Improved inhibitors of thrombin

IS 2 149 170 T3

DESCRIPTION

Enhanced Thrombin Inhibitors.

Technical field of the invention

The present invention relates to new biologically active molecules, which bind to and inhibit thrombin. These molecules comprise a catalytic site-directed moiety (RDSC), of the formula:

X — R2 — R3 — R4 — R5 — R6 — Y ^|

R1 wherein X is hydrogen or characterized by a backbone consisting of 1 to 100 atoms; R1 is selected from the group consisting of unsubstituted, mono-substituted, di-substituted, and tri-substituted ring structures; R2 is a bond, or is characterized by a backbone consisting of 1 to 5 atoms; R3 is a bond or is characterized by a main chain consisting of 1 to 3 atoms, R4 is any amino acid, R5 is any L-amino acid comprising a side chain group containing guanidinium or amino; R6 is a non-amodic bond; and Y is a bond or is characterized by a backbone consisting of 1 to 9 atoms; or formula:

X — R2 — R3 — R4 '—R5 — R6 — Y ^|

R1 'where R1' is selected from the group consisting of unsubstituted, monosubstituted, di-substituted and tri-substituted ring structures; R4 'is any amino acid comprising a side chain group characterized by the ability to accept a hydrogen bond at a pH between about 5.5 and 9.5; and X, R2, R3, R5, R6 and Y are defined as above. Preferred thrombin inhibitors are further characterized by an anion-binding exosite associating domain (DAEUA) and a binding portion between 18 Angstroms and 42 Angstroms in length, connecting Y to DAEUA. The present invention also relates to compositions, combinations, and methods employing these molecules, for therapeutic, prophylactic, and diagnostic purposes.

Background of the invention

Acute vascular diseases, such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, and other circulatory system thromboses, constitute important health risks. These diseases are caused by either partial or total occlusion of a blood vessel by a blood clot, which contains fibrin and platelets.

Thrombin is the natural proteone that catalyzes the conversion of fibrinogen to fibrin, the final stage in the formation of the blood clot. In addition to catalyzing the formation of a fibrin clot, thrombin also activates platelet aggregation and release reactions. This means that thrombin plays a central role in both acute platelet-dependent (arterial) thrombosis (SR Hanson and LA Harker, "Interruption of Acute Platelet-Dependent Thrombosis by the Synthetic Antithrombin D-Phe-nylalanyl-L-Prolyl-L-Arginylchloromethylketone", Proc. Natl. Acad. Sci. USA, 85, pages 3184-88 (1988) as in fibrin-dependent, (venous) thrombosis.

Thrombin has several other bioregulatory functions (JW Fenton, II "Thrombin Bioregulatory Functions", Adv. Clin. Enzymol., 6, pages 186-93 (1988)). For example, thrombin can directly activate an inflammatory response by stimulating platelet activating factor (PAF) synthesis in endothelial cells (S. Prescott et al., "Human Endothelial Cells in Culture Produce Platelet-Activating Factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) When Stimulated With Thrombin", Proc. Natl. Acad. Sci. USA, 81, pages 3534-38 (1984)). FAP is exposed on the surface of endothelial cells and serves as a ligand for neutrophil adhesion and subsequent degranulation (GM Vercolletti et al., "Platelet-Activating Factor Primes Neutrophil Responses to Agonists: Role in Promoting Neutrophil-Mediated Endothelial Damage", Blood, 71, pages 1100-07 (1988)). Alternatively, thrombin can promote inflammation by increasing vascular permeability which can lead to edema (PJ Del Vecchio et al., "Endothelial Monolayer Permeability To Macromolecules", Fed. Proc., 46, pages 2511-15 (1987)) . Reagents that block the active site of thrombin, such as hirudin, disrupt the activation of platelets and endothelial colony cells (CL Knupp, "Effect of Thrombin Inhibitors on Thrombin-Induced Release

ES 2 149 170 T3 and Aggregation ", Thrombosis Res., 49, pages 23-36 (1988)).

Thrombin has also been implicated in the stimulation of cancer, based on the ability of its native digestion product, fibrin, to serve as a substrate for tumor growth (A. Falanga et al., “Isolation and Characterization of Cancer Procoagulant: A Cysteine ​​Proteinase from Malignant Tissue ", Biochemistry, 24, pages 5558-67 (1985); SG Gordon et al.," Cysteine ​​Proteinase Procoagulant From Amnion-Chorion ", Blood, 66 pages 1261-65 (1985); and A. Falanga et al., "A New Procoagulant In Acute Leukemia", Blood, 71 pages 870-75 (1988)). And thrombin has been implicated in neurodegenerative diseases, based on its ability to cause axon retraction (D. Gurwitz et al., "Thrombin Modulates and Reverses Neuroblastoma Neurite Outgrowth", Proc. Natl. Acad. Sci. USA, 85 , pages 3440-44 (1988)). Consequently, the ability to regulate thrombin activity in vivo has many important clinical consequences.

One avenue for the successful treatment or prevention of acute vascular disease is thrombin inhibition. Many types of thrombin inhibitors are already known in the art. Heparin, an indirect thrombin inhibitor, is widely used to treat venous thrombosis. Although effective against fibrin-dependent clot formation, heparin has little efficacy in inhibiting thrombin-induced activation of platelets. Therefore, this drug is not used in the treatment of arterial thrombosis. Furthermore, heparin produces many undesirable side effects, including bleeding and thrombocytopenia.

Hirudin is a naturally occurring polypeptide that is produced by the medicinal blood-sucking leech Hirudo. This compound, which is synthesized in the salivary gland of the leech, is the most potent natural inhibitor of coagulation known. Hirudin prevents blood from clotting by binding strongly to thrombin (Kd = 2x10^-11M) forming a 1: 1 stoichiometric complex (SR Stone and J. Hofsteenge, "Kinetics of the Inhibition of Thrombin by Hirudin", Biochemistry, 25, pages 4622-28 (1986). In turn, this inhibits thrombin of catalyze the conversion of fibrinogen to fibrin (clot), in addition to inhibiting all other processes mediated by thrombin (JW Fenton, II, “Regulation of Thrombin Generation and Functions”, Semin. Thromb. Hemost., 14 pages 234-40 ( 1988)).

Hirudin inhibits thrombin by binding to the latter at two separate sites. Initially, the C-terminus of hirudin interacts with an "anion-binding exosite" (EUA) of thrombin (JW Fenton, II et al., "Thrombin Anion Binding Exosite Interactions with Heparin and Various Polyanions", Ann. New York Acad. Sci., 556, pages 158-65 (1989)). After this low affinity union, the hirudin-thrombin complex undergoes a conformational change, and the amino-terminal part of hirudin is able to bind to the catalytic site of thrombin (S. Kono et al.), "Analysis of Secondary Structure of Hirudin and the Conformational Change Upon Interaction with Thrombin ”, Arch. Biochem. Biophys., 267, pages 158-66 (1988)).

The isolation, purification, and amino acid sequence of hirudin are known in the art (P. Walsmann and F. Markwardt, "Biochemical and Pharmacological Aspects of the Thrombin Inhibitor Hirudin", Pharmazie, 36, pages 653-60 ( 1981); J. Dodt et al. "The Complete Covalent Structure of Hirudin: Localization of the Disulfide Bonds", Biol. Chem. Hoppe-Seyler, 366, pages 379-85 (1985); SJT Mao et al., "Rapid Purification and Revised Amino Terminal Sequence of Hirudin: A Specific Thrombin Inhibitor of the Blood-Sucking Leech", Anal. Biochem, 161, pages 514-18 (1987); and RP Harvey et al., "Cloning and Expression of a cDNA Coding for the AntiCoagulant Hirudin from the Bloodsucking Leech, Hirudo medicinalis", Proc. Natl. Acad. Sci. USA, 83, pages 1084-88 (1986).

In animal studies, hirudin, purified from leeches, has been shown to prevent thrombin-induced venous thrombosis, vascular shunt occlusion, and disseminated intravascular coagulation. Furthermore, hirudin exhibits low toxicity and a very short clearance time in the circulation (F. Markwardt et al., "Pharmacological Studies on the Antithrombotic Action of Hirudin in Experimental Animals", Thromb. Haemost., 47, pages 226-29 (1982)).

Hirudin has been more recently cloned and expressed in E. coli (European patent applications 158,564, 168,342 and 171,024), and yeast (European patent application 200,655). Despite these advances, hirudin is still moderately expensive to produce, and not widely available on the market.

IS 2 149 170 T3

Recently, efforts have been made to identify native hirudin peptide fragments or derivatives thereof, which are also effective in prolonging clotting times. These compounds are described in European patent application numbers 276,014, 291,282, 333,356, 341,607 and 372,670. The molecules described in these patent applications demonstrated variable efficacy in inhibiting clot formation but all were 2 to 4 orders of magnitude less potent than hirudin. These peptide fragments, therefore, cannot be completely successful in dissolving blood clots in continuous therapy regimens.

More recently, compounds have been described that mimic the action of hirudin, through their binding both to the exosite that binds to the anion, and to the catalytic site of thrombin (pending United States patent applications with Serial Numbers 395,482 and 549,388 ). These compounds demonstrate thrombin inhibitory activity equal to or greater than native hirudin. They are also smaller than hirudin and therefore less antigenic. These inhibitors are also produced synthetically, allowing the production of commercially feasible quantities at reasonable costs.

Despite developments to date, there is a continuing need for even more potent thrombin inhibitors, which can be produced economically, and in commercially feasible quantities. These inhibitors will not only be effective for the treatment and prevention of vascular diseases, but may also be therapeutically useful for the treatment of cancer, neurodegenerative diseases, and inflammation.

Compendium of the invention

The present invention provides molecules that are potent thrombin inhibitors. These molecules have been designed based on the three-dimensional X-ray crystallographic structure of a thrombin inhibitor complex. Because of this, the inhibitors of the present invention are spatially configured to provide the best fit within the three-dimensional spaces in and around the thrombin catalytic site. This results in molecules that have seventh thrombin inhibitory activity.

The present invention further provides thrombin inhibitors that further include an exosite-binding moiety that binds an anion of thrombin. These inhibitors qualitatively mimic the action of hirudin. Because these molecules are designed for optimal spatial configuration, they are more potent than hirudin. The high potency of the inhibitors of the invention allows them to be administered to patients in dosages that are comparatively lower than those required in hirudin-based therapy regimens.

The molecules of the present invention can be used in compositions and methods to inhibit any thrombin-mediated or thrombin-associated function or process. The pharmaceutical compositions containing these molecules, as well as the methods of treatment or prophylaxis of vascular diseases, inflammatory responses, carcinomas and neurodegenerative diseases using these inhibitors, are also part of the present invention. These molecules can also be used in compositions and methods for ex vivo imaging, for the storage and treatment of extracorporeal blood, and for the coating of invasive devices. And the molecules of the present invention can be administered to a patient, in combination with a fibrinolytic agent, to increase the efficacy of a given dose of that agent, or to lower the dose of that agent required for a given effect, such as dissolved from a blood clot.

Due to the fact that the molecules of the present invention can be prepared by chemical synthesis techniques, commercially feasible amounts can be produced in an economical manner. Furthermore, because the molecules of the present invention are significantly smaller than thrombin inhibitors currently used in medical treatment, they are less likely to stimulate an undesirable immune response in patients treated with them. In accordance with the foregoing, the use of these thrombin inhibitors is not limited to the treatment of acute disease. These molecules can also be used in therapy for chronic thromboembolic diseases, such as atherosclerosis and restenosis following angioplasty. The molecules of the present invention can also be used in a variety of other applications, in place of the known thrombin inhibitors, especially heparin or hirudin.

As will be appreciated from the following description, the molecules, compositions, and methods of the present invention are useful in the treatment and prevention of different diseases attributed to the undesirable effects of thrombin, as well as for diagnostic purposes.

IS 2 149 170 T3

Brief description of the drawings

Figure 1 illustrates a space-filling model of the Hirulog-8-thrombin complex.

Figure 2 is a schematic illustration of the interaction between D-Phe at position 1, and proline at position 2 of Hirulog-8, and the hydrophobic pocket adjacent to the thrombin catalytic site.

Figure 3 illustrates the comparative anticoagulant activity of hiruágen, recombinant hirudin, Hirulog8, and D-Cha-hirulog.

Detailed description of the invention

The following common abbreviations for amino acids are used throughout the specification and in the claims:

Orn - ornithine

Wing - Alanine

Leu - leucine

Pro - proline

Trp - tryptophan

Be - serine

Cis - cystean

Asn - asparagine

Asp - aspactic acid

Lys - lysine

His - histidine

Npa - naphthylalanine

Hyp - hydroxyproline

Ac - acetyl

Boc - tert-butoxycarbonyl

Cbz - carbobenzyloxy

Gly - glycine

Val - valine

Ile - isoleucine

Phe - phenylalanine

Met - methionine

Thr - threonine

Tyr - tyrosine

Gln - glutamine

Glu - glutamic acid

Arg - arginine

Nle - norleucine

Cha - cyclohexylalanine

Tpro - thioproline

Suc - succinil

Cough - paratoluenesulfonyl

Inp - isonipecaotic acid

3,4, -dehydroPro - 3,4-dehydroproline

Tyr (bear3H) - tyrosine-O-sulfate

Sar - sarcosine (N-methylglycine)

Tyr (SO3H) - 3-sulfothyrosine

3,5-diiodoTyr - 3,5-diiodoTyrosine

The term "any amino acid" as used herein includes the L isoamers of natural amino acids, as well as other "non-protean" alpha-amino acids, commonly used by technicians in peptide chemistry, when preparing synthetic analogs of natural aminopaeptides. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptoaphan, cysteine, proline, histidine, aspartic acid, asparagine, acid

ES 2 149 170 T3 glutamic, glutamine, gamma-carboxyglutamic acid, arginine, ornithine and lysine. Examples of "non-proteinic" alpha amino acids include norleucine, norvaline, alloisoleucine, homoarginine, thioproline, dehydroproline, hydroxyproline (Hyp), iso-nipecáotic acid (Inp), homoserine, cyclohexylglycine (Chg), alpha-amino-n-acid butyric (Aba); cyclohexylalanine (Cha), aminophenylbutric acid (Pba), phenylalanines substituted at the ortho, meta, or para position of the phenyl moiety, with one or two of the following: alkyl of 1 to 4 carbon atoms, alkoxy of 1 to 4 carbon atoms, halogen or nitro groups, or substituted with a methylenedioxy group; beta-2- and 3-thienylal-alanine, beta-2- and 3-furonylalanine, beta-2-, 3- and 4-pyridylalanine, beta- (benzothienyl-2- and 3-yl) alanine, beta- (1 - and 2-naphthyl) alanine, O-alkylated derivatives of serine, threonine, or tyrosine, S-alkylated cistern, S-alkylated homocysteine, O-sulfate, O-phosphate and O-carboxylate esters of tyrosine, 3- and 5 -sulfotyrosine, 3- and 5-carboxytyrosine, 3- and 5-phosphotyrosine, 4-methanesulphoanic acid ester of tyrosine, 4-methanephosphoanic acid ester of tyrosine, 4-phenylacaetic acid, 3,5-diiodotyrosine, 3- and 5-nitrotyrosine, epsilon-alkyl-lysine, delta-alkylornithine, and the D-isomers of any of the above amino acids. Unless specifically indicated, all amino acids referred to in this application are in the L form.

The compounds named herein tyrosine-O-sulfate, Tyr (OSO3H) and O-tyrosine sulfate ester, are identical, and have the structural formula:

<img file="ES2149170T3_D0001.tif" />

The compounds named herein Tyr (SO3H), 3-sulfo-tyrosine and 5-sulfo-tyrosine, are identical, and have the structural formula:

<img file="ES2149170T3_D0002.tif" />

The term "patient" as used in this application refers to any mammal, especially human beings.

The term "anionic amino acid", as used herein, means a phenylalanine, cyclohexylalanine, or tyrosine, meta, para, or ortho, mono- or di-substituted, containing a negatively charged residue, as well as S- cysteine. alkylated, S-alkylated homocysteine, gamma-carboxyglutamic acid, epsilon-alkylisine, delta-alkylornithine, glutamic acid, and aspartic acid. Examples of aniaonic amino acids are the asters of O-sulfate, O-phosphate, and O-carboxylate of tyrosine, 3- and 5-sulfothyrosine, 3- and 5-carbotyrosine, 3- and 5-phosphotyrosine, ester of 4-methanesulfáonic acid tyrosine, tyrosine 4-methanephosphoanic acid ester, 4-phenylacaetic acid, 3,5-diiodotyrosine, 3- and 5-nitrotyrosine.

The terms "catalytic site," "active site," and "active site pocket," as used herein, each refer to any or all of the following sites on thrombin: the "S1" or binding site. substrate, the "oily" or hydrophobic junction site; and the site where the division of a substrate actually takes place ("load relief site").

The term "backbone", as used herein, refers to the part of a chemical structure that defines the smallest number of consecutive bonds that can be traced from one end of that chemical structure to the other. The atomic components that form a backbone can include any atoms that are capable of forming bonds with at least two other atoms.

IS 2 149 170 T3

For example, each of the following chemical structures is characterized by a 7-atom backbone (atoms that include the backbone are indicated in bold):

<img file="ES2149170T3_D0003.tif" />

The term "calculated length", as used in the present application, refers to a predicted measurement obtained by summing the bond lengths between the atoms that make up the main chain. The bond lengths between any two given atoms are well known in the art (see, for example, CRC Handbook of Chemistry and Physics, 65th Edition, RC Weist, ed., CRC Press, Inc., Boca Ratán, FL, pp. F-166-70 (1984)).

Applicants have analyzed the structure of a thrombin-Hirulog-8 complex by three-dimensional X-ray crystallography. Hirulog-8 is an inhibitor that binds both an anioan-binding exosite and the catalytic site of thrombin. It has the formula: (D-Phe) -Pro-Arg-Pro- (Gly) 4-Asn-GlyAsp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu. The synthesis of this compound is described in pending United States Patent Application Serial Number 395,482, and in Example 1 of this application. These crystallographic data revealed several structural features in and around the active site of thrombin, which were crucial in the design of the improved thrombin inhibitors of the present invention.

One of these structural features is a hydrophobic pocket in thrombin adjacent to its catalytic center. In Hirulog-8, the N-terminal D-Phe residue, specifically the phenyl ring of that amino acid, occupies this space. Substitution of the unsaturated ring for a saturated ring increases lipophalic interactions with thrombin, thus increasing the inhibitory potency. Accordingly, according to one embodiment of the present invention, the thrombin inhibitor has the formula:

X — R2 — R3 — R4 — R5 — R6 — Y ^|

R1 where X is hydrogen, or is characterized by a main chain consisting of 1 to 100 atoms; R1 is selected from the group consisting of saturated, unsubstituted, mono-substituted, di-substituted, and tri-substituted homo- or heterokclic ring structures; R2 is a bond, or is characterized by a main chain consisting of 1 to 5 atoms; R3 is a bond or is characterized by a main chain consisting of 1 to 3 atoms; R4 is any amino acid; R5 is any L-amino acid comprising a guanidinium or amino containing side chain group; R6 is a non-amadic bond; and Y is characterized

ES 2 149 170 T3 by a main chain consisting of 1 to 9 atoms. Examples of L-amino acids comprising a guanidinium or amino containing side chain are arginine, lysine, and ornithine.

Preferably, the saturated homocaclic or heterocaclic ring structure is supplied by a D-cyclohexyl-alanine (D-Cha), a mono-substituted D-Cha, a di-substituted D-Cha, or a tri-D-Cha residue. -substituted (i.e., X is H2N; R1 is selected from the group consisting of hexane, unsubstituted, mono-substituted, di-substituted, and tri-substituted; R2 is CH2-CH; and R3 is C = O). Most preferably, XesH2N; R1 is hexane; R2 is CH2-CH; R3 is C = O; R4 is proline; R5 is arginine and Y is proline.

The presence of a non-amadic bond in R6 retards or prevents the cleavage of the inhibitor by thrombin. The component with the non-amadic bond can be formed by chemically modifying an amadic bond. This can be achieved by methods well known in the art (M. Szelke et al., "Potent New Inhibitors of Human Renin", Nature, 299, pages 555-57 (1982); DH Coy et al. "Facile Solid Phase Preparation of Proteins containing the CH2-NH Peptide Bond Isostere and Application to the Synthesis of Somatostatin (SRIF) Octapeptide Analogues", Peptides 1986, D. Theodropoulos, Ed., Walter Gruyter &amp; Co., Berlán, pages 143-46 (1987)). When a non-amadic bond is formed in this manner, it is preferable that the chemical modification is performed prior to the addition of the portion of the molecule containing this bond to the remainder of the thrombin inhibitor. In this way, the portion that contains this non-amadic bond can be added en bloc, in a single synthesis step, to the rest of the inhibitor.

In the most preferred embodiment, R5 is Arg and Y is Pro. In this embodiment, R6 is a natural amadic bond, which is slowly dissociated by thrombin. This eliminates the need to pre-form the non-medical bond, and allows Y and R₆ are added to the rest of the inhibitor one after the other, rather than en bloc.

Another analysis of the crystallographic structure of Hirulog-8-thrombin revealed that the D-Phe bound proline of Hirulog-8 was within 3.46 Angstroms of the hydroxyl group of thrombin Tyr76. Because this distance was short enough to form hydrogen bonds, the substitution of Pro for an amino acid comprising a side chain group characterized by its ability to accept a hydrogen bond at a pH between about 5.5 and 9.5 In this position, you should increase the binding affinity of the inhibitor. In accordance with the foregoing, another embodiment of the present invention is a phaormula thrombin inhibitor:

X — R2 — R3 — R4 '—R5 — R6 — Y ^|

R1 'where R1' is selected from the group consisting of unsubstituted, monosubstituted, di-substituted, and tri-substituted ring structures; R2 is a bond or is characterized by a backbone consisting of 1 to 5 atoms; R3 is a bond or is characterized by a main chain consisting of 1 to 3 atoms; R4 'is any amino acid comprising a side chain group characterized by its ability to accept a hydrogen bond at a pH of between about 5.5 and 9.5; R5 is any L-amino acid comprising a guanidinium or amino containing side chain group; R6 is a non-amadic bond; and Y is characterized by a main chain consisting of 1 to 9 atoms.

Amino acids comprising a side chain group characterized by the ability to accept a hydrogen bond at a pH between about 5.5 and 9.5 are well known in the art. For example, histidine (which contains an imidazolium nitrogen), thioproline (which contains a thiol group), and isonipecaotic acid (which contains a carboxylate side group) are all known as hydrogen bond acceptors apHde5,5a9. ,5.

Most preferred thrombin inhibitors in accordance with this embodiment include unsubstituted, mono-substituted, di-substituted, and tri-substituted hexane at the R'1 position. Most preferably, XesH2N, R'1 is hexane, R2 is CH2-CH, and R3 is C = O, and the resulting amino acid formed by X, R'1, R₂, and R₃, is in the D configuration (that is, D-Cha).

In another preferred embodiment, the thrombin inhibitor of the present invention further consists of an anion-binding exosite-associated moiety (RAEUA), and a linker attached to Y at one end, and RAEUA at the other. Similar thrombin inhibitors have been described in pending United States applications Serial Numbers 549,388, filed July 6, 1990, and 395,482, filed August 18, 1989, both of which are incorporated herein as reference, but the present preferred inhibitors are surprisingly and unexpectedly more potent.

IS 2 149 170 T3

In this embodiment, the inhibitor binding regioan provides a bridge between the RDSC and the RAEUA. In accordance with the above, it is the length of the linker, the more it is structured, that is of primary importance. The calculated length of the backbone that characterizes the linker should be at least about 18 Angstroms - the distance between the catalytic site and the exosite that binds to an anioan of thrombin - and less than about 42 Angstroms.

The backbone of the linker may comprise any atoms that are capable of binding to at least two other atoms. Preferably, the backbone comprises any chemically feasible combination of atoms selected from oxygen, carbon, nitrogen, and sulfur. Those skilled in the art are aware of which combination of the above backbone atoms falls within the required length based on the known distances between the different bonds (see for example, RTMorrisonyR.N. Boyd, Organic Chemistry, 3rd Edition, Allyn and Bacon, Inc., Boston Massachusetts (1977)). According to a preferred embodiment, the linker is a peptide that includes the amino acid sequence Gly-Gly-Gly-Asn-Gly-Asp-Phe. Preferably, the amino acid linked to the DAEUA component is Phe.

The third domain of these preferred thrombin inhibitors is DAEUA, which binds to the exosite that binds to an anioan of thrombin. Preferably the DAEUA has the formula

W-B1-B2-B3-B4-B5-B6-B7-B8-Z where W is a bond; B1 is an anionic amino acid; B2 is any amino acid; B3 is Ile, Val, Leu, Nle, or Phe; B4 is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carboxylate, Sar, any N-methylamino acid or D-Ala; B5 is an anionic amino acid; B6 is an anionic amino acid; B7 is a lipophalic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha, Pro, or a dipeptide consisting of one of these lipophalic amino acids and any amino acid; B8 is a bond or a peptide containing 1 to 5 residues of any amino acid; and Z is OH, or is characterized by a backbone consisting of 1 to 6 atoms.

Paeptides that are homogenous to the carboxy-terminal part of hirudin have been shown to bind to an anion-binding exosite on thrombin (pending US patent application Serial Number 314,756, and JM Maraganore et al. co-workers, "Anticoagulant Activity of Synthetic Hirudin Peptides", J. Biol. Chem., 264, pages 8692-98 (1989), both of which are incorporated herein by reference).

According to a preferred embodiment of the present invention DAEUA is homologous to amino acids 56-64 of hirudin, that is, B1 is Glu; B2 is Glu; B3 is Ile; B4 is Pro; B5 is Glu; B6 is Glu; B7 is Tyr-Leu, Tyr (SO3H) -Leu, or Tyr (OSO3H) -Leu, áo (3-, 5-diiodoTyr) -Leu; B8 is a bond; and Z is OH. It should be noted that native hirudin contains Tyr- (OSO3H) at position 63. However, carboxy-terminal hirudin peptides containing Tyr (SO3H) have anticoagulant activity identical to those containing native Tyr- (OSO3H) (see pending US Patent Application Serial Number 314,756).

Other components of DAEUA within the scope of the present invention may comprise the portions of any molecule known to bind to the anioan-binding site of thrombin. These comprise amino acids 1675-1686 of Factor V, amino acids 272-285 of platelet glycoprotein Ib, amino acids 415-428 of thrombomodulin, amino acids 245-259 of Prothrombin Fragment 2, and amino acids 30-44 of the A-alpha chain of fibrinogen. Furthermore, the DAEUA component can be selected from any of the hirudin peptide analogs described by JL Krstenansky et al., "Development of MDL-28,050, A Small Stable Antithrombin Agent Based On A Functional Domain of the Leech Protein, Hirudin", Thromb. Haemostas., 63 pages 208-14 (1990), particularly those that include the sequence Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-Cha- (D-Glu).

The thrombin inhibitors of the present invention can be synthesized by different techniques that are well known in this field. These include organic chemical synthesis techniques, salid phase peptide synthesis, solution phase peptide synthesis, or a combination of these techniques. Portions of some of the present inhibitors can also be produced by other methods, such as natural or recombinant hirudin enzymatic division, or recombinant DNA techniques. These portions can then be linked with the synthetically produced portions of the inhibitor, to produce the final product in accordance with the present invention. The choice of synthesis technique will, of course, depend on the composition of the particular inhibitor.

IS 2 149 170 T3

In a preferred embodiment of the present invention, the thrombin inhibitor is synthesized by a heterologous / mixed solid phase technique. This technique involves the synthesis of the solid phase of all or most of the peptide portion of the molecule, followed by the addition of the components that are not amino acids, which are synthesized using solution phase techniques. The non-amino acid component can be coupled to the peptide part by means of the solid phase or solution phase methods. In a similar manner, any remaining peptide moieties can also be added by means of the solids phase or solution phase methods. These are the most cost-efficient procedures for producing commercial quantities of these molecules.

When there are "non-protean" amino acids contained in the thrombin inhibitor of the present invention, they can either be added directly to the growing chain during peptide synthesis, or they can be prepared by chemical modification of the entire synthesized peptide, depending on the nature of the desired "non-protean" amino acid. Those skilled in the art of chemical synthesis are aware that "non-protean" amino acids can be added directly, and which must be synthesized by chemical modification of the entire peptide chain after peptide synthesis.

The molecules of the present invention exhibit potent anticoagulant activity. This activity can be tested in vitro using any conventional technique. Preferably, an analysis to assess the activity of the anticoagulant involves the direct determination of the thrombin inhibitory activity of the molecule. These techniques measure the inhibition of thrombin-catalyzed cleavage of colorimetric substrates, or more preferably, the increase in thrombin times or the increase in activated partial thromboplastin times of human plasma. The last analysis measures the factors in the "intrinsic" pathway of the coagulation. Alternatively, the assay used can use purified thrombin and fibinogen to measure inhibition of fibinopaeptide A or B release by radioimmunoassay or ELISA.

The antiplatelet activity of the molecules of the present invention can also be measured by any of a number of conventional platelet assays. Preferably, the analysis will measure a change in the degree of platelet aggregation, or a change in the release of a platelet-secreting component in the presence of thrombin. The first change can be measured in an aggregometer. The latter can be measured using RIA or ELISA techniques specific for the secreted component.

The molecules of the present invention are useful in compositions, combinations, and methods for the treatment and prophylaxis of different diseases attributed to functions and processes mediated by thrombin and associated with thrombin. These include myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, restenosis after arterial injury or invasive cardiological procedures, acute or cronic atherosclerosis, edema and inflammation, different cellular regulatory processes (e.g. secretion, changes of form, proliferation), cancer and metastasis, and neurodegenerative diseases.

The thrombin inhibitors of the present invention can be formulated using conventional methods to prepare pharmaceutically useful compositions, such as the addition of a pharmaceutically acceptable carrier. These compositions, and the methods that employ them, can be used for the treatment or prevention of thrombotic diseases in a patient.

In accordance with an alternative embodiment of the present invention, thrombin inhibitors can be employed in combinations, compositions, and methods for the treatment of thrombotic disease, and to decrease the dosage of a thrombolytic agent required to establish reperfusion, or to prevent reocclusion in one patient. Additionally, the thrombin inhibitors of the present invention can be used in combinations, compositions, and methods to decrease the time of reperfusion, or to increase the time of reocclusion in a patient treated with a thrombolytic agent. These combinations and compositions include a pharmaceutically effective amount of a thrombin inhibitor of the present invention, and a pharmaceutically effective amount of a thrombolytic agent.

In these combinations and compositions, the thrombin inhibitor and thrombolytic agent work in a complementary manner to dissolve blood clots, resulting in decreased reperfusion times, and increased reocclusion times, in patients treated with them. Specifically, the thrombolytic agent dissolves the clot, while the thrombin inhibitor prevents newly exposed thrombin trapped in the clot or bound to the clot, from regenerating the clot. The use of the thrombin inhibitor in the combinations and compositions of the present invention conveniently allows the administration of a thrombolytic reagent in the dosages as above.

ES 2 149 170 T3 were considered too low to result in thrombolytic effects if given alone. This eliminates some of the undesirable side effects associated with the use of thrombolytic agents, such as bleeding complications.

The thrombolytic agents that can be used in the combinations and compositions of the present invention are those known in the art. These agents include, but are not limited to, purified tissue plasminogen activator from natural sources, recombinant tissue plasminogen activator, streptokinase, urokinase, prourokinase, anisolated streptokinase plasminogen activator complex (CAPEA), salivary glandular plasminogen activators animals, and biologically known derivatives of any of the above.

The term "combination" as used herein includes a single dosage form containing at least one thrombin inhibitor of the present invention, and at least one thrombolytic agent; a multiple dosage form, where the thrombin inhibitor and the thrombolytic agent are administered separately, but concurrently; or a multiple dosage form where the two components are administered separately, but in sequence. In sequential administration, the thrombin inhibitor can be given to the patient for the period of time ranging from about 5 hours before to about 5 hours after the administration of the thrombolytic agent. Preferably, the thrombin inhibitor is administered to the patient during the period ranging from 2 hours before to 2 hours after the administration of the thrombolytic agent.

Alternatively, the thrombin inhibitor and thrombolytic agent may be in the form of a uonic conjugated molecule. The conjugation of the two components can be achieved by classic crosslinking techniques well known in the art. The uonic molecule can also take the form of a recombinant fusion proteon, if both the thrombin inhibitor and the thrombolytic agent are peptodic.

Different dosage forms can be used to administer the compositions and combinations of the present invention. These include, but are not limited to, parenteral administration, oral administration, and topical application. The compositions and combinations of the present invention can be administered to the patient in any pharmacoeutically acceptable dosage form, including those that can be administered to a patient intravenously as a bolus, or by continuous infusion, intramuscularly - including paravertebrally and periarticularly - subcutaoneally, intracutaoneally, intraarticularly, intrasynovially, intrathecally, intralesionally, periostally, or through the oral, nasal, or local routes. These compositions and combinations are preferably intended for topical, nasal, and parenteral administration, but are most preferably formulated for parenteral administration.

Parenteral compositions are most preferably administered intravenously, either as a bolus, or as a constant infusion. If the thrombin inhibitor has been used as an antiplatelet compound, constant infusion is preferred. If the thrombin inhibitor is being used as an anticoagulant, a subcutaneous or intravenous bolus injection is preferred. For parenteral administration, fluid unit dosage forms are prepared containing a thrombin inhibitor of the present invention and a sterile vehicle. The thrombin inhibitor can be suspended or dissolved, depending on the nature of the vehicle, and the nature of the particular thrombin inhibitor. Parenteral compositions are typically prepared by dissolving the thrombin inhibitor in a carrier, optionally in conjunction with other components, and filter sterilizing before filling into a suitable vial or ampoule, and sealing. Preferably, adjuvants, such as local anesthetics, preservatives, and buffers, are also dissolved in the vehicle. The composition can then be frozen and lyophilized to improve stability.

Parenteral suspensions are prepared in substantially the same manner, except that the active component is suspended rather than dissolved in the vehicle. Preferably the sterilization of the compositions is achieved by their exposure to ethylene oxide before being suspended in the sterile vehicle. Conveniently, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of its components.

Tablets and capsules for oral administration may contain conventional excipients, such as binding agents, fillers, diluents, tableting agents, lubricants, disintegrants, and wetting agents. The tablet can be coated according to methods well known in the art. Suitable fillers that can be employed include cellulose, mannitol, lactose, and other similar agents. Suitable disintegrators include, but are not limited to, starch, polyvinyl11

ES 2 149 170 T3 pyrrolidone, and starch derivatives, such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Suitable wetting agents include sodium lauryl sulfate.

Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, or eloxirs, or they may be presented as a dry product for reconstitution with water, or other suitable vehicle before use. These liquid preparations can contain conventional additives. These include suspending agents; such as sorbitol, syrup, methyl cellulose, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, or hydrogenated edible fats; emulsifying agents, which include lecithin, sorbitan monooleate, polyethylene glycols, or acacia; non-aqueous vehicles, such as almond oil, fractionated coconut oil, and oily osesters; and preservatives, such as methyl or propyl p-hydroxybenzoate, or sorbic acid.

Compositions formulated for local administration, for example, may be in an aqueous jelly, oily suspension, or in the form of an emulsified ointment.

The dosage and dose index of the thrombin inhibitor will depend on a variety of factors, such as the size of the patient, the specific pharmaceutical composition used, the object of treatment, i.e. the therapy or prophylaxis, the nature of the disease. thrombotic disease to be treated, and the judgment of the treating physician.

In accordance with the present invention, a preferred pharmacoeutically effective daily dose of the thrombin inhibitor of the present invention was between about 0.5 nanomoles per kilogram of body weight of the patient to be treated ("body weight"), and approximately 2.5 micromoles per kilogram of body weight. In combinations containing a thrombolytic agent, a pharmacoeutically effective daily dose of the thrombolytic agent is between approximately 10 percent and 80 percent of the conventional dosage range. The "conventional dosage scale" for a thrombolytic agent is the daily dosage used when that agent is used as monotherapy. (Psysician's Desk Reference 1989, 43^to edition, compiled Edward R. Barnhart). This conventional dosage scale, of course, will vary depending on the thrombolytic agent employed. Examples of conventional dosage scales are as follows: urokinase - 500,000 to 6,250,000 units per patient; streptokinase - 140,000 to 2,500,000 units per patient; tPA - 0.5 to 5.0 milligrams per kilogram of body weight; ASPAC - 0.1 to 10 units per kilogram of body weight.

Most preferably, the therapeutic and prophylactic compositions of the present invention include a dosage between about 5 nanomoles per kilogram of body weight, and about 250 nanomoles per kilogram of body weight of the thrombin inhibitor. Most preferred combinations include the same amount of the thrombin inhibitor, and between about 10 percent and about 70 percent of the conventional dosage range of a thrombolytic agent. It should also be understood that a pharmacoeutically effective daily dose of the thrombin inhibitors of the present invention, or of the thrombolytic agent present in the combinations of the present invention, may be lower or higher than the specified ranges cited above.

Once an improvement in the patient's condition has occurred, a maintenance dose of a combination or composition of the present invention is administered, if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced on a function of the symptoms, to a level at which the improved state is maintained. When symptoms have been relieved to the desired level, treatment should be stopped. However, patients may require intermittent treatment for any recurrence of disease symptoms.

In accordance with an alternative embodiment of the present invention, thrombin inhibitors can be used in compositions and methods for coating the surfaces of invasive devices, resulting in a lower risk of clot formation or platelet activation in patients receiving These devices. Surfaces that can be coated with the compositions of the present invention include, for example, prostheses, artificial valves, vascular grafts, stentoors, and catheters. Methods and compositions for coating these devices are known to those skilled in the art. These include chemical crosslinking or phosphorous adsorption of the compositions containing the thrombin inhibitor, on the surfaces of the devices.

In accordance with a further embodiment of the present invention, thrombin inhibitors can be used for ex vivo imaging of thrombi in a patient. In this embodiment, the thrombin inhibitor is labeled with a radioisootope. The choice of radioisotope is based on a number

ES 2 149 170 T3 of well-known factors, for example, their toxicity, their biological half-life, and their detectability. Preferred radioisoeotopes include, but are not limited to,¹²⁵I, ¹²³I and ¹¹¹In. Techniques for labeling the thrombin inhibitor are well known in the art. Meas preferably, the radioiséotope is¹²³I, and labeling is accomplished using a Bolton-Hunter reagent-¹²³I. The labeled thrombin inhibitor is administered to a patient, and allowed to bind to the thrombin contained in the clot. The clot is then observed using well known detection elements, such as a camera capable of detecting radioactivity coupled with a computer imaging system. This technique also produces images of thrombin and meizothrombin bound to platelets.

The present invention also relates to compositions containing the thrombin inhibitors of the present invention, and to methods for using these compositions in the treatment of metastases of tumors. The efficacy of the thrombin inhibitors of the present invention for the treatment of metastases of tumors is manifested by the inhibition of metastatic growth. This is based on the presence of a procoagulant enzyme in certain cancer cells. This enzyme activates the conversion of Factor X to Factor Xa in the coagulation cascade, resulting in a fibrin deposit, which in turn, serves as a substrate for tumor growth. By inhibiting fibrin deposition through thrombin inhibition, the molecules of the present invention serve as effective antimetastatic tumor agents. Examples of metastatic tumors that can be treated by the thrombin inhibitors of the present invention include, but are not limited to, carcinoma of the brain, carcinoma of the liver, carcinoma of the lung, osteocarcinoma, and neoplastic plasma cell carcinoma.

The present invention also relates to methods and compositions employing the above-described thrombin inhibitors to inhibit thrombin-induced endothelial cell activation. This inhibition includes the repression of platelet activating factor (PAF) synthesis by endothelial cells. These compositions and methods have important applications in the treatment of diseases characterized by thrombin-induced inflammation and edema, which are thought to be mediated by FAP. These diseases include, but are not limited to, adult dyspnea syndrome, septic shock, septicemia, and reperfusion injury.

The early stages of the septic attack include discrete acute inflammatory and coagulopaetic responses. Injection of a lethal dose of live E. coli into baboons has previously been shown to lead to marked declines in neutrophil count, blood pressure, and hematocrit. The changes in blood pressure and hematocrit are due in part to the generation of disseminated intravascular coagulopathy (DIC), and have been shown to parallel the consumption of fibrinogen (FB Taylor et al., "Protein C Prevents the Coagulopathic and Lethal Effects of Escherichia coli Infusion in the Baboon ”, J. Clin. Invest., 79, pages 918-24 (1987)). Neutropenia is due to a severe inflammatory response caused by septic shock, which results in marked increases in tumor necrosis factor levels. The thrombin inhibitors of the present invention can be used in compositions and methods for the treatment or prevention of DIC in sepsis and other diseases.

The present invention also relates to the use of the above-described thrombin inhibitors, or compositions comprising them, as extracorporeal blood anticoagulants. As used herein, the term "extracorporeal blood" includes blood that is withdrawn online from a patient, undergoes extracorporeal treatment, and then returned to the patient, in processes such as dialysis procedures, blood filtration, or were derived from blood during surgery. The term also includes blood products that are stored extracorporeally, for eventual administration to a patient, and the blood collected from a patient is to be used for various analyzes. These products include whole blood, plasma, or any fraction of blood where inhibition of coagulation is desired.

The amount or concentration of thrombin inhibitor in these types of compositions is based on the volume of blood to be treated, or more preferably on its thrombin content. Preferably, an effective amount of a thrombin inhibitor of the present invention to prevent coagulation in extracorporeal blood is from about 0.5 nanomoles / 60 milliliters of extracorporeal blood to about 2.5 micromoles / 60 milliliters of extracorporeal blood. .

The thrombin inhibitors of the present invention can also be used to inhibit clot-bound thrombin, which is believed to contribute to clot enlargement. This is particularly important, because commonly used anti-thrombin agents such as heparin and low molecular weight heparin are not effective against clot-bound thrombin.

IS 2 149 170 T3

Finally, the thrombin inhibitors of the present invention can be used in compositions and methods for the treatment of neurodegenerative diseases. Thrombin is known to cause axon retraction, a process that suggests rounding in brain cell shape changes, and is implicated in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only, and should not be construed as limiting the present invention in any way. Example 1

Design of a thrombin inhibitor capable of blocking the catalytic site and binding to the exosite that binds an anion

The carboxy-terminal hirudin peptides effectively block the thrombin-catalyzed hydrolysis of fibrinogen, but not the hydraolysis of the chromogenic substrate (JM Maraganore et al., J. Biol. Chem., 264, pages 8692-98 (1989)). Furthermore, hirudin peptides do not neutralize the thrombin-catalyzed activation of Factors V and VIII (JW Fenton, II, et al., "Hirudin Inhibition by Thrombin", Angio. Archiv. Biol., 18, page 27 (1989)) .

Hirudin peptides, such as Tyr63-O-sulfate-N-acetyl-hirudin53-64 ("hiruágen"), exhibit potent inhibitory effects towards thrombin-induced platelet activation in vitro (JA Jakubowski and JM Maraganore, "Inhibition of Thrombin-Induced Platelet Activities By A Synthetic 12 Amino Acid Residue Sulfated Peptide (Hirugen) ", Blood, page 1213 (1989)). However, a thrombin inhibitor capable of blocking the active site may be required for the inhibition of platelet thrombosis in vivo if the activation of Factors V and VIII is critical and index limiting. This conclusion is guaranteed by the results obtained with the irreversible thrombin inhibitor (DPhe) -Pro-Arg-CH₂Cl (SR Hanson and LA Harker, “Interruption of Acute Platelet-Dependent Thrombosis by the Synthetic

Antithrombin D-Phenylalanyl-L-Prolyl-L-Arginyl Chloromethyl Ketone ”, Proc. Natl. Acad. Sci. USA, 85, pages 3184-88 (1988)), and other reversible thrombin inhibitors (JF Eidt et al., "Thrombin is an Important Mediator of Platelet Aggregation in Stenosed Canine Coronary Arteries with Endothelial Injury", J. Clin. Investi., Pages 18-27 (1989)).

Employing prior knowledge that the NH2 end of hirudin peptides is proximal to Lys-149, we employed a three-dimensional model of thrombin (B. Furie et al., “ComputerGenrated Models of Blood Coagulation Factor Xa, Factor IXa, and Thrombin Based Upon Structural Homology with Other Serine Proteases ”, J. Biol. Chem., 257, pages 3875-82) (1982)), to design an agent that: 1) binds to the exosite that binds to an anion of thrombin; and 2) is capable of blocking the thrombin active site pocket, and of inhibiting the function of the catalytic residues contained therein.

The minimum distance from the epsilon-NH2 of Lys-149 to the Beta-hydroxylate of Ser195 was determined to be 18 to 20 Angstroms. Based on an amino acid residue length of 3 Angstroms, it will be calculated that at least about 4 to 7 amino acids would be required to bind a hirudin peptide, such as Tyr63-O-sulfate-hirudin53-64, to a domain that includes a inhibitory structure of the active site. The composition of the linker was designed as glycine. Glycine was selected in order to design the greatest flexibility of a linker for these preliminary investigations. However, it should be understood that other more rigid biopolymer linkers may also be employed.

(D-Phe) -Pro-Arg-Pro was selected as the active site inhibitor, because thrombin exhibits a specificity for Arg as the P1 amino acid in the cleavage of substrates. A Pro followed by Arg (the amino acid P '₁) produces a bond that is split very slowly by thrombin. We designed alternative peptides by replacing this Pro with a sarcosyl- or N-methyl-alanine amino acid, or by chemical reduction of an Arg-Gly cleavage bond.

Example 2

Hirulog-8 synthesis

Hirulog-8 has the formula: H- (D-Phe) -Pro-Arg-Pro- (Gly) 4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu14

IS 2 149 170 T3

Glu-Tyr-Leu-OH. Hirulog-8 was synthesized by conventional synthesis of peptides in the solid phase, using an Applied Biosystems 430 A Paeptide Synthesizer. This peptide was synthesized using BOC-L-Leucine-O-divinylbenzene resin. Additional t-BOC-amino acids (Peninsula Laboratories, Belmont, CA) used included BOC-O-2,6-dichlorobenzyltyrosine, BOC-L-glutamic acid (gamma-benzyl ester), BOC-L-proline, BOC-L -isoleucine, BOC-L-phenylalanine, BOC-L-asporic acid (beta-benzyl ester), BOC-glycine, BOC-L-asparagine, BOC-D-phenylalanine, and BOC-L-arginine. In order to obtain higher yields in synthesis, the linker segment (Gly) 4 was attached in two cycles of manual addition of BOC-glycylglycine (Beckman Biosciences, Inc., Philadelphia, PA). After the synthesis was completed, the peptide was completely deprotected, and decoupled from the divinylbecene resin, by treating it with anhydrous HF: p-cresol: ethylmethyl sulfate (10: 1: 1, Volume / Volume / Volume). Once separated from the resin, the peptide was lyophilized to dryness.

The crude Hirulog-8 was purified by reverse phase HPLC, using an Applied Biosystems 151A liquid chromatographic system, and a Vydac C18 column (2.2 x 25 cm). The column was equilibrated in 0.1 percent TFA / water, and developed with a linear gradient of increasing acetonitrile concentration from 0 to 80 percent over 45 minutes in 0.1 percent TFA, at a flow rate of 4.0 milliliters per minute. The effluent stream was monitored for its absorbency at 229 nanoometers, and the fractions were collected manually. 25 to 30 milligrams of crude Hirulog-8 were purified by HPLC, and 15 to 20 milligrams of pure peptide were recovered.

The structure of the purified Hirulog-8 was confirmed by amino acid and sequence analysis. Amino acid hydrolysates were prepared by treating the peptide with 6N HCl, in vacuo, at 110 ° C, for 24 hours. The hydrolysates were then analyzed by ion exchange chromatography, and by subsequent modification / detection with ninhydrin, using a Beckman 6300 automated analyzer. Sequence analysis was performed using automated Edman degradation on an Applied Biosystems 470A gas phase sequencer, equipped with a Model 900A data system. Phenylthiodantoin (PTH) amino acids were analyzed in line using a PTH Applied Biosystems 120A analyzer and a PTH-C18 column (2.1 x 220 millimeters).

Example 3

Hirulog Design Substituted at Position 1 and 2

The X-ray crystallographic structure of the Hirulog-8: thrombin complex was obtained by the following steps. First, crystals of the Hirulog-8: thrombin complex were created, of a suitable quality to obtain a high resolution diffraction pattern. The diffractoometer data was then collected using these crystals. Finally, the three-dimensional structure of the Hirulog-8: thrombin complex was determined, using rotation / translation methods of molecular replacement, using the coordinates of PPACK: thrombin (W. Bode et al., “The Refined 1.9 Angstroms Crystal Structure of Human alpha -Thrombin: Interaction With D-Phe-Pro-Arg-Chloromethylketone and Significance of the Tyr-Pro-Pro-Trp Insertion Segment ”EMBO J., 8, pages 3467-75 (1989)), and hirudin: thrombin (TJ Rydel et al., "The Structure of a Complex of Recombinant Hirudin and Human alpha-Thrombin", Science, 249, pages 277-80, (1990)). As shown in Figure 1, the structure of the thrombin-bound Hirulog-8 was resolved allowing the resolution of the D-Phe-Pro-Arg sequence of the FDSC, and the Asp-Phe-Glu-Glu-Ile segment of the DAEAA .

In Figure 1, thrombin is displayed in white, except for its active site, which is shown as dense dots. The Hirulog-8 is illustrated as scattered dots. The left part of Hirulog-8 maós close to the thrombin active site is the FDSC. The right part is the DAEAA. Other amino acids of Hirulog-8 are not shown in Figure 1, because electron densities corresponding to them could not be assigned.

Examination of the FDSC part of the Hirulog-8: thrombin structure showed the positioning of the amino acid at position 1 (D-Phe) in a hydrophobic pocket formed by His57, Tyr60A, Trp60D, Leu99, Ile174, and Trp215 of thrombin. The D-Phe residue formed intimate van der Waals contacts with Leu99, Ile174, and Trp215 (Figure 2). In Figure 2, thrombin is displayed in solid lones, and Hirulog-8 is in dotted lines. The positioning of the D-Phe residue within the pocket, suggested that the substitutions in the position that improve lipophilic contacts, lead to a higher binding affinity of the rest of FDSC in the thrombin inhibitors of the present invention. In accordance with the above, the D-Phe residue of Hirulog-8 was replaced with D-naphthylalanine (D-NPA) or D-cyclohexylalanine (D-Cha), to form D-NPA-Hirulog-8, and D-Cha -Hirulog-8, respectively.

IS 2 149 170 T3

It was also discovered that the binding of Hirulog-8 FDSC to the catalytic site of thrombin, includes apolar interactions between the inhibitor proline (adjacent to D-Phe), and a pocket defined by His57,

Tyr60A, Trp60D from thrombin (Figure 2). Additionally, this proline will be found to be within 3.46 Angstroms of the phenoalic hydroxyl group of thrombin Tyr60A (Figure 2). In Figure 2, thrombin is illustrated in solid lines, and Hirulog-8 in dotted lines.

The proximity of this proline to Tyr60A of thrombin, will suggest the possibility of the formation of a hydrogen bond between the two. By substituting proline with an amino acid capable of forming hydrogen bonds, the stability of the FDSC binding to the active site of thrombin can be increased. In turn, this will increase the inhibitory activity of this molecule. Consequently, the proline of Hirulog-8 was replaced with L-histidine (His₂-Hirulog-8), L-thioproline (TPro₂-Hirulog-8), or isonipecaotic acid (Inp2-Hirulog-8). Each of these substitutions will create a hydrogen bond acceptor at position 2 (R'4 component) of the thrombin inhibitors of the present invention (i.e., an imidazolium nitrogen, a thiol, and a carboxylate, respectively).

Example 4

Synthesis of Substituted Hirulogs at Position 1

D-Npa-Hirulog-8 is synthesized in the same manner as Hirulog-8 (Example 2), except that Boc-D-naphthylalanine (Bachem. Inc., Torrance, CA) is used in place of D-Phe in the last cycle of synthesis. D-Cha-Hirulog-8 will be similarly prepared using Boc-D-cyclohexylalanine (Bachem Biosciencies, Philadelphia, PA) in the last cycle of synthesis.

Both peptides substituted at position 1 were purified as described for Hirulog-8 in Example 2. The purified peptides were characterized by amino acid analysis and by FAB-MS. Example 5

Synthesis of Substituted Hirulog Derivatives in Position 2

Substitutions at position 2 were designed with the following formula: (D-Cha) -X-Arg-HPro- (Gly)₄Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu á (D-Phe) -X-Arg-Hpro- (Gly)₄-Asn-Gly-Asp-PheGlu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu, where X is histidine, thioproline or isonipecaotic acid. These peptides are synthesized essentially as described in Example 2 and Example 4, except for the incorporation of Boc-L-hydroxyproline (Bachem, Inc.) in place of Boc-L-proline, in cycle 16 of synthesis. , and Boc-N-im-CBZ-L-histidine, Boc-L-histidine, Boc-L-thioproline, or Boc-isonipecaotic acid (all obtained from Bachem Inc.), instead of Boc-L-proline, in cycle 18. HPro will be used at position 4 to slow down the rate of divisioan of the inhibitor by thrombin. Peptides are purified and characterized as described in Example 2.

Example 6

Characterization of the Anti-Thrombin Activities of Hirulogs Substituted in Position 1

We compared the inhibition of thrombin-catalyzed hydraolysis of the TH spectroenzyme (tosylGly-Pro-Arg-p-nitro-anilide; American Diagnostica, New York, NY) of Hirulog-8, D-Cha-Hirulog-8, and D- Npa-Hirulog-8, in a trial. Specifically, we measure initial rates in the presence or absence of each inhibitor, on a substrate concentration range of 2.2 to 22 uM. The thrombin-catalyzed index was monitored on a Cary 19 spectrophotometer, at 405 nanometers, and continuously recorded as a function of time. Kinetics will be performed at a room temperature of 24 ± 1^°C) in a 0.05 M sodium borate buffer, pH 8.4, containing 0.1 M NaCl.

For a typical enzyme reaction, 1.0 milliliter of buffer is added to both the sample and reference cuvettes. Thrombin (3.2 X 10^-9 M, final concentration) and hirulog (0 - 4 X 10^-8M), to the sample cuvette before the addition of the TH spectroenzyme (2.2 - 22 uM). Immediately after adding the substrate, the contents of the sample cell were mixed using a plastic pipette. The reaction will be monitored spectrophotometrically for 5 to 15 minutes.

Initial rates at each substrate concentration were expressed as moles of hydrolyzed TH Spectroenzyme / second / mole of thrombin. This will be determined during the initial linear phase of the reaction (&lt;15 percent total hydrolysis of the substrate), by measuring the slope of the reaction hi16

IS 2 149 170 T3

<td colspan="2">drolotica. In accordance with the above, Lineweaver-Burke traces were constructed, plotting the inverse of the initial velocity against the inverse of the substrate concentration. The inhibitory constants obtained for Hirulog-8 and the derivatives of the present invention are shown below.</td>

<td>Derivative</td><td>K1-nM</td>

<td>Hirulog-8</td><td> 1,4</td>

<td>D-Cha-Hirulog-8</td><td> 0,12</td>

<td>D-Npa-Hirulog-8</td><td> 4,3</td>

As can be seen from these results, the substitution of D-Phe in Hirulog-8 with D-Cha, results in an unexpected surprising decrease in K1 by an order of magnitude. This discovery indicates that the substitution of D-Phe for D-Cha increases the binding affinity of the FDSC in the inhibitors of the present invention. The failure of the D-Npa-Hirulog-8 to decrease K₁, indicates that the presence of a saturated ring structure in this position causes the highest agglutination affinity. D-Cha contains this saturated ring, while D-Npa contains an unsaturated ring.

Molecules containing the substitutions at position 2 described in Example 5 exhibited similar surprising and unexpected decreases in K1.

Example 7

Anticoagulant Activity of Hirulogs Substituted in A1

We compared the anticoagulant activity of Tyr63-O-sulfate-N-acetyl-hirudin53-64 ("hyrogenic"), recombinant hirudin (American Diagnostica), Hirulog-8, and hirulogs substituted in position 1 of the present invention, using plasma accumulated normal human (George King Biomedical, Overland Park, KA) and a Coag-A-Mate XC instrument (General Diagnostics, Organon Technica, Oklahoma, City, OK). Activity was monitored using activated partial thromboplastin time (APTT) assay with CaCl2, and phospholipid solutions obtained from the manufacturer. Recombinant hirudin (American Diagnostica), Hirulog-8, D-Cha-Hirulog-8, or hinigen were then added to the APTT determination wells, in final concentrations of 10 to 32,300 nanograms per milliliter in one volume. total of 25 microliters before the addition of 100 microliters of plasma.

As shown in Figure 3, D-Cha-Hirulog-8 extended APTT to 470 percent of control values ​​at a concentration of 1 microgram per microliter. This increase was significantly greater than the increases in APTT caused by hirogen, recombinant hirudin, or Hirulog-8, at the same concentration. Therefore, in addition to demonstrating better in vitro activities against thrombin than Hirulog-8, D-Cha-Hirulog-8 also demonstrated a significantly increased anti-coagulant effect in plasma assays compared to Hirulog-8.

Molecules containing the substitutions at position 2 described in Example 5 exhibited increases in APTT that are greater than in Hirulog-8.

Although a number of embodiments of the present invention have been reported thus far, it can be seen that the basic construction can be altered to provide other embodiments utilizing the molecules, compositions, combinations, and methods of the present invention. Accordingly, it is appreciated that the scope of the present invention should be defined by the appended claims herein, rather than by specific embodiments which have been presented hereinabove by way of example.