the following claims. For example, included in the invention are polypeptides containing two or more domains, each of which contains the peptide sequence of the invention, connected by three or more amino acids which are capable of forming a loop (e.g., Pro-Arg-Arg) . Such polypeptides are capable of folding in a unimolecular rather than multimolecular manner to form a three-dimensional helical bundle of peptide domains which may interact with and neutralize heparin. The invention also includes peptides which are produced by recombinant techniques. For example, DNA sequences encoding the peptide can be generated using standard methods well known to those skilled in the art (see for example, Ausebel et al.. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989) . These DNA sequences can be expressed using any appropriate expression system (either prokaryotic or eukaryotic) . For example, the DNA encoding the peptide is carried on a vector operably linked to control signals capable of effecting expression in the prokaryotic host. If desired, the coding sequence may contain, at its 5' end, a sequence encoding any of the known signal sequences capable of effecting secretion of the expressed protein into the periplasmic space of the host cell, thereby facilitating recovery of the protein and subsequent purification. Prokaryotes most frequently used are various strains of E. coli; however, other microbial strains may also be used. Plasmid vectors are used which contain replication origins, selectable markers, and control sequences derived from a species

compatible with the microbial host. For example, E. coli may be transformed using derivatives of pBR322, a plasmid constructed by Bolivar et al. (Gene 2: 95, 1977) using fragments derived from three naturally-occurring plasmids, two isolated from species of Salmonella , and one isolated from E. coli. pBR322 contains genes from ampicillin and tetracycline resistance, and thus provides multiple selectable markers which can be either retained or destroyed in constructing the desired expression vector. Commonly used prokaryotic control sequences

(also referred to as "regulatory elements") are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences. Promoters commonly used to direct protein expression include the beta-lactamase

(penicillinase) , the lactose (lac) (Chang et al., Nature 198: 1056, 1977) and the tryptophan (Trp) promoter systems (Goeddel et al., Nucl. Acids Res. 8.: 4057, 1980) as well as the lambda-derived P _L promoter and N-gene ribosome binding site (Simatake et al., Nature 292:128. 1981) .

The invention also includes analogs of the peptides of the invention. Preferred analogs include peptides whose sequences differ by amino acid sequence differences or by modifications which do not destroy the analog's biological activity as measured by the heparin neutralization assays described herein. Modifications include in vivo or in vitro chemical derivatization of polypeptides, e.g, acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., by exposing the polypeptide to glycosylating affecting enzymes from cells that normally provide such processing, e.g., mammalian glycosylation enzymes. Also embraced are versions of the same primary amino acid sequence that have phosphorylated amino acid residues, e.g.,

phosphotyrosine, phosphoserine, or phosphothreonine; and analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non- naturally occurring or synthetic amino acids, e.g., β or γ amino acids. Preferred analogs also include peptides which are modified for the purpose of increasing peptide stability, e.g., one or more desaturated peptide bonds, or non-peptide bonds.

SEOUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: James Boyd

(ii) TITLE OF INVENTION: HEPARIN NEUTRALIZATION WITH

MUTIMERIC PEPTIDES

(ϋi) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson

(B) STREET: 225 Franklin Street

(C) CITY: Boston

(D) STATE: Massachusetts

(E) COUNTRY: U.S.A.

(F) ZIP: 02110-2804

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5" Diskette, 1.44 Mb

(B) COMPUTER: IBM PS/2 Model 50Z or 55SX

(C) OPERATING SYSTEM: MS-DOS (Version 5.0)

(D) SOFTWARE: WordPerfect (Version 5.1)

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(Vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: U.S.S.N. 07/932,456

(B) FILING DATE: August 17, 1992

( iii) ATTORNEY/AGENT INFORMATION:

(A) NAME: PAUL T. CLARK

(B) REGISTRATION NUMBER: 30,162

(C) REFERENCE/DOCKET NUMBER:00231/066WO1

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (617) 542-5070

(B) TELEFAX: (6lV) 542-8906

(C) TELEX: 200154

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18

(B) TYPE: amino

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Leu Ala Ala Leu Lys Lys lie Leu Lys Lys Leu Leu Glu Ser Leu Gly

5 10 15

Gly Cys

(2) INFORMATION FOR SEQUENCE IDENTIFICATION NUMBER: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Leu Ala Ala Leu Lys Lys Leu Leu Lys Lys Ala Glu Ser Leu Gly Gly

5 10 15

Gly Cys