WO1990015615A1

WO1990015615A1 - The phospholipid binding domain of factor viii

Info

Publication number: WO1990015615A1
Application number: PCT/US1990/003456
Authority: WO
Inventors: Theodore S. Zimmerman; Carol A. Fulcher; Richard A. Houghten; Paul A. Foster
Original assignee: Scripps Clinic And Research Foundation
Priority date: 1989-06-20
Filing date: 1990-06-19
Publication date: 1990-12-27
Also published as: IE902211A1; PT94427A; AU5930890A

Abstract

Peptides that inhibit the binding of Factor VIII to phospholipid which can be used for preventing thrombosis are disclosed. Methods for preparing those peptides are also disclosed.

Description

THE PHOSPHOLIPID BINDING DOMAIN OF FACTOR VIII . BACKGROUND OF THE INVENTION 1) Field of the Invention

This invention relates to a polypeptide - sequence located on the C2 domain of human Factor

VIII which inhibits Factor VIII binding to phospholipid.

2) Background of the Invention

Anticoagulants and antithrombotic agents are used as drugs to treat and prevent intravascular clotting disorders, currently a major cause of death and disease in Western society.

The process of clot formation (thrombus formation) is only incompletely understood. Upon injury to the vascular bed, a complicated sequence of reactions is initiated in order to prevent fluid loss. The first response is the activation of platelets, which adhere to the wound and undergo a series of reactions. Those reactions include the attraction of other platelets to the site, the release of a number of organic compounds and proteins, and the formation of a thrombogenic surface for the activation of the blood coagulation cascade. Through that combined sequence of reactions, a platelet plug is formed sealing the wound. The platelet plug is stabilized by the formation of fibrin threads around the plug preventing unwanted fluid loss. The platelet plug and fibrin matrix are subsequently slowly dissolved as the wound is repaired^'. For a general review, see Hemostasis and

10 Thrombosis, Bloom and Thomas, Eds., Churchill, Livingstone, NY (1981) .

A crucial Factor in the arrest of bleeding

,c_; is the activation of the coagulation cascade in order to stabilize the initial platelet plug. The coagulation cascade consists of over a dozen interacting proteins present in plasma as well as

20 released and/or activated cellular proteins. Davie, et al. , Ann. Rev. Biochem, 44, 799 (1975) ; Jackson, et al. , Ann, Rev. Biochem. 49, 767 (1980) . Each step in the cascade involves the activation of a specific

25 inactive (zymogen) form of a protease to the catalytically active form. By international agreement Wright, J. Am. Med. Assoc. 170, 325 (1959) each protein of the cascade has been assigned a Roman 30 numeral designation. The activated form of each is represented by the Roman numeral followed by a subscript "a". The activated form of the protease at each step of the cascade catalytically activates the protease involved in the subseφαent step in the cascade. In this manner a small initial stimulus resulting in the activation of a protein at the beginning of the cascade is catalytically amplified at each step such that the final outcome is the formation of a burst of thrombin, with the resulting thrombin catalyzed conversion of the soluble protein fibrinogen into its insoluble form, fibrin. Fibrin has the property of self-aggregating into threads or fibers which function to stabilize the platelet plug such that the plug is not easily dislodged. Figure l summarizes the interactions of some of the proteins involved in blood coagulation. The lack or efficiency of any of the proteins involved in the cascade would result in a blockage of the . . . . . propagation of the initial stimulus for the production of fibrin. In the middle of the cascade represented in Figure 1 is a step wherein an activated Factor IX (Factor IXa) , Factor Villa, phospholipid complex initiates the conversion of Factor X to its activated form, Factor Xa. Indeed, the assembly of a Factor Villa, Factor IXa, phospholipid complex is required for effective activation of Factor X.

The activation of Factor X by Factor IXa in the presence of Factor Villa and phospholipid is extremely significant since the two most common

10 hemophilia disorders have been determined to be caused by the decreased functioning of either Factor VIII (hemophilia A or classic hemophilia) or Factor

, r IXa (hemophilia B) . Approximately 80 percent of hemophilia disorders are due to a deficiency of Factor VIII. The clinical manifestation in both types of disorders are the same: a lack of

20 sufficient fibrin formation required for platelet plug stabilization, resulting in a plug which is easily dislodged with subsequent rebleeding at the site.

25

Additionally, concurrent with clot formation, fibrinolytic enzymes having the ability to break down the fibrin polymer are working to counteract the formation of the clot and dissolve the 30 clot.

Therefore, there are at least three steps at which antithrombotic or anticoagulant drugs may work: (1) prevention of platelet aggregation, (2) prevention of the formation of the fibrin polymer, and (3) enhancement of fibrinolytic activity.

The present invention is related to the second type of action.

10

The polypeptide of the subject invention is capable of inhibiting the association between Factor VIII and phospholipid. Thus, the polypeptide of the , c subject invention is potentially effective in preventing formation of a Factor Villa, Factor IXa, phospholipid complex, and therefore, potentially effective in inhibiting the activation of Factor X. 0 As a result, the clotting cascade is inhibited and clot formation is significantly impaired.

The utilization of such a polypeptide could not be realized until the phospholipid binding site 5 of human Factor VIII was located. The inventors of the subject invention have discovered that the phospholipid binding domain of human Factor VIII comprises amino acid residues 2303-2332 as defined by the sequence TRYLRIHPQSWVHQIALRMEVLGCEAQDLY. The amino acid numbering is that of Vehar et al. , "Structure of Human Factor VIII", Nature, Vol. 312:337-342 (1984). The polypeptide may be produced recombinantly or synthetically or derived from a human plasma source.

Although there are several drugs which act as anticoagulants on the basis that they ultimately prevent the formation of the fibrin polymer, none of them act by inhibiting the Factor Villa, Factor IXa, phospholipid complex. Known anticoagulation agents such as heparin, the coumarins, and the indanedione drugs interfere with the formation of cross-linked fibrin. While the point of action and the mechanism of those drugs is not completely understood, some undesirable side effects are known. Heparin has been known to occasionally cause severe thrombocytopenia (decrease in the number of platelets) . The orally administered coumarins can cause nonspecific dermatitis and even skin necrosis. The indanediones (e.g. phenindione) have antithyroid activity and can cause liver and kidney damage. U.S. Patent No. 4,455,290 to Olexa et al, discloses a purified polypeptide isolated from fibrinogen Fragment D that acts by directly inhibiting fibrin monomer polymerization.

U.S. Patent No. 4,801,576 to Fritz et al. discloses a purified modified hirudin polypeptide capable of directly inhibiting the action of thrombin, thereby inhibiting coagulation.

SUMMARY OF THE INVENTION

Broadly stated, the present invention comprises peptides characterized in that they inhibit Factor VIII - phospholipid binding. Accordingly, it is an object of this invention to provide a method of preventing the formation of thrombi that utilizes a polypeptide capable of inhibiting a Factor VIII - phospholipid complex, and thereby, inhibit the activation of Factor X.

The present invention comprises a polypeptide which inhibits the binding of human Factor VIII to phospholipid having the amino acid sequence TRYLRIHPQSWVHQIALRMEVLGCEAQDLY or a sequential subset thereof. The invention further comprises a human Factor VIII polypeptide which inhibits binding of human Factor VIII to phospholipid having the amino acid sequence TRYLRIHPQSWVHQIALRMEVLGCEAQDLY or a sequential subset thereof, said amino acid sequence corresponding to amino acid residues 2303-2332 or a sequential subset thereof of the human Factor VIII sequence.

10

The invention further comprises therapeutic compositions comprising an effective amount of a polypeptide which inhibits binding of human Factor ι c VIII to phospholipid having the polypeptide sequence TRYLRIHPQSWVHQIALRMEVLGCEAQDLY or a sequential subset thereof in association with a pharmaceutically acceptable carrier.

20 The invention further comprises a method of inhibiting thrombosis in a patient suffering therefrom, comprising administering to such patient an effective amount of a therapeutic composition

25 containing a polypeptide which inhibits binding of human Factor VIII to phospholipid having the sequence

TRYLRIHPQSWVHQIALRMEVLGCEAQDLY or a sequential subset thereof in association with a pharmaceutical carrier therefore.

DETAILED DESCRIPTION OF THE INVENTION

The peptides of the present invention have utility in the study of the binding of Factor VIII to phospholipid, and the formation of Factor the Villa, Factor IXa, phospholipid complex. In view of the peptides " potential ability to inhibit the activation

10 of Factor X by this complex, they have potential utility wherever it is desirable to retard or prevent the formation of a thrombus or clot in the blood , c (i.e. anti-thrombotic activity) .

In the peptide structures described herein, each amino acid residue can be in the (L) or the (D) configuration unless specified otherwise. 0 The peptides are preferably prepared using solid phase synthesis, such as that described by Merrifield, J. Am. Chem. Soc. 85,2149 (1964), although other equivalent chemical syntheses known in 5 the art can also be used, such as the syntheses of

Houghten, Proc. Natl. Acad. Sci. 82:5132 (1985) .

Solid-phase synthesis is commenced from the

C-terminus of the peptide by coupling a protected amino acid to a suitable resin, as generally set forth in U.S. Pat. No. 4,244,946, issued Jan. 21, 1982 to Rivier et al., the disclosure of which is incorporated herein by reference. Examples of syntheses of this general type are set forth in U.S. Pat. Nos. 4,305,872 and 4,316,891. Discussion of the solid-phase synthesis of a 41-residue polypeptide is set forth in Science, 213, 1394-1397 (September 1981) in an article by Vale et al. , which refers to a more detailed discussion of the synthesis, which appears in an article by Marke et al. in J. Am. Chem. Soc. ; 103, 3178 (1981).

In synthesizing the polypeptide, valine or serine having its alpha-amino group suitably protected (and, with serine, having its hydroxymethyl side chain protected) is coupled to a chlorometh lated polystyrene resin or the like. After removal of the alpha-amino protecting group, as by using trifluoroacetic acid in methylene chloride, the next step in the synthesis is ready to proceed. Other standard cleaving reagents and conditions for the removal of specific amino protecting groups may be used, as described in the open literature. The remaining alpha-amino- and side-chain-protected amino acids are then coupled stepwise in the desired order to obtain an intermediate compound connected to the resin. As an alternative to adding each amino acid separately in the synthesis, some of them may be coupled to one another prior to the addition to the growing solid-phase chain. The selection of the appropriate

10 coupling reagents is within the skill of the art.

Common to chemical syntheses of peptides is the protection of the labile side-chain groups of the

, c various amino acid moieties with suitable protecting groups at that site until the group is ultimately removed after the chain has been completely assembled. Also common is the protection of the 0 alpha-amino group on an amino acid or a fragment while that entity reacts at the carboxyl group, followed by the selective removal of the alpha-amino-protecting group to allow subsequent 5 reaction to take place at that location. Accordingly, it is common that, as a step in the synthesis, an intermediate compound is produced which includes each of the amino acid residues located in 0 the desired sequence in the peptide chain with various of these residues having side-chain protecting groups. These protecting groups are then commonly removed substantially at the same time so as to produce the desired resultant product following purification.

After the desired amino acid sequence has been completed, the intermediate peptide is removed from the resin support by treatment with a reagent, such as liquid HF, which not only cleaves the peptide from the resin, but also cleaves all the remaining side-chain-protecting groups. The polypeptide can then be purified by gel permeation followed by semipreparative HPLC, as described in Rivier et al., Peptides: Structure and Biological Function (1979) pp. 125-128. A purity of at least 93% or higher (based upon all peptides present) is reasonably obtainable and is preferred for clinical testing and/or use. Purity of 98% is practical; however, for certain in vitro applications, lower purity may be acceptable. Accordingly, the polypeptide is considered useful when it is in substantially pure form which, for purposes of this application, means at least about 50 weight percent, based upon all peptides present.

Alternatively, to obtain the Factor VIII polypeptide from human plasma, any plasma or plasma concentrate containing human Factor VIII can be employed. The novel anticoagulant polypeptide can be prepared from human Factor VIII which has been ultrapurified in accordance with the process described in U.S. Reissue Pat. No. 32,011 issued Oct. 22, 1985, the disclosure of which is hereby incorporated herein by reference. To make the desired polypeptide by recombinant DNA techniques, the portion of the gene for human Factor VIII that codes for the fragment of interest is cloned, inserted into a cell, and used to express the fragment. For a description of the gene and how to use it, see Gitschier, J. , et al., "Characterization of the human Factor VIII gene".

Nature, Vol. 312, pp. 326-330; Wood, William I., et al., "Expression of active human Factor VIII from recombinant DNA clones", Nature, Vol. 312, pp.

330-337; and Toole, John J., et al., "Molecular cloning of a cDNA encoding human antihemophilic Factor" , Nature Vol . 312 , pp. 342-347 .

The peptides of the present invention can be formulated into pharmaceutical preparations for therapeutic, diagnostic, or other uses. To prepare them for intravenous administration, the compositions are dissolved in water containing physiologically compatible substances such as sodium chloride (e.g. 0.1-2.0 M) , glycine, and the like and having a buffered pH compatible with physiological conditions. The amount to administer will depend on the activity of the particular compound administered, which can readily be determined by those of ordinary skill in the art. A technique for determining the effective amount to administer is described herein. For purposes of this disclosure, accepted shorthand designations of the amino acids have been used. A complete listing is provided herein below:

One and three-letter Amino Acid Abbreviations:

A Ala Alanine

C Cys Cysteine

D Asp Aspartic Acid

E Glu Glutamic Acid

F Phe Phenylalanine

G Gly Glycine H His Histidine

I He Isoleucine

K Lys Lysine

L Leu Leucine

M Met Methionine

N Asn Asparagine

P Pro Proline

Q Gin Glutamine

R Arg Arginine

S Ser Serine

T Thr Threonine

V Val Valine

W Trp Tryptophan

Y Tyr Tyrosine

EXAMPLE I Inhibition of Phospholipid Binding to Factor

VIII

L-a-Phosphatidyl-L-Serine (Sigma) was dissolved in methanol at a concentration of 3 ug/ l. 100 ul of this solution was placed into each well of a 96 well tissue culture plate (Linbro) and allowed to air dry at room temperature. The wells were then blocked with 200 ul of a solution of 0.05 M Tris, 0.15 M NaCl, 0.5% (W/V) gelatin, pH 7.2 (blocking buffer), for 1 hour at 37^βC. The wells were then washed with 200 ul of 0.01 M phosphate buffer, 0.15 M NaCl, 0.05% (v/v) Tween 20 (washing buffer) two times. 100 ul of purified human Factor VIII diluted to a final concentration of 1 U/ml in 0.05 M i idazole, 0.15 M NaCl, 3 mM CaCl2, pH 7.0, in the presence or absence of synthetic Factor VIII peptides in concentrations ranging from 1-100 uM was placed in each well and incubated for 2 hours at 37°C. After washing with 200 ul of washing buffer three times, 100 ul of a 5 ug/ml solution of monoclonal anti-Factor VIII light chain antibody in blocking buffer was added to each well and incubated for 1 hour at 37°C. After washing with 200 ul of washing buffer three times, 100 ul of a 1:2000 dilution of peroxidase labeled goat anti-mouse IgG (Zymed) in blocking buffer was added to each well and incubated for 1 hour at 37'C. After washing three times with 200 ul of washing buffer, O-Phenylenediamine substrate (Zymed) dissolved in 0.1 M citric acid,

0.03% hydrogen peroxide pH 5.0 was added to each well and incubated at room temperature for 5 minutes before the reaction was quenched with 100 ul of 2 M

H2SO . The color change in each well was read on a

MR 600 Microplate reader (Dynatech) .

In this system, the absence of color indicated the blocking of Factor VIII binding to phosphatidylserine by synthetic Factor VIII peptide. In a typical experiment. Factor VIII in the absence of synthetic Factor VIII peptide bound to the immobilized phosphatidylserine and produced an absorbance reading of 1.30. Incubation of Factor VIII with peptide 2305-2332 at a concentration of 10 uM resulted in a 84% reduction in the amount of Factor VIII bound and in a 99% reduction at a concentration of 100 uM. Similarly at a concentration of 100 uM, peptide 2303-2317 produced a 99% reduction in Factor VIII binding and peptide 2308-2322 produced a 95% reduction in Factor VIII binding to phosphatidylserine. Overlapping and flanking peptides, 2293-2307, and 2288-2302 at a concentration of 100 uM produced a less than 10% reduction in Factor VIII binding.

Claims

WHAT IS CLAIMED IS:

1. A polypeptide which inhibits the binding of human Factor VIII to phospholipid having the following amino acid sequence:

TRYLRIHPQSWVHQIALRMEVLGCEAQDLY.

2. A polypeptide according to claim 1 consisting essentially of the following amino acid sequence:

TRYLRIHPQSWVHQIALRMEVLGCEAQDLY.

3. A polypeptide which inhibits the binding of human Factor VIII to phospholipid having an amino acid sequence which is a sequential subset of the following amino acid sequence:

TRYLRIHPQSWVHQIALRMEVLGCEAQDLY.

4. A polypeptide which inhibits the binding of human Factor VIII to phospholipid consisting essentially of an amino acid sequence which is a sequential subset of the following amino acid sequence:

TRYLRIHPQSWVHQIALRMEVLGCEAQDLY.

5. A polypeptide according to claim 4 wherein the polypeptide consists essentially of the following amino acid sequence:

YLRIHPQSWVHQIALRMEVLGCEAQDLY

6. A polypeptide according to claim 4 wherein the polypeptide consists essentially of the following amino acid sequence:

TRYLRIHPQSWVHQI

7. A polypeptide according to claim 4 wherein the polypeptide consists essentially of the following amino acid sequence:

IHPQSWVHQIALRME. 8. A human Factor VIII polypeptide which inhibits binding of human Factor VIII to phospholipid having the following amino acid sequence:

TRYLRIHPQSWVHQIALRMEVLGCEAQDLY;

said amino acid sequence corresponding to amino acid residues 2303-2332 of the human Factor VIII sequence.

9. A human Factor VIII polypeptide which inhibits the binding of human Factor VIII to phospholipid consisting essentially of the following amino acid sequence:

TRYLRIHPQSWVHQIALRMEVLGCEAQDLY;

said amino acid sequence corresponding to amino acid residues 2303-2332 of human Factor VIII sequence.

10. A human Factor VIII polypeptide which inhibits the binding of human Factor VIII to phospholipid having an amino acid sequence which is a sequential subset of the following amino acid sequence:

TRYLRIHPQSWVHQIALRMEVLGCEAQDLY ,

said amino acid sequence corresponding to a sequential subset of amino acid residues 2303-2332 of the human Factor VIII sequence.

11. A human Factor VIII polypeptide which inhibits the binding of human Factor VIII to phospholipid consisting essentially of an amino acid sequence which is a sequential subset of the following amino acid sequence:

TRYLRIHPQSWVHQIALRMEVLGCEAQDLY;

12. A human Factor VIII polypeptide according to claim 11 wherein the Factor VIII polypeptide consists essentially of the following amino acid sequence:

YLRIHPQSWVHQIALRMEVLGCEAQDLY.

said amino acid sequence corresponding to amino acid resides 2305-2332 of the human Factor VIII sequence.

^'13. A human Factor VIII polypeptide according to claim 11 wherein the Factor VIII polypeptide has the following amino acid sequence:

TRYLRIHPQSWVHQI

said amino acid sequence corresponding to amino acid resides 2303-2317 of the human Factor VIII sequence.

14. A human Factor VIII polypeptide according to claim 11 wherein the Factor VIII polypeptide has the following amino acid sequence:

IHPQSWVHQIALRME

said amino acid sequence corresponding to amino acid resides 2308-2322 of the human Factor VIII sequence.

15. A therapeutic composition comprising an amount of a polypeptide according to claim 1 effective to inhibit thrombosis in a patient, in association with a pharmaceutically acceptable carrier.

16. A therapeutic composition comprising an amount of a polypeptide according to claim 2 effective to inhibit thrombosis in a patient, in association with a pharmaceutically acceptable carrier.

17. A therapeutic composition comprising an amount of a polypeptide according to claim 3 effective to inhibit thrombosis in a patient in association with pharmaceutically acceptable carrier.

18. A therapeutic composition comprising an amount of one or more polypeptides according to claims 4, 5, 6 , or 7 effective to inhibit thrombosis in a patient, in association with a pharmaceutically acceptable carrier.

19. A therapeutic composition comprising an amount of a polypeptide according to claim 8 effective to inhibit thrombosis in a patient, in association with a pharmaceutically acceptable carrier.

20. A therapeutic composition comprising an amount of a polypeptide according to claim 9 effective to inhibit thrombosis a patient, in association with a pharmaceutically acceptable carrier.

21. A therapeutic composition comprising an amount of a polypeptide according to claim 10 effective to inhibit thrombosis in a patient, in association with a pharmaceutically acceptable carrier.

22. A therapeutic composition comprising an amount of one or more polypeptides according to claims 11, 12, 13 or 14 effective to inhibit " thrombosis in a patient, in association with a pharmaceutically acceptable carrier.

23. A method of inhibiting thrombosis in a patient suffering therefrom, comprising administering to such patient an effective amount of a therapeutic composition according to claim 15.

24. A method of inhibiting thrombosis in a patient suffering therefrom, comprising administering to such patient an effective amount of a therapeutic composition according to claim 16.

25. A method of inhibiting thrombosis in a patient suffering therefrom, comprising administering to such patient an effective amount of a therapeutic composition according to claim 17.

26. A method of inhibiting thrombosis in a patient suffering therefrom, comprising administering to such patient an effective amount of a therapeutic composition according to claim 18. ° 27. A method of inhibiting thrombosis in a patient suffering therefrom, comprising administering to such patient an effective amount of a therapeutic composition according to claim 19.

28. A method of inhibiting thrombosis in a patient suffering therefrom, comprising administering to such patient an effective amount of a therapeutic composition according to claim 20.

29. A method of inhibiting thrombosis in a 5 patient suffering therefrom, comprising administering to such patient an effective amount of a therapeutic composition according to claim 21.

30. A method of inhibiting thrombosis in a patient suffering therefrom, comprising administering to such patient an effective amount of a therapeutic composition according to claim 22.