BG61670B2 - Thrombin inhibitors - Google Patents

Abstract

The invention relates to new biologically active moleculeswhich inhibit the thrombin and are bound to it. The moleculesinclude a component directed against the catalytic place (CSDM)having the formulawhere X means hydrogen or has a basic chain including from 1 to100 atoms; R1 is from the group consisting of unsubstituted, mono-di- and trisubstituted saturated ring structures; R2 is a bond orhas a basic chain consisting from 1 to 5 atoms, R3 is a bond or isa basic chain consisting from 1 to 3 atoms; R4 is any amino acid;R5 is every L-amino acid including guanidine- or aminocontainigroup in the side chain; R6 is non-amide bond, and Y has a basicchain consisting from 1 to 9 atoms, or having the formulawhere R1 means the group consisting of unsubstituted mono-, di-and trisubstituted ring structures; R4 is any amino acid includinga group in its side chain capable for forming hydrogen bond at pHfrom 5.5 to 9.5; and X, R2, R3, R5, R6 & Y are defined asindicated. The preferable thrombin inhibitors also have domain forassociation with anionbinding exoplace (ABEAM) and linking sectorof length between 18A and 42A connecting Y with ABEAM. Theinvention also relates to preparations, combinations, etc. and tomethods using molecules for therapeutical, prophylactic anddiagnostic purposes.

Description

Technical field

The present invention relates to novel biologically active molecules that bind to and inhibit thrombin. These molecules include a (b) catalytic site directed amount (CSDM) of the formula XR ₂ -R ₃ -R ₄ -R ₅ -R ₆ -Y

I in which X is hydrogen or is a chain consisting of 1 to 100 atoms; R 1 is selected from the group consisting of unsubstituted, mono- and di-substituted saturated ring structures; R ₂ is a bond or a chain consisting of 1 to 5 atoms; R ₃ is a bond or a chain consisting of 1 to 3 atoms; R ₄ is any amino acid; R ₃ is any L-amino acid that includes a guanidine or amino group in the side chain; R ₆ is a non-amide bond; and Y is a chain consisting of 1 to 9 atoms; or the formula:

XRRRRRY | 2 3 4 5 0 in which R 1 is selected from the group consisting of unsubstituted, mono-, di- and tri-substituted ring structures; R ₄ is any amino acid including in its side chain a group characterized by the ability to form a hydrogen bond at a pH between 5.5 and 9.5; and X, R ₂ , R ₃ , R ₃ , R ₆ and Y are defined as above. Preferred thrombin inhibitors are further characterized by an anion binding exome site association domain (ABEAM) and a linker region of length between ΑδΑ and 42Λ that binds Y to ABEAM. This invention also relates to preparations, combinations and methods that utilize 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 artery occlusion and other thrombosis of the circulatory system pose major health risks. Such diseases are caused by partial or complete obstruction of a blood vessel with a blood clot containing fibrin and platelets (platelets).

Thrombin is a natural protein that catalyzes the conversion of fibrinogen to fibrin - the final step in blood clotting. In addition to catalyzing the formation of fibrin clots, thrombin also activates platelet aggregation and releasing reactions. This means that thrombin plays a central role as in acute thrombocyte-dependent (arterial) thrombosis [S.R. Hanson and L.A. Harker, “Interruption of Acute Platelet-Dependent Thrombosis by Synthetic Antithrombin D-Phenylalanyl-L-Prolyl-L-Aroinylchliromethylketone”, Proc, Natl. Acad. Sci. USA, 85, pp. 3184-88 (1988)] and in fibrin-dependent (venous) thrombosis.

Thrombin has some other bioregulatory roles [J.W. Fenton, II, Thrombin Bioregulatoru Functions, Adv. Clin. Enzymol., 6, pp. 186-93 (1988)]. For example, it can directly activate the 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 (l-alkyl-2acetyl- sn-glycero-3-phosphocholine) When Stimulated With Trombin ”, Proc. Natl. Acad. Sci. USA, 81, pp. 3534-38 (1984)]. PAF is located on the surface of endothelial cells and serves as a ligand for neutrophil adhesion and subsequent degranulation [G.M. Vercolletti et al., “Platelet-Activating Factor Primes Neutrophil Responses to Agonists: Role and Promoting Neutrophil-Mediated Endothelial Damage,” Blood, 71, pp. 1100-07 (1988)]. Alternatively, thrombin can cause inflammation by increasing vascular permeability, which can lead to edema (a) [PJ. Del Vecchio et al., “Endothelial Monolayer Permeability To Macromolecules,” Fed. Proc., 46, pp. 2511-15 (1987)]. Reagents that block the thrombin active site, such as hirudin, suspend the activation of platelets and endothelial cells [C.L. Knupp, “Effect of Thrombin Inhibitors on Thrombin-Induced Release and Aggregation,” Thrombosis Res., 49, pp. 23-36 (1988)].

Thrombin is also involved in carcinogenesis due to the ability of its native degraded product, fibrin, to serve as a

6 (6 70 Tumor Growth Substrate [A. Falanga el;> L, “Isolation and Characterization of Cancer Procoagulant: A Cystein Proteinase from Malignant Tissue”, Biochemistry, 24, pp. 5558-67 (1985); SG Gordon et al. ., "Cysteine Proteinase Procoagulant From Amnion-Chorion", Blood, 66, pp. 1261-65 (1985); and A. Falanga et al., "A New Procoagulant In Acute Leukemia", Blood, 71, pp. 870- 75 (1988)] Thrombin is also implicated in neurodegenerative diseases based on its ability to cause neurite retraction] D. Gurwitz et al., “Thrombin Modulates and Reverses Neuroblastoma Neurite Outgrowth”, Proc. Natl. Acad. Sci. 85, pp. 3440-44 (1988)] Therefore, the ability to regulate in vivo thrombin activity has many important clinical applications.

Thrombin inhibition is an approach for the successful treatment and prevention of acute vascular diseases. Many types of thrombin inhibitors are already known. Heparin, an indirect thrombin inhibitor, is widely used for the treatment of venous thrombosis. Although effective against fibrin-dependent coagulation, heparin is poorly effective in inhibiting thrombin-induced platelet activation. Therefore, this medicine is not used for the treatment of arterial thrombosis. Moreover, heparin causes a number of undesirable side effects, including haemorrhages (bleeding) and thrombocytopenia.

Hirudin is a natural polypeptide that is produced by the blood-sucking leech Hirudo medicinalis. This compound, which is synthesized in the salivary gland of the leech, is the most effective natural inhibitor of coagulation, which is known. Hirudin protects blood from coagulation by binding tightly to thrombin (K ^ XIO ^ M) is a 1: 1 stoichiometric complex [S.R. Stone and J. Hofsteenge, “Kinetics of Thrombin Inhibition by Hirudin”, Biochemistry, 25, pp. 4622-28 (1986)]. This practically inhibits thrombin catalyzed conversion of fibrinogen to fibrin (clot) and also inhibits all other thrombin mediated processes [J.W. Fenton, II, “Regulation of Thrombin Generation and Functions”, Semin. Thromb. Hemost., 14, pp. 234-40 (1988)].

Hirudin inhibits thrombin by binding to the latter at two separate sites.

Initially, the C-terminus of hirudin interacts with the aniol-binding exome (ABE) in thrombin [J.W. Fenton, II et al., “Thrombin Anion Binding Exosite Interactions with Heparin and Various Polyanions, Ann. New York Acad. Sci., 556, pp. 158-65 (1989)]. After this low affinity binding, the hirudinthrombin complex undergoes a conformational change and the amino-terminal thrombin region acquires the ability to bind to the thrombin catalytic site [S. Kono et al., “Analysis of the Secondary Structure of Hirudin and the Conformational Change Upon Interaction with Thrombin”, Arch. Biochem. Biophys., 267, pp. 158-66 (1988)].

The isolation, purification and amino acid sequence of thrombin are known [P. Walsmann and F. Markwardt, “Biochemical and Pharmacological Aspects of the Thrombin Hirudin Inhibitor”, Pharmazie, 36, pp. 653-60 (1981); J. Dodt et al., “The Complete Covalent Structure of Hirudin: Localization of Disulfide Bonds,” Biol. Chem. Hoppe-Seyler, 366, pp. 37985 (1985); S.J.T. Mao et al., “Rapid Purification and Revised Amino Terminal Sequence of Hirudin: A Specific Thrombin Inhibitor of Blood-Sucking Leech”, Anal. Biochem., 161, pp. 514-18 (1987); and R.P. Harvey et al., “Cloning and Expression of a cDNA Coding for Anti-Coagulant Hirudin from Blood-Sucking Leech, Hirudo Medicinalis”, Proc. Natl. Acad. Sci. USA, 83, pp. 1084-88 (1986)].

In animal studies, leeches purified from leeches effectively prevented venous thrombosis, obstruction of vascular shunts, and thrombin-induced disseminated intravascular coagulopathy. In addition, hirudin exhibits low toxicity and very short circulation clearance] F. Markwardt et al., “Pharmacological Studies on Antithrombotic Action of Hirudin in Experimental Animals,” Thromb. Haemost., 47, pp. 226-29 (1982)].

Most recently, hirudin was cloned and expressed in E. coli [EP 158 564, EP 168 342 and EP 171 024] and in yeast EP 200 655. Despite these achievements, hirudin is still moderately expensive to manufacture and is not commercially available. is widespread.

Efforts have recently been made to identify peptide fragments of native hirudin or derivatives thereof, and the blood efficiently prolongs the time of blood61670 stoirance. Such compounds are described in EP 276 014, EP 291 982, EP 333 356, EP 341 607 and EP 372 670. The molecules described in these patent applications show different efficacy in inhibiting blood clotting, but all are 2 to 4 orders of magnitude slightly active from hirudin. therefore, such peptide fragments may not be fully satisfactory for dissolution of blood clots in subsequent (on-going) treatment regimens.

Compounds have been described that mimic the action of hirudin by binding both to the anion-binding exome and the catalytic site of thrombin (US 395 482, now abandoned and US 5 196 404). These compounds exhibit thrombin-suppressive activity equal to or greater than native hirudin. They are also smaller than hirudin and therefore less antigenic. These inhibitors are also manufactured synthetically, which enables the production of commercially available quantities at reasonable prices.

Despite its achievements, there is still a need for more active thrombin inhibitors, which can be produced cheaply and in commercially available quantities. Such inhibitors would not only be effective in treating or preventing vascular disease, but could also find therapeutic utility in the treatment of cancer, neurodegenerative diseases and inflammation.

SUMMARY OF THE INVENTION

According to the present invention, molecules are provided that are potential thrombin inhibitors. These molecules are designed on the basis of the three-dimensional X-ray crystallographic structure of a thrombin inhibitor complex. Therefore, the inhibitors of the present invention are of a spatial configuration that provides the best fit in the three-dimensional space in and around the catalytic site of thrombin. As a result, molecules having optimal thrombin-inhibitory activity are obtained.

The invention also provides thrombin inhibitors, which further comprise an ingredient that binds to the anion-binding exome of thrombin. These inhibitors mimic the action of chlrudine. Because these molecules are intended to have an optimal spatial configuration, they are more active than hirudin. The high activity of the inhibitors of the invention allows them to be administered at relatively lower doses than those required in hirudin-based treatment regimens.

The molecules of the invention may be used in preparations and methods for inhibiting any thrombin-mediated or thrombin-associated function or process. The scope of the invention also encompasses pharmaceutical preparations containing these molecules, as well as methods for treating or preventing vascular diseases, inflammations, cancers and neurodegenerative diseases using these inhibitors. These molecules can also be used in ex vivo scintigraphy preparations and methods, for the storage and treatment of extracorporeal blood and for the cover of invasive devices. The molecules of the invention may be introduced into the patient in combination with a fibrinolytic agent in order to increase or decrease the efficacy of a given dose of that agent required for a given effect, for example to dissolve a blood clot.

Due to the fact that the molecules of the present invention can be prepared by chemical synthesis techniques, it is possible to produce commercially available quantities cheaply. Moreover, since the molecules of the present invention are significantly smaller than the thrombin inhibitors used for medical treatment, they are less likely to stimulate an undesired immune response in the patients treated with them. Accordingly, the use of these thrombin inhibitors is not limited to the treatment of an acute illness. These molecules can also be used in the treatment of chronic thromboembolic disorders, such as atherosclerosis and restenosis after angioplasty. The molecules of the present invention can also be used in a variety of other applications in place of known thrombin inhibitors, in particular heparin and hirudin.

As will be apparent from the description, the molecules, preparations and methods of this invention are useful for treating and preventing various diseases due to undesirable effects of thrombin, as well as for diagnostic purposes.

Short description of the figures

Figure 1 shows the spatial model of the Hirulog-8-thrombin complex;

Figure 2 schematically shows the interaction between D-Phe at position 1 and proOrp-ornithine

Ala - alanine Leu - leucine Pro - proline Trp - tryptophan Ser - serine Cys - cysteine Asn - asparagine Asp - aspartic acid Lys - lysine His - histidine Npa - naphthylalanine Nur - hydroxyproline Ac - acetyl

BOC is tert-butoxycarbonyl

Cbz - carbobenzyloxy

3,4, -dehydroPro-3,4, dehydroproline Tyr (OSO ₃ H) -thyrosine-Osulfate 3-, 5-diiodoTyr-3-, 5-fluorophenyl 3HH

The terms each amino acid or any amino acid are interchangeable and as used herein include natural amino acid isomers as well as other non-protein α-amino acids commonly used by peptide chemist when preparing synthetic analogs natural aminopeptides. Natural amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, γ-carbamide, γ-carbamide lysine. Examples of non-allin at position 2 of Hirulog-8 and the hydrophobic furrow adjacent to the catalytic site of thrombin and Figure 3 shows the comparative anticoagulant activity of surgeon, recombinant hirudin, surgeon-8 and D-Cha-surgeon.

Detailed description of the invention

The following commonly used amino acid abbreviations are used throughout the specification and claims:

Gly - glycine

Vai - valine lie - isoleucine

Phe - phenylalanine

Met - methionine

Thr - threonine

Tug is tyrosine

Gin is glutamine

Glu - glutamic acid

Arg - arginine

Nle - norleucine

Cha is cyclohexylalanine

TPo - thioproline

Sue - succinyl

Tos is paratoluenesulfonyl

Inp - isonipecotinic acid

Sar - sarcosine (N-methylglycine)

Tyr (SO ₃ H) -3-sulfothyrosine α-amino acids include norleucine, norvaline, alloisoleucine, homoarginine, thioproline, dehydroproline, hydroxyproline (Nor), isonipecotinic acid (Inp), homoserine, cyclohexylglycine (Chyhohexylglycine (Ch) -butyric acid (Aba), cyclohexylalanine (Cha), aminophenylbutyric acid (Pba), phenylalanines substituted in the ortho-, meta- or para-positions of the phenyl nucleus by one or two of the following groups: (C 1 -C ₄ ) alkyl, (C _{(-C 4)} alkoxy, halogen or nitro groups or substituted with a methylenedioxy group: β-2- and 3-thienyl alanine-alanine, β-2- and 3-furanilalani , β2-, 3- and 4-pyridylalanine, P- (benzothienyl-2- and 3-yl) alanine, β- (1- and 2-naphthyl) alanine, O-alkyl titrated derivatives of serine, trsonine or tyrosine, S- alkylated cysteine, S-alkylated homocysteine, O-sulfate, O-phosphate and carboxylate esters of tyrosine, 3- and 5-sulfothyrosine, 3- and 5-carboxytyrosine, 3- and

5-phosphotyrosine, 4-methanesulfonate tyrosine ester, 4-methanesulfonate tyrosine ester, 4-phenylacetic acid, 3,5-diiodothyrosine, 3- and 5-nitrotyrosine, ε-alkyllysine, Δ-alkylornithine and D-isomers of each the above amino acids. All amino acids cited in this application are in L-form, unless specifically noted otherwise.

The compounds referred to herein as tyrosine-O-sulfate, Tyr (OSO ₃ H) and tyrosine O-sulfate ester are identical and have the structural formula:

Soon ι SN-SN. Nit,

The compounds referred to herein as Tyr (SO ₃ H), 3-sulfothyrosine and 5-sulfothyrosine are identical and have the structural formula:

SOj-H *

The term patient as used in this application refers to all mammals, in particular humans.

The term anionic amino acid as used herein means methate, para ortho-, mono- or disubstituted phenylalanine, cyclohexylalanine or tyrosine containing a negatively charged group, as well as S-alkylated cysteine, S-alkylated homocysteine, γ-carboxyglutarboxylate, -alkyllysine, Δalkylornithine, glutamic acid and aspartic acid. Examples of anionic amino acids are the O-sulfate, O-phosphate and carboxylate esters of tyrosine, 3- and 5-sulfothyrosine, 3- and 5-carbothyrosine, 3- and 5-phosphotyrosine, 4-methanesulfonate tyrosine ester, 4-methanesphosphine -phenylacetic acid, 3,5-diiodothyrosine and 3- and 5-nitrotyrosine.

The terms catalytic site, active site and active site furrow as used, each refer to all or any of the following thrombin sites: substrate-binding or S, site; hydrophobic binding or grease site; and the place where the decomposition of the substrate (the site of load shifting) actually takes place.

The term basic chain as used herein refers to a section of a chemical 5 that defines the smallest number of sequential bonds that can be traced from one end to the other of this chemical structure. The atoms that make up this chain can include any 10 atoms that are able to form bonds with at least two other atoms.

For example, each of the following chemical structures is characterized by a basic chain of 7 atoms.

II

The term calculated length in the description refers to a predetermined length obtained by summing the lengths of the bonds between the atoms that form the 35 basic chain. The bond lengths between any two given atoms are well known [see, e.g., CRC Handbook of Chemistry and Physics, 65th Edition, R.C. Weist, ed., CRC Press, Inc., Boca Raton, Fla., Pp. F-16640 70 (1984)].

Applicants have analyzed the structure of the thrombin-Hirulog-8- complex by three-dimensional X-ray crystallography. The surgeon-8 is an inhibitor that binds to both the ani45 on-binding exome and the catalytic site of thrombin. It has the formula: (D-Phe) -Pro-Arg-Pro- (Gly) ₄ -Asn-Gly-Asp-PheGlu-Glu-Ile-Pro-Glu-Tyr-Leu. The synthesis of this compound is described in US 395 482, now 50 abandoned, and in Example 2 of this application. These crystallographic data reveal some of the structural features in and around the active thrombin meat meson that are central to the design of the improved thrombin inhibitors of the invention.

One of these structural features is the hydrophobic thrombin furrow adjacent to its catalytic center. In Hirulog-8, the N-terminal D-Phe moiety, especially the phenyl ring of this amino acid, occupies this place. Substitution of the unsaturated ring with a saturated one enhances the lipophilic interactions with thrombin, thereby increasing the inhibitory potential. Therefore, according to one application of the present invention, the thrombin inhibitor has the formula:

XR ₂ -R ₃ -R ₄ -R _J -R ₆ -Y in which X is hydrogen or a basic chain consisting of 1 to 100 atoms; R 1 is selected from the group consisting of unsubstituted, mono-, di- and tri-substituted saturated homocyclic or heterocyclic ring structures; R ₂ is a bond or a basic chain consisting of 1 to 5 atoms; R ₃ is a bond or a basic chain consisting of 1 to 3 atoms; R ₄ is any amino acid; R ₅ is any L-amino acid that includes a guanidine or amino group in the side chain; R ₆ is a non-amide bond; and Y is a basic chain consisting of 1 to 9 atoms. Examples of L-amino acids that include guanidine- or amino-containing side chains are arginine, lysine and ornithine.

It is preferred that the saturated homocyclic or heterocyclic ring structure be supplied from D-cyclohexylalanine (DCha), monosubstituted D-Cha, disubstituted D-Cha, or trisubstituted D-Cha residue (for example, X is Η ₂ Ν; R ₍ is CH ₂ -CH and R ₃ is C-O) Preferably X is H ₂ N; R 1 is cyclohexane; R ₂ is CH ₂ CH; R ₃ is C 1 O; R ₄ is proline; R ₅ is arginine and Y is proline.

The presence of a non-amide bond at R ₆ slows or inhibits the breakdown of the thrombin inhibitor. The non-amide bond component may be formed by chemical modification of the amide bond. This can be achieved by methods that are well known [M. Szelke et al., “Potent New Inhibitors of Human Renin,” Nature, 299, pp. 555-57 (1982); DH Coy et al., “Facile Solid Phase

Proteins preparation containinig the CH ₂ -NH Peptide Bond Isosierc and Application to the Synthesis of Somatostatin (SRIF) Octapeptide Analogues ”, Peptides 1986, D. Theodropoulos, Ed., Walter Gruyter & Co., Berlin, pp. 143-46 (1987)]. When the non-amide bond is thus formed, it is preferable that the chemical modification is carried out before the portion of the molecule containing this bond is added to the remainder of the thrombin inhibitor. Thus, the portion containing this non-amide bond can be added en bloc in a single synthetic step to the rest of the inhibitor.

In the most preferred application, R _s is Arg and Y is Pro. In this annex, R ₆ is a natural imide bond that is slowly broken by thrombin. This avoids the need to pre-form a non-amide bond and allows Y and R ₆ to be added to the rest of the inhibitor more sequentially than en bloc.

Further analysis of the crystallographic structure of the Hirulog-8-thrombin shows that the proline-D-Phe bond of Hirulog-8 is within 3.4bA of the hydroxyl group of Tug76 in thrombin. As this distance is close enough to form a hydrogen bond, the substitution of Pro at this position with an amino acid comprising in its side chain a group characterized by the ability to form a hydrogen bond at a pH of between about 5.5 and 9.5 , would increase the binding affinity of the inhibitor. Accordingly, another application of this invention is a thrombin inhibitor of the formula:

X-R, -R, -R-R.-R.-Y

3 4 5 o

I

R. ' in which R 1 'is selected from the group consisting of unsubstituted, mono-, di- and tri-substituted saturated ring structures; R ₂ is a bond or a basic chain consisting of 1 to 5 atoms; R ₃ is a bond or a basic chain consisting of 1 to 3 atoms; R ₄ is any amino acid, including in the side chain group, characterized by the ability to form a hydrogen bond at a pH between about 5.5 and 9.5; R ₅ is any L-amino acid that includes a guanidine- or amino-side group; R ₆ is a non-amide bond;

and Y having a basic chain consisting of 1 to 9 atoms.

The amino acids containing in their side chain a group forming a hydrogen bond at a pH of between about 5.5 and 9.5 are well known. For example, histidine containing imidazole nitrogen, thioproline containing a thiol group, and isonepecotinic acid containing a carboxylate side group are known hydrogen bond acceptors at pH 5.5 to 9.5.

More preferred thrombin inhibitors according to this annex include unsubstituted, mono-, di-, and trisubstituted at the position R < 2 > Preferably X is H ₂ N, R ₍ 'is cyclohexane, R ₂ is CH ₂ -CH and R ₃ is C = O, with the resulting amino acid formed by X, R 1', R ₂ and R ₃ being in the D-configuration ( i.e., D-Cha).

In another preferred application, the thrombin inhibitor according to the invention further comprises an anion binding exomyel association component (ABEAM) and a linker linked on one side to Y and ABEAM on the other. Similar thrombin inhibitors are described in US 549 388, filed July 6, 1990, and US 5,196,404; and US 395 482, filed Aug. 18, 1989, now abandoned. Both applications have been included in the literature, but the inhibitors described surprisingly and unexpectedly prove to be more active.

In this application, the inhibitor linker region provides a bridge between CSDM and ABEAM. Accordingly, the length of the linker rather than its structure is of paramount importance. The calculated length of the main chain characterizing the linker should be at least about 18A - the distance between the catalytic site and the anionic binding exome of thrombin - and less than about 42A.

The linker main chain may include any atoms capable of binding at least two other atoms. The basic chain consists predominantly of any chemically possible combination of atoms selected from oxygen, carbon, nitrogen and sulfur. It is known which combination of the above atoms in the main chain falls within the required length, based on the distance between different bonds [see, for example, R.T. Morrison and R.N. Boyd,

Organic Chemistry, 3rd Edi'.ion. Allyn and Bacon. Inc., Boston, Mass.; (1977)]. According to a preferred embodiment, the linker is a peptide that includes the amino acid sequence Gly-Gly-Gly-Asn-Glv-Asp-Phe. Preferably, the amino acid associated with the ABEAM component is Phe.

The third domain of these preferred thrombin inhibitors is ABEAM, which binds to the anion-binding thrombin exosome. It is preferred that ABEAM has the formula:

^w - ^B r ^B ₂ -B ₃ -B ₄ -B ₅ -B ₆ -B ₇ -B ₈ -Z;

in which W is a bond; B (is an anionic amino acid; B ₂ is any amino acid; B ₃ is Le, Val, Leu, Nle or Phe; B ₄ is Pro, Hyp, 3,4-dehydro-R 20, thiazolidine-4-carboxylate, Sar, any N-methyl amino acid or D-Ala; B ₅ is an anionic amino acid; B ₆ is an anionic amino acid; B ₇ is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, Le, Val, Cha, Pro or dipeptide consisting of one of these lipophilic amino acids and any amino acid; B ₈ is a bond or peptide containing from one to five residues of any amino acid; and Z is OH or characterized by a basic chain, p oyashta of between 1 and 6 carbon atoms.

Peptides that are homologous to the carboxyterminal region of hirudin bind to the anion-binding exomyst on thrombin [US 314 756 and J. M. Maraganore et al., “Anticoagulant Activity of Synthetic Hirudin Peptides”, J. Biol. Chem., 264, pp. 8692-98 (1989); both of which are incorporated herein by reference].

According to a preferred application of this invention, ABEAM is homologous to the amino acids 56-64 of hirudin, i.e., B _| is Glu; B ₂ is Glu; B ₃ is Le; B ₄ is Pro; B ₅ is Glu; B ₆ is Glu; B ₇ is Tyr-Leu, Tyr (SO ₃ H) -Leu, Tyr (OSO ₃ H) -Leu or (3-, 5-diode Tug) -Eee; B ₈ is a bond; and Z is OH. Native hirudin contains Tyr (OSO ₃ H) at position 63. Carboxyterminal hirudin peptides, however, which contain Tyr (SO ₃ H), have the same anticoagulant activity as those containing native Tyr (OSO ₃ H) [US 314 756].

Other ABEAM components within the scope of this invention may include those regions of any molecule that are known to bind to the anion-binding site of thrombin. These include amino acids 1675-1686 of Factor V, amino acids 272-285 of platelet glycoprotein 1b, amino acids 415-428 of thrombomodulin, amino acids 245-259 of the prothrombin Fragment 2, and amino acids 30 to 44 of AA. In addition, the ABEAM component can be selected from any of the hirudin peptide analogs described by J.L. Krstenansky et al, “Development of MDL28,050, A Small Stable Antithrombin Agent Based on a Leech Protein Functional Domain, Hirudin”, Thromb. Haemostas., 63, pp. 208-14 (1990), especially those comprising the Asp-Tyr-Glu-Pro-Jle-Pro-Glu-GluAla-Cha- (D-Glu) sequence.

The thrombin inhibitors of the present invention can be synthesized by various methods that are well known. These include the method of organic chemical synthesis, solid phase peptide synthesis, solution-phase peptide synthesis, or a combination of these methods. Sections of some of the inhibitors of this invention may also be prepared by other methods, such as enzymatic cleavage of natural or recombinant hirudin or by recombinant DNA techniques. These portions can then be coupled to the synthetically produced portions of the inhibitor to obtain the final product according to the invention. The choice of synthesis method will, of course, depend on the composition of the particular inhibitor.

According to a preferred application of this invention, the thrombin inhibitor is synthesized by mixed heterologous / solid phase synthesis involving solid-phase synthesis of all or most of the peptide region of the molecule, followed by the addition of non-amino acid components. The non-amino acid can be attached to the peptide region by solid-phase or solution-phase methods. Similarly, all other peptide regions can also be added by solid-phase or solution-phase methods. This creates the most efficient methods for the cheap production of industrial quantities of these molecules.

When non-protein amino acids are contained in the thrombin inhibitor of this invention. they can be added either directly to the growing strand during peptide synthesis or obtained by chemical modification of the fully synthesized peptide depending on the nature of the desired non-protein amino acid. Chemical synthesis specialists are well aware of which non-protein amino acids can be added directly and which need to be synthesized by chemical modification of the finished peptide chain after peptide synthesis.

The molecules of the present invention exhibit potential anticoagulant activity. The latter can be tested in vitro using any conventional method. Preferably, the anticoagulant activity test involves direct determination of the thrombin inhibitory activity of the molecule. By such a method, the inhibition of thrombin-catalyzed degradation of colorimetric substrates is measured, or preferably the rise in thrombin times or the increase of activated partial thrombo platelet times in human plasma. The latter test measures factors in the immanent coagulation pathway. Alternatively, the test administered may use purified thrombin or fibrinogen to measure the inhibition of the release of fibrinopeptides A or B by radioimmunoassay or ELISA (enzyme-linked immunosorbent assay).

The antiplatelet activity of the molecules of this invention can also be measured by any of a series of conventional platelet tests. It is preferred that the test measures a change in the degree of platelet aggregation or a change in the secretion of platelet components by the presence of thrombin. The former can be measured in an aggregator. The latter can be measured using RIA (radioimmunoassay) or ELISA-specific techniques for the secreted component.

The molecules of the present invention are useful in formulations, combination preparations and methods for the treatment and prevention of various diseases due to thrombin-mediated or thrombin-associated functions and processes. These include myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, obstruction of the peripheral arteries, restenosis after arterial or post-invasive cardiac procedures, acute or chronic atherosclerosis, elements (s) and inflammation (cells) e.g., secretion, changes in form, proliferation), cancer and metastases, neurodegenerative diseases.

The thrombin inhibitors of the present invention can be incorporated into medicaments using conventional methods for the preparation of pharmaceutically acceptable formulations, such as the addition of a pharmaceutically acceptable carrier. These preparations and methods for their use can be used to treat or prevent thrombotic diseases.

According to an alternative application of the present invention, thrombin inhibitors may be used in combination and other preparations and methods for treating thrombotic diseases and for reducing the dose of thrombolytic agent necessary to establish reperfusion or to stop reocclusion. In addition, thrombin inhibitors according to the invention can be used in combination and other preparations, and methods for reducing reperfusion time or increasing the time of reocclusion in a patient treated with a thrombolytic agent. The compositions include a pharmaceutically effective amount of a thrombin inhibitor according to the present invention and a pharmaceutically effective amount of a thrombolytic agent.

In these combination and other preparations, the thrombin inhibitor and the thrombolytic agent act in a complementary way to dissolve blood clots, leading to a decrease in reperfusion time and prolongation of reocclusion times in the patient being treated. In particular, the thrombolytic agent dissolves the clot, while the thrombin inhibitor prevents the newly discovered clot or related thrombin from regenerating the clot. The use of the thrombin inhibitor in combination preparations and other preparations of this invention has the advantage of allowing the administration of a thrombolytic reagent at doses previously thought to be too low to lead to thrombolytic effects if administered alone. . This avoids 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 combination preparations and the compositions of the present invention are known. Such agents include tissue plasminogen activator, purified from natural sources, recombinant tissue plasminogen activator, streptokinase, urokinase, prourokinase, anisolated streptokinase-plasminogen activator complex (ASPAC), plasminogen activin (plasminogen activin, plasminogen activin (ASPAC), plasminogen activin .

The term combination preparation as used herein includes a single dosage form containing at least one thrombin inhibitor of this invention and at least one thrombolytic agent; a multiple dosage form in which the thrombin inhibitor and the thrombolytic agent are administered separately but competitively; or a multiple dosage form in which the two components are administered separately but sequentially. In sequential administration, the thrombin inhibitor may be administered over a period ranging from about 5 hours before up to about 5 hours after administration of the thrombolytic agent. It is preferred that the thrombin inhibitor is introduced into the patient over a period ranging from 2 hours before 2 hours after administration of the thrombolytic agent.

Alternatively, the thrombin inhibitor and the thrombolytic agent may be in the form of a single, conjugated molecule. Conjugation of the two components can be achieved by standard cross-linking techniques that are well known. The single molecule may also take the form of a recombinant fusion protein if the thrombin inhibitor and the thrombolytic agent are peptides.

The formulations, including the combination preparations of the invention, are administered parenterally, orally and topically. They are administered to the patient in any pharmaceutically acceptable dosage form, including those which may be administered to the patient intravenously at a single time (bolus) or by continuous infusion (intramuscular), intramuscular λ I 6 7 L including paravertebral and psiarticular subcutaneous, intracutaneous, intracutaneous intra-articular, intrasynovial, intrathecal, intralesional, periosteal or by oral, nasal or topical methods. It is preferred that the preparations, including the combined ones, be adapted for topical, nasal, oral and parenteral administration, preferably for preparation for parenteral administration.

It is most advisable to administer the parenteral preparations intravenously either as a single bolus or as a continuous infusion. If a thrombin inhibitor is used as an antiplatelet compound, a subcutaneous or intravenous single injection is preferred. For parenteral administration, single dose liquid formulations containing the thrombin inhibitor of the present invention and a sterile vehicle are prepared. The thrombin inhibitor may be suspended or dissolved depending on the nature of the vehicle and the nature of the particular thrombin inhibitor. Parenteral preparations are usually prepared by dissolving the thrombin inhibitor in a vehicle, possibly together with other components, sterilized by filtration before filling suitable vials or ampoules and sealing. It is also preferred to dissolve adjuvants such as topical anesthetics, preservatives and buffering agents in the vehicle. The preparation may then be frozen or lyophilized to improve stability.

Parenteral suspensions are prepared in the same way, except that the active ingredient is suspended rather than dissolved in the vehicle. Preferably, sterilization of the preparations is achieved by treatment with ethylene oxide prior to suspension in a sterile vehicle. The benefit of incorporating surfactants or wetting agents into the formulation is to facilitate the even distribution of its components.

Tablets or capsules for oral administration may contain conventional excipients, such as adhesive agents, fillers, solvents, tabletting agents, lubricants, disintegrants and wetting agents. The tablet may be coated according to well known methods. Suitable excipients that may be used include cellulose, mannitol, lactose and the like. Suitable disintegrants may include starch, polyvinylpyrrolidone, 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 elixirs or may be presented as dry reconstitution products with water or other suitable vehicle. Such liquid preparations may contain conventional additives. The latter include suspending agents; such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel, hydrogenated edible fats; emulsifying agents that include lithium, sorbitan monooleate, polyethylene glycols or Senegalese resin; non-aqueous vehicles such as almond oil, fractionated coconut oil and fatty esters; and preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid.

Topical formulations may, for example, be in aqueous jelly, an oil suspension or in the form of an emulsified ointment.

The dosage and dosage regimen of the thrombin inhibitor will depend on various factors such as the patient's weight, the specific pharmaceutical preparation being used, the purpose of treatment, i.e., therapy or prophylaxis, the nature of the thrombotic disease being treated, and the judgment of the treating physician.

According to the present invention, a pharmaceutically effective daily dose of the thrombin inhibitor of this invention is preferably between 0.5 and 2.5 nmol / kg of body weight of the patient. In preparations containing a thrombolytic agent, the pharmaceutically effective daily dose of thrombolytic is between about 10 and 80% of the conventional dose. The conventional dose of thrombolytic agent is the daily dose used when the agent is administered in monotherapy. [Physician's Desk

61670 '

References 1989, 43rd Edition, Edward R. Barnhart, publisher]. This conventional dose varies depending on the thrombolytic agent used. Examples of conventional doses are the following: urokinase - 500,000 to 6,250,000 units / patient; streptokinase - 140,000 to 2,500,000 units / patient; tPA - 0.5 to 5.0 mg / kg body weight; ASPAC - 0.1 to 10 units / kg body weight.

It is most preferred that the therapeutic and prophylactic compositions of the present invention include a dose of thrombin inhibitor between about 5 nmol / kg body weight and about 250 nmol / kg body weight. Most preferred formulations include the same amount of thrombin inhibitor and between 10 and 70% of the conventional dose of thrombolytic agent. It should also be understood that the daily pharmaceutically effective dose of both the thrombin inhibitor of this invention and the thrombolytic agent, in the combination preparations of the invention, may be below or above the specific limits cited above.

Once an improvement in the patient's condition has occurred, a maintenance dose of a combination or other preparation of the invention is administered, if necessary. Subsequently, the dose or frequency of administration, or both, may be reduced as a function of symptoms to a level where the improved state is maintained. Once the symptoms have resolved to the desired level, treatment should be discontinued. However, intermittent treatment may be necessary after any recurrence of disease symptoms.

According to an alternative application of this invention, thrombin inhibitors can be used in preparations and methods to cover the surfaces of invasive devices, which results in a lower risk of clot formation or platelet activation in patients introducing such devices. Surfaces that can be coated with the compositions according to the invention include, for example, prostheses, artificial valves, grafts and catheters. Methods and preparations for covering these surfaces are known to those skilled in the art. These include chemical cross-linking and physical adsorption of thrombi new inhibitor preparations onto the surfaces of the devices

According to a further application of the present invention, thrombin inhibitors can be used for ex vivo thrombus scintigraphy in a patient. In this annex, the thrombin inhibitor is labeled with a radioisotope. The choice of radioisotope is based on a number of well known factors, for example, toxicity, biological half-life, detectability. Preferred radioisotopes include, but are not limited to, ¹²⁵ l, ¹²³ l and ¹¹ ppm. Thrombin inhibitor labeling techniques are well known. It is most preferred that the radioisotope is ^123l and the marking is carried out using the ^I23 -Bolton-Hunter reagent. The labeled thrombin inhibitor is introduced into the patient and allowed to bind to the clot contained in the clot. The clot is then monitored using well-known detection methods, such as a camera capable of detecting radioactivity associated with a computer image acquisition system. This technique also gives an idea of platelet-bound thrombin and mesotrombin.

The present invention also relates to preparations containing thrombin inhibitors according to the invention and to methods of using such preparations for the treatment of tumor metastases. The efficacy of thrombin inhibitors in the treatment of tumor metastases is demonstrated by the inhibition of metastatic growth. This is based on the presence of procoagulant enzyme in certain cancer cells. This enzyme activates the conversion of Factor X into Factor Xa in the coagulation cascade, leading to the deposition of fibrin, which actually serves as a substrate for tumor growth. By inhibiting fibrin deposition by inhibiting thrombin, the molecules of the invention serve as effective agents against metastatic tumors. Examples of metastatic tumors that can be treated with the thrombin inhibitors of this invention include, but are not limited to, brain cancer, liver cancer, lung cancer, osteocarcinoma, and plasmacytoma.

The invention also relates to methods and preparations using the above described nits thrombin inhibitors to inhibit thrombin-induced activation of endothelial cells. This inhibition involves the repression of platelet activating factor (PAF) synthesis by endothelial cells. These preparations and methods have important applications in the treatment of diseases characterized by thrombin-induced inflammation and edema (a) that are thought to be mediated by PAF. Such diseases include adult respiratory distress syndrome, septic shock, septicemia and reperfusion injury.

The early stages of septic shock include discrete, acute inflammatory and coagulopathic responses. Injection of lethal doses of lethal doses of live E. coli results in a dramatic decrease in neutrophil count, blood pressure and hemocrit. Changes in blood pressure and hemocrit are due in part to the development of Disseminated Intravascular Coagulopathy (DIC) and have been shown to develop in parallel with fibrinogen consumption [F.B. Taylor et al., “Protein C Prevents the Coagulopathic and Lethal Effects of Escherichia coli Infusion in the Baboon,” J. Clin. Invest., 79, pp. 918-25 (1987)]. Neutropenia is the result of severe inflammatory responses caused by septic shock that lead to a sharp rise in tumor necrosis factor level. The thrombin inhibitors of the invention can be used in preparations and methods for treating or preventing DIC in septicemia and other diseases.

The invention also relates to the use of the inhibitors described above, or preparations thereof, as anticoagulants for extracorporeal blood. As used herein, the term "extracorporeal blood" includes blood removed in-line by a patient undergoing extracorporeal treatment and then reintroduced into the patient through dialysis procedures, blood filtration, or blood bypass during surgery. The term also includes blood products that are stored extracorporeally for eventual introduction into a patient, as well as blood taken from a patient for use in various tests. Such products include total blood, plasma or any other blood fraction that suppresses coagulation suppression.

The amount or concentration of thrombin inhibitor in these types of preparations is based on the volume of blood to be treated, in particular the thrombin content. It is preferred that the effective amount of thrombin inhibitor according to the invention for preventing coagulation in extracorporeal blood be from about 0.5 nmol / 60 ml extracorporeal blood to about 2.5 pmol / 60 ml extracorporeal blood.

The thrombin inhibitors of the invention can also be used to inhibit thrombin associated with a clot and contribute to the growth of a clot. This is especially important since commonly used antithrombin agents, such as heparin and low molecular weight heparin, are ineffective against clot bound thrombin.

The thrombin inhibitors of the invention can be used in preparations and methods for the treatment of neurodegenerative diseases. Thrombin is known to cause neurite retraction, a process that is thought to be related to changes in the shape of brain cells in the direction of their rounding, and is involved in neurodegenerative diseases such as Alzheimer's and Parkinson's disease.

The following examples illustrate the invention without limiting its scope.

Examples of carrying out the invention

Example 1. Design of a thrombin inhibitor capable of blocking the catalytic site and binding to the anion-binding exome.

The carboxyterminal hirudin peptides block thrombin-catalyzed hydrolysis of fibrinogen but not hydrolysis of chromogenic substrates [J.M. Maraganore et al., J. Biol. Chem., 264, pp. 869298 (1989)]. Moreover, the hirudin peptides do not neutralize the thrombin catalyzed activation of Factors V and VIII [J.W. Fenton, 11 et al., “Hirudin Inhibition by Thrombin,” Angio. Archiv. Biol., 18, p. 27 (1989)].

Hirudin peptides, such as Tug _and -O-sulph.sub.3 - acetylchirudin I ₆₄ ("hirugen"), show potential inhibitory effects of thrombin cell activation by 61670 in vitro [JA Jakubowski and JM Maraganore, "Inhibition of Thrombin" Platelet Activities By A Synthetic 12 Amino Acid Residue Sulfated Peptide (Hirugen) ”, Blood, 5 p. 1213 (1989)]. However, thrombin inhibitor capable of blocking the active site may be required to inhibit platelet thrombosis in vivo if the activation of Factors V and VIII is critical and constitutes a rate-determining step. This conclusion is justified by the results obtained with the irreversible thrombin inhibitor (D-Phe) -ProArg-CH ₂ C1 [SR Hanson and LA Harker, “Interruption of Acute Platelet-Dependent 15 Trombosis by the Synthetic Antithrombin DPhenylalanyl-L-Prolyl-L -Arginyl Chloromethyl Ketone ”, Proc. Natl. Acad. Sci. USA, 85, pp. 318488 (1988)] and other reversible thrombin inhibitors [JF Eidt et al., “Thrombin is an 20 Important Mediator of Platelet Aggregation in Stenosed Canine Coronary Arteries with Endothelial Injury”, J. Clin. Invest., 84, pp. 18-27 (1989)].

Knowing that the NH 2 terminus of hirudi-25 peptides is close to Lys-149, a three-dimensional thrombin model is used [B. Furie, et al., “Computer-Generated Models of Blood Coagulation Factor Xa, Factor IXa, and Thrombin Based Upon Structural Homology with Other Serine 30 Proteases”,. J. Biol. Chem., 257, pp. 3875-82 (1982)] to design an agent that: 1) binds to the anion-binding exome of thrombin; and 2) is able to block the furrow at the active site in thrombin by inhibiting 35 the function of the catalytic residues present therein.

The minimum distance from Lys-149 ε-ΝΗ ₂ to the β-hydroxyl of Ser195 - 18-2θΑ is determined. Based on a length of 40 3A / amino acid residue, it is estimated that at least 4-7 amino acids will be required to attach a hirudin peptide, such as Tug _63- O-sulfate-hirudin _{53 64} , to a domain comprising structure for inhibition of the 45 active site. The linker is intended to be composed of glycine. Glycine is selected to maximize linker flexibility in preliminary studies. However, it should be known that other, 50 more rigid biopolymer linkers may be used.

Sequence selected (D-Phe)

Pro-Arg-Pro as an inhibitor of the active site, because upon substrate hydrolysis, thrombin exhibits Arg specificity as the amino acid P _r Pro following Arg (the amino acid P 2) forms a bond that is very slowly cleaved by thrombin. Alternative peptides are designed by replacing this Pro (residue) with the amino acid sarcosyl- or N-methylalanine or by chemical reduction of the Arg-Gly cleavage bond.

Example 2. Synthesis of Surgeon-8.

The surgeon-8 has the formula: H- (D-Phe) Pro-Arg-Pro- (Gly ₄ ) -Asn-Gly-Asp-Phe-Glu-Glulle-Pro-Glu-Glu-Tyr-Leu-OH. It is synthesized by conventional solid phase peptide synthesis using the Applied Biosystems 430 A. peptide synthesizer. This peptide is synthesized using the BOC-L-leucine-O-divinylbenzene resin. Other t-BOC-amino acids used (Peninsula Laboratories, Belmont, Calif.) Include BOC0-2,6-dichlorobenzyltyrosine, BOC-L-glutamic acid (gamma-benzyl ester), BOCL-proline, BOC-L-isoleucine, BOC- L-phenylalanine, BOC-L-aspartic acid (β-benzyl ester), BOC-glycine, BOC-L-asparagine, BOC-D-phenylalanine and BOC-L-arginine. To achieve higher yields from the synthesis, the (Cl ₄ ) _4- linker segment is joined in two cycles by manually adding BOC-glycylglycine (Beckman Biosciences, Inc., Philadelphia, Pa.). After synthesis was complete, the peptide was completely unblocked and separated from the divinylbenzene resin by treatment with anhydrous NB: p-cresol: ethylmethyl sulfate (10: 1: 1, v / v / v). After removal from the resin, the peptide was lyophilized to dryness.

The crude Hirulog-8 was purified by reverse phase HPLC (high performance liquid chromatography) using Applied Biosystems 151A liquid chromatography system and a Vydac C ₁₈ column (2.2X25 cm). The column was equilibrated with 0.1% TFA / water and eluted with a linear gradient of increasing acetonitrile concentration from 0 to 80% in 0.1% TFA for 45 minutes at a flow rate of 4.0 ml / min. The eluent flux was counted for absorption at 229 nm and the fractions were collected manually. Purify 25-30 mg of crude Hirulog-8 by HPI.C to give 15-20 mg of pure peptide.

J 6'0

The structure of the purified Hirulog-8 was confirmed by amino acid sequence analyzes. Amino acid hydrolysis is obtained by treating the peptide with 6N HCl in vacuo at 110 ° C. The hydrolysates were then analyzed by ion exchange chromatography and subsequent ninhydrin derivatization / detection using the Beckman 6300 automated analyzer. Sequence analysis was performed by automated Edman decomposition in an Applied Biosystems 470A gas-phase sequencer equipped with the Model 900 data system. Phenylthiohydanthionic acids (PTH) were analyzed online using the Applied Biosystems 120A PTH analyzer and the PTHC ₁₈ column (2.1X220 cm).

Example 3. Design of Surgeons substituted at positions 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 of appropriate quality are obtained to obtain a high resolution diffraction profile. Then, using these crystals, the diffractometry data were collected. Finally, the three-dimensional structure of the Hirulog-8: thrombin complex is determined using molecular substitution rotation / translation methods using the coordinates of PPACK: thrombin [W. Bode et al., “The Refined 1,9A Crystal Structures of Human α-Thrombin: Interactio With D-Phe-Pro-Arg-Chloromethylethylone and Significance of the Tyr-Pro-Pro-Trp Insertion Segment”, EMBO J., 8 , pp. 4367-75 (1989)] and the hirudin: thrombin complexes [T.J. Rydel et al., “The Structure of a Recombinant Hirudin and Human a-Thrombin Complex,” Science, 249, pp. 277-80 (1990)]. As shown in Figure 1, the structure of Thrombin-bound Chirulog-8 was read by reading the D-Phe-Pro-Arg sequence of CSDM and the ABEAM segment Asp-Phe-Glu-Glu-Ile.

In FIG. 1 Thrombin is presented in white except for its active site, which is indicated by dense points. The surgeon-8 is indicated by the scatter points. The region of Hirulog-8 on the left hand closest to the active site of thrombin is CSDM. The section on the right is ABEAM. The other amino acids of Hirulog-8 are not shown in Fig. and their corresponding electronic densities cannot be determined.

Examination of the CSDM region of the Hirulog-8 structure: thrombin showed the location of the first-position amino acid (D-Phe) in the hydrophobic furrow formed by His57, TugOA, Trp60D, Leu99, 11e174 and Trp215 (Figure 2). In Figure 2, the thrombin is represented by continuous lines and the Hirulogist 8 by dashed lines. The arrangement of the D-Phe residue in the groove suggests that substitutions at position 1 that enhance lipophilic contacts would lead to increased binding affinity of the CSDM component in the thrombin inhibitors of the invention. Accordingly, the D-Phe residue of Chirulog-8 or D-naphthalalanine (D-NPA) or D-cyclohexylalanine (DCha) is replaced to form O-1PRA-Chirulog-8 and D-Cha-Xnpynor-8 , respectively.

It was also found that the binding of the CSDM of Hirulog-8 to the catalytic site of thrombin involves apolar interactions between the first proline of the inhibitor (adjacent to D-Phe) and a groove defined by His57, TugBOA and Tyr60D of thrombin (Figure 2). In addition, this proline was found to be within 3.4bA of the phenolic hydroxyl group of thrombin TugBOA (Figure 2). In Figure 2, thrombin is indicated by continuous lines and Hirulog-8 is indicated by broken lines.

The proximity of this proline to ThugboA thrombin suggests the possibility of forming a hydrogen bond between them. By replacing proline with an amino acid capable of forming hydrogen bonds, the stability of binding of CSDM to the active site of thrombin can be enhanced. This would in practice increase the inhibitory activity of such a molecule. Therefore, proline of Chirulog-8 is replaced by L-histidine (H15 ₂ -Hirulog-8), L-thioproline (TP20 ₂ Chirulog-8) or isonipecotinic acid (1pr ₂ -Hirulog-8). Each of these substitutions introduces a hydrogen bond acceptor at position 2 (K ' ₄ -component) of the thrombin inhibitors of this invention (i.e., imidazole nitrogen, thiol and carboxylate, respectively).

Example 4. Synthesis of Surgeons substituted at position 1.

O-p-Hirulog-8 was synthesized in the same way as Hirulog-8 (Example 2), except that in the last D-Phe synthesis cycle, Boc-D-naphthylalanine was used (Bachem Inc. , Torrance, Calif.). DCIA-Surgeon-8 was similarly prepared using Boc-D-cyclohexylalanine (Bachem Biosciences, Philadelphia, Pa) in the final synthesis step.

The two 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 FAB-MS.

Example 5. Synthesis of Chirulog Derivatives Substituted at Position 2.

Substitutions at position 2 are designed by the formula: (D-Cha) -X-Arg-HPro- (Gly ₄ ) -AsnGly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu or (D -Cha) -X-Arg-HPro- (Gly ₄ ) -Asn-Gly-AspPhe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu, in which X is histidine, thioproline or isonipecotinic acid. These peptides were essentially synthesized as described in Examples 2 and 4, except for the inclusion of Boc-L-hydroxyproline (Bachem, Inc.) instead of Boc-L-proline in synthesis cycle 16 and Boc-N-im -CBZ-Lhistidine, Boc-L-thioproline or Boc-isonipecotinic acid (all obtained from Bachem, Inc.) instead of Boc-L-proline in cycle 18. HPO is used at position 4 to slow the rate of rupture of the inhibitor from thrombin. The peptides were purified and characterized as described in Example 2.

Example 6. Characterization of the antithrombin activity of the substituents at position 1 Surgeons.

The test compares the inhibition of thrombin catalyzed hydrolysis of Spectrosim TH (tosyl-Ou-Pr-N-p-nitroanilide; American Diagnostica, New York, NY) by Hirulog-8, O-Cb-Hirulog-8 and D Npa-XHpy.ior-8. In particular, the initial velocities in the presence or absence of each inhibitor are measured within the substrate concentrations of 2.2 to 22 μΜ. The thrombin-catalyzed rate was recorded in a Cary 19 spectrophotometer at 405 nm and recorded as a continuous function of time. The kinetics were carried out at room temperature (25 ° ± 1 ° C) in 0.05 M sodium borate buffer, pH 8.4 containing 0.1 M NaCl.

In typical enzymatic reaction in both cells. 1.0 ml of buffer are added to the sample and the control. Thrombin (3.2x10'M final concentration) and surgeon (0-4x10 ⁸ .M) were added to the sample cuvate prior to the addition of Spectrosim TH (2.2-22 μΜ). Immediately after the substrate was added, the contents of the sample cells were mixed using a plastic pipette. The reaction was read spectrophotometrically for 5-15 minutes.

The initial rates for each substrate concentration are expressed as moles of hydrolyzed Spectrosim TH / s / mol thrombin. These are determined during the initial linear phase of the reaction (<15% of total substrate hydrolysis) by measuring the slope of the hydrolytic reaction. Lineweaver-Burke graphs are plotted accordingly by plotting the reciprocal of the initial velocity as a function of the reciprocal of the substrate concentration. Below are the inhibition constants obtained for the Hirulog-8 and derivatives of the present invention.

The derivative K _{p is} PM

Surgeon-8 1.4 0-CIA-Surgeon-8 0.12 D-Npa-Xnpynor-8 4.3 As you can see from these results-

you, replacing D-Phe in Chirulog-8 with DCha leads to a surprising and unexpected decrease in Kj by one order. This finding shows that the replacement of D-Phe with D-Cha. enhances CSDM binding affinity in inhibitors of this invention. The refusal of D-Npa-XHpynor-8 to lower K1 indicates that the presence of a saturated ring structure at this position causes an increased binding affinity. D-Cha contains such a saturated ring, while D-Npa contains an unsaturated ring.

Molecules Containing in Item 2 The substitutions described in Example 5 will exhibit similar unexpected reductions in K.

EXAMPLE 7 Anticoagulant Activity of Aj-Substituted Surgeons.

Compare the anticoagulant activity of Tyr ₆₃ -O-sulphate-N-acetyl-hirudin _R ( "hirugen"), recombinant hirudin (American Diagnostica), Hirulog-8 and substituted in position 1 hirulozi of the present invention, using a unified normal human plasma (George King Biomedical, Overland Park, Kans.) And Coag-A-Mate XC (General Diagnostics, Organon Technica, Oklahoma City, Okla.). Activity was assayed using the test of the activated partial thromboplastin time (APTT) with SaS1 ₂ and phospholipid solutions obtained from the manufacturer. Recombinant hirudin (American Diagnostica), Surgeon-8, O-Cba-Surgeon-8 or surgeon are then added to the wells for the determination of APTT to final concentrations of 10 to 32 300 ng / ml in a total volume of 25 μΐ before adding 100 μΐ of plasma.

As shown in Fig. 3, D-Cha-Surgeon-8 prolonged APTT up to 470% of control values at a concentration of 1 mg / μ /. This increase is significantly greater than the increase in APTT caused by hirugen, recombinant hirudin and Hirulog-8 at the same concentration. Thus, in addition to the demonstrated increased in vitro antithrombin activity against Hirulog-8, D-ChaHirulog-8 also showed a significantly increased anticoagulant effect in plasma tests relative to Hirulog-8.

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

Since a number of applications of the invention have been presented so far, it is apparent that the basic construction can be modified to provide other applications using the molecules, preparations, combination preparations and methods described.

Claims (8)

A thrombin inhibitor targeting the catalytic site, characterized by the formula: x-r ^ -r.-r ^ -y I wherein X is HN; R _I is cyclohexyl; R ₂ is CH ₂ -CH: R ₃ is C = O; R ₄ is Pro or any amino acid comprising a side chain group characterized by the capacity to accept a hydrogen bond at a pH of between 5.5 and 9.5; R ₃ is any L-amino acid which 5 includes a guanidine or amino group in the side chain; R _e is a non-amide bond that slows or inhibits the rupture of the thrombin inhibitor; and Y is selected from the group consisting of sarcosine, N-methylal10nin, hydroxyproline and any natural La amino acid; and the amino acid defined by components X, R _p Rj and R is in the D configuration. A thrombin inhibitor according to claim 1, which has the sequence of amino acids: D-Cha-Rgo-Arg-Pro The thrombin inhibitor according to claim 1, wherein R ₄ is selected from the group consisting of histidine, thioproline and isoniapicotinic acid. The thrombin inhibitor according to claim 1, wherein the inhibitor is radiolabelled. 5. Fibrin or platelet thrombosis scintigraphy preparation in a patient, characterized in that it comprises a pharmaceutically acceptable buffer and a thrombin inhibitor according to claim 4. 6. A method for scintigraphy of fibrin or platelet thrombus in a patient, characterized in that it comprises the steps of: a) administering to the patient a preparation according to claim 5; and b) using ex vivo detection methods to monitor the thrombin inhibitor present in the preparation. 7. The method of claim 6, wherein it is administered to humans. The thrombin inhibitor according to claim 4, wherein the radioisotope is selected from the group consisting of ¹²³ I, ¹²⁵ I and II.