JP2005538936A - Dimeric TF antagonist - Google Patents

Abstract

Dimeric FVII polypeptide. And it binds simultaneously and inhibits two TF molecules.

Description

The present invention relates to novel compounds that bind to TF and inhibit activity. The invention also relates to pharmaceutical compositions comprising the novel compounds and to the treatment or prevention of thrombotic or coagulopathy related diseases or disorders including vascular diseases and inflammatory reactions.

BACKGROUND OF THE INVENTION Blood coagulation is a process that consists of complex interactions of various blood components or factors, which ultimately cause fibrin clots. The blood components that normally participate in what is called the coagulation “cascade” are enzyme precursors or enzyme precursors, enzymatically inactive proteins, which are themselves activated coagulation factors It is converted into a proteolytic enzyme by the action of an activator. Coagulation factors that have undergone such conversion are commonly referred to as “active factors” and are indicated by adding the letter “a” to the name of the coagulation factor (eg, FVIIa).

  Activated factor X (FXa) is required to form a fibrin clot and thereby stop bleeding. FXa is required to convert fibrinogen to fibrin as the final step in converting prothrombin to thrombin and then forming a fibrin clot. There are two systems or pathways that promote factor X (FX) activation. “Endogenous pathway” refers to a reaction that causes thrombin formation by utilizing factors present only in plasma. A series of protease-mediated activations I finally produce factor Xa and, in conjunction with factor VII Ia, cleave FX into FXa. In the “exogenous pathway” of blood clotting, factor VIIa (FVIIa) and its cofactor tissue factor (TF) produce the same proteolysis. Although the relative contribution of the two coagulation pathways in hemostasis is unknown, it has recently been found that FVIIa and TF play an important role in the initiation of blood coagulation.

  FVIIa is a two-chain, 50 kilodalton (kDa) vitamin K-dependent plasma serine protease that participates in the complex regulation of hemostasis in vivo. FVIIa is produced by proteolysis of a single peptide bond from its single-chain proenzyme factor VII (FVII), which is present in plasma at a concentration of about 0.5 g / ml. The enzyme precursor is catalytically inactive. Conversion of the enzyme precursor FVII to an activated double-stranded molecule occurs by breaking internal peptide bonds. In human FVII, the cleavage site is Arg152-Ile153.

  In the presence of calcium ions, FVIIa binds with high affinity to exposed TF (essential membrane protein). TF acts as a cofactor for FVIIa and enhances the proteolytic activation of its substrates FVII, Factor IX, and FX.

  TF is a 263 amino acid residue glycoprotein composed of a 219-residue extracellular domain, a single transmembrane region, and a short cytoplasmic domain. TF is a membrane bound protein and does not normally circulate in active plasma.

  For example, selectively blocking or inhibiting the patient's coagulation cascade during renal perfusion, or preventing or treating a host of deep vein thrombosis, sepsis, DIC, atherosclerosis, and other medical disorders Is often desired. Typically, anticoagulants such as heparin, coumarin, coumarin derivatives, indandione derivatives, thrombin inhibitors, or FXa inhibitors have been used. For example, heparin treatment or extracorporeal treatment with citrate ions may be used during perfusion to prevent clotting during the course of treatment. Heparin is also used to prevent deep vein thrombosis in patients undergoing surgery. However, treatment with heparin and other anticoagulants will have undesirable side effects. Useful anticoagulants generally act throughout the body, rather than specifically at the site of injury, ie where the coagulation cascade is active. For example, heparin will cause severe bleeding. In addition, heparin has a half-life of about 80 minutes, is cleared rapidly from the blood and needs to be administered frequently. Heparin acts as a cofactor for antithrombin III (ATIII), and ATIII is rapidly depleted in DIC treatment, so it is often difficult to maintain appropriate heparin doses and maintain ATIII and heparin levels Monitoring is necessary. Also, heparin is ineffective when ATIII depletion is extreme. In addition, prolonged use of heparin could increase platelet aggregation and reduce platelet count and was also associated with the development of osteoporosis. Indandione derivatives will also have toxic side effects.

  Other known anticoagulants include thrombin derived from blood-sucking organisms and FXa inhibitors. Antithrombin, hirudin, hirulog, and hirugen are recombinant proteins or peptides derived from leach Hirudo medicinalis, but the FXa inhibitor antistatin and The recombinant derivative rTAP is a tick-derived protein. Inhibitors of platelet aggregation such as monoclonal antibodies or synthetic peptides interfere with the platelet receptor GPIIb / IIIa and are also effective as a blood coagulation inhibitor.

  Bleeding complications are observed as an undesired major disadvantage of antithrombin, anti-FXa, and antiplatelet drugs. This side effect is strongly reduced or eliminated by inhibitors of TF / FVIIa activity. Such anticoagulants are the physiological inhibitors TFPI (inhibitor of tissue factor pathway) and inactivated FVII (FVIIai) (which are catalytically inactive and thus convert FX to FXa It is FVIIa modified in such a way that it can bind to TF and compete with active endogenous FVIIa).

  International patent applications WO 92/15686, WO 94/27631, WO 96/12800, WO 97/47651 relate to FVIIai and their use. International patent applications WO 90/03390, WO 95/00541, WO 96/18653, and EP 500800 are peptides derived from FVIIa having TF / FVIIa antagonist activity. Is described. WO 01/21661 relates to divalent inhibitors of FVII and FXa. WO 02/02764 relates to high molecular weight derivatives of vitamin K dependent polypeptides.

  HuZ and GarenA (2001) Proc. Natl. Acad. Sci. USA 97; 9221-9225; HuZ and GarenA (1999) Proc. Natl. Acad. Sci. USA 96; 8161-8166, and WO 0102439 are human. It relates to immune complexes that bind to TF but do not initiate blood clotting, including the Fc region of IgG1 immunoglobulins and mutant FVII polypeptides.

  In addition, International Patent Application Publication No. WO 98/03632 describes a bivalent agonist having affinity for one or more G-coupled receptors, Burgess, LE et al., Proc. Natl. Acad. Sci. USA 96, 8348-8352 (July 1999) describes "a potent selective non-peptide inhibitor of human lung tryptase".

  There remains a need in the art for improved compositions that have anticoagulant activity at relatively low doses and that do not produce undesirable side effects. The present invention provides anticoagulants that act specifically on TF-initiated processes at the site of injury at relatively low doses.

DESCRIPTION OF THE INVENTION The present invention relates to a dimeric FVII polypeptide that is bivalent and has binding activity and possibly increased in vivo half-life by simultaneously inhibiting two TF molecules. Thus, lower concentrations of TF antagonist can be used compared to monovalent FVIIai molecules. In one particular embodiment of the invention, two molecules of FVIIa are conjugated with a divalent chloromethyl ketone inhibitor consisting of two chloromethyl ketone moieties linked together.

  An inactivated dimeric FVII polypeptide consists of two FVII polypeptides A and D joined by a suitable binding moiety (LM), said LM being chemically conjugated to the FVII polypeptide Yes. Compared to inhibitors that target only one TF site, dimeric inhibitors provide additional effects with respect to higher binding affinity and capacity due to the binding activity effect.

  In order to achieve multiple binding, proper design of TF dimer antagonists is essential. The functional affinity of such antagonists is dependent on the overall concentration, but in particular on the spatial localization of FVII polypeptides A and D, both divalent FVIIai molecules for TF This is advantageous for binding of motifs. After binding of one FVII polypeptide to TF, the diffusion of the partner connected to it is constrained within a radius equal to the length of the spacer connecting the two FVII polypeptides. This constraining effect increases the local concentration of FVII polypeptide that binds to the second TF site.

  The term “TF antagonist” or “TF antagonists” as used herein refers to any compound that binds directly to TF and inhibits the conversion of FX to FXa in an FXa production assay. It is intended to mean. One example of a TF antagonist is FVIIai.

  The term “TF dimer antagonist” or “TF dimer antagonist” as used herein shall mean any TF antagonist comprising two binding sites for TF. Intended.

  Bivalent TF inhibitors contain the following three different motifs that can be modulated to achieve preferentially repressing characteristics: two FVII polypeptides and a linker moiety that connects the two FVII polypeptides (LM). LM should not be considered as the only spacer connecting two FVII polypeptides. To obtain the highest affinity, LM can be used to optimize the solubility, bioavailability, and binding properties of the divalent TF inhibitor.

  An object of the present invention is to provide a method for inhibiting TF-mediated coagulation activity by providing a compound having pharmacological activity as a TF antagonist.

  The term “TF-mediated clotting activity” refers to TF-initiated clotting through the formation of the TF / FVIIa complex and its activation from FIX and factor X to FIXa and FXa, respectively.

  The compounds are useful for the prevention or treatment of TF related diseases or disorders including vascular diseases and inflammatory reactions.

Abbreviations used throughout the specification include:
TF tissue factor
FVIIa Activated Factor VII
FXa factor Xa Activated factor X
FVII Enzyme Precursor (single chain, non-active) Factor VII
FX enzyme precursor (single chain, inactive) is factor X
TF / FVIIa Complex between TF and FVIIa
TF / FVIIa / FXa Complex formed by FVIIa, TF, and FXa
Pt prothrombin time
APTT activated partial thromboplastin time
SP serine protease
FFR Phe-Phe-Arg or D-Phe-Phe-Arg
D-FFR D-Phe-Phe-Arg
dimer form of FFR, two FFRs joined together by dim-FFR binding moieties
dim-FFR-FVIIa dim-FFR bound to factor VIIa, preferably in the active site
dim-FFR-cmk FFR-cmk joined together by a linking moiety, dimer form of two FFR-cmk
FFR-cmk, FFR-CMK Phe-Phe-Arg chloromethyl ketone or D-Phe-Phe-Arg chloromethyl ketone
D-FFR-cmk D-Phe-Phe-Arg chloromethyl ketone
D-FFR-CMK
DPTA-dim-FFR-cmk Diethylenetriaminepentaacetic anhydride-dimFFR-cmk
EGR Glu-Gly-Arg or D-Glu-Gly-Arg
D-EGR D-Glu-Gly-Arg
EGR-cmk, EGR-CMK Glu-Gly-Arg chloromethyl ketone or D-Glu-Gly-Arg chloromethyl ketone
K _I the dissociation constant, the inhibition constant name "D" of the enzyme inhibitor complex is in the immediately preceding character in as amino acids described above (abreviation) When the amino acid, it is understood that unnatural d- enantiomer Should be.

Terms such as “active site”, as used herein with respect to FVIIa, are termed by Schecter, I. and Berger, A., (1967) Biochem. Biophys. Res. Commun. 7: 157-162. Refers to the substrate binding site of the catalyst and enzyme precursor containing the “S ₁ ” site of FVIIa as defined.

  A TF / FVIIa-mediated or related process or event, or a process or event associated with TF-mediated clotting activity is any event that requires the presence of TF / FVIIa.

  Such processes or events include fibrin formation leading to thrombus formation; platelet deposition; platelet deposition and thrombus formation, release of chemotactic and mitogenic factors, and vascular smooth muscle cells to the intima of arterial segments Proliferation of vascular wall smooth muscle cells (SMC), such as intimal hyperplasia or restenosis, considered to be caused by complex interactions of biological processes including migration and proliferation; and undergoing coronary thrombolysis, for example Including, but not limited to, adverse events related to post-ischemic reperfusion, such as in patients with acute myocardial infarction.

  The avascular resumption phenomenon, the lack of uniform perfusion to the microvasculature of previously ischemic tissue, was first described by Krug et al. (Circ. Res. 1966; 19: 57-62).

General mechanism clot formation, by ^{Ganong, Medical Physiology, 13 th ed} ., Lange, Los Altos Calif., And is outlined in the review of pp 411-414 (1987). Clotting requires that two processes meet, the production of thrombin that induces platelet aggregation and the formation of fibrin that stabilizes the platelet plug. This process involves several steps, each requiring the presence of separate enzyme precursors and precursor factors. The process ends with fibrin cross-linking and thrombus formation. Fibrinogen is converted to fibrin by the action of thrombin. Thrombin is then formed by proteolytic cleavage of prothrombin. This proteolysis is performed by FXa that binds to the surface of activated platelets and cleaves prothrombin in the presence of FVa and calcium. TF / FVIIa is required for FX proteolytic activity by the extrinsic coagulation pathway. Thus, TF / FVIIa-mediated or related processes, or TF-mediated clotting activity, includes any step of the clotting cascade from the formation of the TF / FVIIa complex to the formation of a fibrin platelet clot, This requires that TF / FVIIa is initially present. For example, the TF / FVIIa complex initiates an extrinsic pathway by activation of FXa to FVIIa, FIX to FIXa, and even FX to FVII. TF / FVIIa-mediated or related processes, or TF-mediated clotting activity, are described in Roy, S. for the conversion of FX to FXa in the presence of TF / FVIIa and other necessary reagents. , (1991) J. Biol. Chem. 266: 4665-4668, and O'Brien, D. et al. (1988) J. Clin. Invest. 82: 206-212. Can be conveniently measured using the method.

  The term “a disease or disorder associated with TF” as used herein means any disease or disorder in which TF is involved. This includes deep vein thrombosis, arterial thrombosis, postoperative thrombosis, coronary artery bypass graft (CABG), percutaneous percutaneous coronary artery reconstruction (PTCA), stroke, cancer, tumor metastasis, angiogenesis , Ischemia / reperfusion, arthritis including rheumatoid arthritis, thrombolysis, arteriosclerosis and restenosis after angioplasty, acute and chronic signs such as inflammation, septic shock, sepsis, hypotension, adult Such as prophylactic treatment of mammals with atherosclerotic blood vessels at risk for respiratory distress syndrome (ARDS), disseminated intravascular coagulation syndrome (DIC), pulmonary embolism, platelet deposition, myocardial infarction, or thrombosis, Diseases or disorders associated with TF-mediated coagulation activity, diseases such as thrombotic or coagulopathy related disorders, or diseases such as inflammatory reactions, and fibrin formation including acute and chronic signs Chronic thromboembolic diseases or disorders, and including other diseases, but not limited to. Diseases or disorders associated with TF are not limited to in vivo coagulopathy disorders such as those named above, but in-line from the patient in processes such as dialysis procedures, hemofiltration, or blood bypass during surgery. Includes in vitro TF / FVIIa related processes, such as clotting that would be caused by extracorporeal circulation of blood, including removed blood.

  “Treatment” is an effective treatment of a therapeutically active compound of the present invention for the purpose of preventing the onset of any symptom or disease state, or for the purpose of curing or alleviating such a symptom or disease state that has already developed. It means to administer an amount. Thus, the term “treatment” is meant to include prophylactic treatment.

  In a first aspect, the invention is a compound having the formula A- (LM) -D, wherein A and D are FVII polypeptides that bind to TF; and LM is less than 30,000 Daltons A linker moiety having a molecular weight of: and the compound is a compound of wtFVIIai- (DPTA-dim-FFR-cmk) -wtFVIIai or FVIIai (Q10E32)-(DPTA-dim-FFR-cmk) -FVIIai (Q10E32) It relates to compounds that inhibit TF activity, provided that they are not compounds having the formula.

  It should be understood that portion A is chemically bonded to LM and chemically bonded to portion D. A and D may be the same FVII polypeptide joined together by an LM moiety, or A and D may be different FVII polypeptides.

  The term “FVII polypeptide” or “FVII polypeptide” as used herein means, but is not limited to, Factor VII as well as the equivalent of Factor VII. Factor VII equivalents have been chemically modified compared to human factor VII and / or altered in one or more amino acid sequences compared to human factor VII (ie, a factor VII variant) And / or include, but are not limited to, Factor VII polypeptides that include amino acid sequence truncations as compared to human Factor VII (ie, Factor VII fragments). Such equivalents may exhibit different properties including stability, phospholipid binding, altered specific activity and the like compared to human Factor VII.

  The term "Factor VII" refers to these uncleaved (enzyme precursor) forms of Factor VII polypeptides as well as their respective bioactive forms, which will be referred to as Factor VIIa It is contemplated to include those that have been processed by hydrolysis. Typically, Factor VII is cleaved between residues 152 and 153 to yield Factor VIIa. The term “Factor VII” also refers to wild-type human Factor VII (disclosed in US Pat. No. 4,784,950) and other species such as bovine, pig, dog, mouse, and salmon Factor VII. It is contemplated to include, but is not limited to, a polypeptide having the amino acid sequence 1-406 of wild type factor VII derived from This further includes natural allelic variations of Factor VII that may exist and occur from one individual to another. Also, the extent and site of glycosylation or other post-translational modifications will vary depending on the host cell chosen and the nature of the host cell's environment.

  As used herein, a “Factor VII equivalent” is a polypeptide that exhibits substantially the same or improved biological activity as compared to wild type human Factor VII, as well as wild type humans. Including, but not limited to, a polypeptide in which Factor VIIa biological activity is substantially modified or reduced compared to the activity of Factor VIIa. These polypeptides include chemically modified Factor VII or Factor VIIa, and Factor VII variants in which a specific amino acid sequence change has been introduced to modify or disrupt the biological activity of the polypeptide. Including, but not limited to.

  The term “tissue factor activity” or “TF activity” as used herein means the activity of TF as measured in an FXa production assay.

  The term “FXa production assay”, as used herein, means any assay where the activity of FX is measured in a sample comprising TF, FVIIa, FX, calcium and phospholipid. Intended. An example of an FXa production assay is described in Assay 2.

  In a second aspect, the present invention is a method for reducing TF activity, said method comprising contacting a compound having formula A- (LM) -D with a TF expressing cell, wherein , A and D are FVII polypeptides that bind to TF; and LM is a linker moiety having a molecular weight of less than 30,000 daltons; and said compound is wtFVIIai- (DPTA-dim-FFR-cmk) It relates to a method for inhibiting TF activity, provided that it is not a compound having the formula -wtFVIIai or FVIIai (Q10E32)-(DPTA-dim-FFR-cmk) -FVIIai (Q10E32).

  In a third aspect, the present invention provides a pharmaceutical composition comprising an amount of a compound having the formula A- (LM) -D and a pharmaceutically acceptable carrier or excipient, wherein A And D is an FVII polypeptide that binds to TF; and LM is a linker moiety having a molecular weight of less than 30,000 daltons; and said compound has the compound wtFVIIai- (DPTA-dim-FFR-cmk) -wtFVIIai Alternatively, the present invention relates to a composition that inhibits TF activity, provided that the compound is not a compound having the formula of FVIIai (Q10E32)-(DPTA-dim-FFR-cmk) -FVIIai (Q10E32).

  In a further aspect, the present invention is a compound for use as a medicament having the formula A- (LM) -D, wherein A and D are FVII polypeptides that bind to TF; and LM is A linker moiety having a molecular weight of less than 30,000 daltons; and said compound is a compound having a compound weight of wtFVIIai- (DPTA-dim-FFR-cmk) -wtFVIIai or FVIIai (Q10E32)-(DPTA-dim-FFR-cmk) -FVIIai The present invention relates to a compound that inhibits TF activity on condition that the compound is not a compound having the formula (Q10E32).

  In a further aspect, the invention is the use of a compound having the formula A- (LM) -D, wherein A and D are FVII polypeptides that bind to TF; and LM is less than 30,000 Daltons A linker moiety having a molecular weight of: and the compound is a compound of wtFVIIai- (DPTA-dim-FFR-cmk) -wtFVIIai or FVIIai (Q10E32)-(DPTA-dim-FFR-cmk) -FVIIai (Q10E32) It relates to the use of inhibiting TF activity, provided that it is not a compound having the formula.

  In a further aspect, the present invention is a method for the prevention or treatment of a TF related disease or disorder in a mammal, said method comprising an effective amount of at least one compound having the formula A- (LM) -D. Wherein A and D are FVII polypeptides that bind to TF; and LM is a linker moiety having a molecular weight of less than 30,000 daltons; and said compound is a compound of wtFVIIai -(DPTA-dim-FFR-cmk) -wtFVIIai or FVIIai (Q10E32)-(DPTA-dim-FFR-cmk) -FVIIai (Q10E32) .

  In a further aspect, the invention is a compound having the formula A- (LM) -D, wherein A and D are FVII polypeptides that bind to TF; and LM has a molecular weight of less than 30,000 Daltons And said compound relates to a compound that inhibits TF activity.

  In a further aspect, the present invention is a method for reducing TF activity comprising contacting a compound having formula A- (LM) -D with a TF expressing cell, wherein A And D are FVII polypeptides that bind to TF; and LM is a linker moiety having a molecular weight of less than 30,000 daltons; and the compounds relate to those that inhibit TF activity.

  In a further aspect, the invention provides a pharmaceutical composition comprising an amount of a compound having formula A- (LM) -D and a pharmaceutically acceptable carrier or excipient, wherein A and D Is a FVII polypeptide that binds to TF; and LM is a linker moiety having a molecular weight of less than 30,000 daltons; and said compound relates to a composition that inhibits TF activity.

  In a further aspect, the present invention is a compound for use as a medicament having the formula A- (LM) -D, wherein A and D are FVII polypeptides that bind to TF; and LM is A linker moiety having a molecular weight of less than 30,000 daltons; and said compound relates to a compound that inhibits TF activity.

  In a further aspect, the invention is the use of a compound having the formula A- (LM) -D, wherein A and D are FVII polypeptides that bind to TF; and LM is less than 30,000 Daltons A linker moiety having a molecular weight of; and said compound relates to a use that inhibits TF activity.

  In a further aspect, the present invention is a method for the prevention or treatment of a TF related disease or disorder in a mammal, said method comprising an effective amount of at least one compound having the formula A- (LM) -D. Wherein A and D are FVII polypeptides that bind to TF; and LM is a linker moiety having a molecular weight of less than 30,000 daltons; and said compound exhibits TF activity It relates to a method of inhibiting.

  In one embodiment of the compound having the formula A- (LM) -D, the binding affinity of the compound for TF is higher than the binding affinity for TF of either A or D alone. In certain embodiments, the binding affinity of the compound for TF is two times higher than the binding affinity for TF of either A or D alone. In certain embodiments, the binding affinity of the compound for TF is three times higher than the binding affinity for TF of either A or D alone. In certain embodiments, the binding affinity of the compound for TF is 5 times higher than the binding affinity for TF of either A or D alone. In certain embodiments, the binding affinity of the compound for TF is 10 times higher than the binding affinity for TF of either A or D alone.

  In a further embodiment of the invention, A and D of the compound having the formula A- (LM) -D are human recombinant FVIIa.

  In a further embodiment of the invention, A and D of the compound having the formula A- (LM) -D are human recombinant FVIIai.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight of less than 20,000 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight of less than 10,000 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight of less than 5,000 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight higher than 1,380 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight higher than 1,400 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight higher than 1,600 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight higher than 1,800 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight higher than 2,000 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight greater than 3,000 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight higher than 4,000 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight greater than 5,000 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D comprises an amino acid sequence. In certain embodiments, the compound having the formula A- (LM) -D only includes the amino acid sequence.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a leucine zipper.

The term “leucine zipper” or “LZ” as used herein is the amino acid sequence of the pattern (H ¹ · P · P · H ² · P · P · P) n, wherein H is a residue selected from Leu, Ile, Val, or Asn, and P is Ser, Thr, Asn, Gln, Asp, Glu, Lys, Arg, His, Gly, Ala, Val, Leu, Ile Refers to any amino acid sequence that is a residue selected from Phe, and Tyr and where n ≧ 2.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D comprises a leucine zipper.

In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is the amino acid sequence (H ¹ · P · P · H ² · P · P · P) _n , wherein H is , Leu, Ile, Val, or Asn, P is Ser, Thr, Asn, Gln, Asp, Glu, Lys, Arg, His, Gly, Ala, Val, Leu, Ile, Phe And any amino acid sequence that is a residue selected from Tyr and n ≧ 2.

In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is the amino acid sequence (H ¹ · P · P · H ² · P · P · P) _n , wherein H is , Leu, Ile, Val, and P is a residue selected from Ser, Thr, Asn, Gln, Asp, Glu, Lys, Arg, His, and n ≧ 4 Contains an array.

In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is the amino acid sequence (H ¹ · P · P · H ² · P · P · P) _n , wherein H is , Leu residues, and P is a residue selected from Ser, Thr, Asn, Gln, Asp, Glu, Lys, Arg, His, and includes an amino acid sequence where n ≧ 4.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D independently comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 6.

In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is an amino acid sequence (Gly-Gly-Gly-Gly-Ser) _n , wherein n is any of 1-10 An amino acid sequence that is an integer of In one embodiment, n is 1-5. In a further embodiment, n is 3.

  In a further embodiment of the invention the compound having the formula A- (LM) -D is not an immune complex. It should be understood that the compounds of the present invention should preferably not mediate a cytolytic response of the immune system.

In a further embodiment of the invention, the LM of the compound having the formula A- (LM) -D is linear or branched C _1-50 -alkyl, linear or branched C _2-50 -alkenyl, linear Or branched C _2-50 -alkynyl, 1-50 member straight or branched chain containing carbon and at least one N, O, or S atom in the chain, C _3-8 cycloalkyl, carbon in the ring and at least Selected from the group consisting of 3- to 8-membered cyclic rings containing one N, O, or S atom, aryl, heteroaryl, amino acid, the structure optionally substituted with one or more of the following groups Including molecules such as: H, hydroxy, phenyl, phenoxy, benzyl, thienyl, oxo, amino, C _1-4 -alkyl, CONH ₂ , CSNH ₂ , C _1-4 monoalkylamino, C _1-4 dialkylamino, acylamino, sulfonyl, carboxy, carboxamido, halogeno, C _1-6 Al Alkoxy, C _1-6 alkylthio, trifluoromethyl, alkoxycarbonyl, haloalkyl. The LM may be linear or branched and may contain one or more double bonds or triple bonds. The LM may contain one or more heteroatom-like N, O, or S. It should be understood that an LM can include multiple classes of the group described above, as well as multiple members within a class. Where the LM comprises multiple classes of groups, such LMs are preferably obtained by connecting different units via these functional groups. Methods for forming such bonds include standard organic synthesis and are well known to those skilled in the art.

  In a further embodiment of the invention, the LM of the compound having the formula A- (LM) -D is independently Phe-Phe-Arg chloromethyl ketone, Phe-Phe-Arg chloromethyl ketone, D-Phe-Phe-Arg Chloromethyl ketone, D-Phe-Phe-Arg chloromethyl ketone, Phe-Pro-Arg chloromethyl ketone, D-Phe-Pro-Arg chloromethyl ketone, Phe-Pro-Arg chloromethyl ketone, D-Phe-Pro- A divalent chloromethyl ketone inhibitor comprising two monomers selected from the group comprising Arg chloromethyl ketone, L-Glu-Gly-Arg chloromethyl ketone, and D-Glu-Gly-Arg chloromethyl ketone; Including.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a divalent FVIIa inhibitor.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D comprises one FVIIa inhibitor.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D comprises two FVIIa inhibitors.

  In a further embodiment of the invention, the LM of the compound having the formula A- (LM) -D is bis-octanedioate ({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2- Phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide.

  In a further embodiment of the invention, the LM of the compound having the formula A- (LM) -D is bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) 10,12-docosadiindioate. ) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide).

  In a further embodiment of the invention, the LM of the compound having the formula A- (LM) -D is bis-docosanedioic acid bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2- Phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide).

  In a further embodiment of the invention, the LM of the compound having the formula A- (LM) -D is icosanedioic acid bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2- Phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide).

  In a further embodiment of the invention, the LM of the compound having the formula A- (LM) -D is bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-octadecadioic acid] -2- Phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide).

  In a further embodiment of the invention, the LM of the compound having the formula A- (LM) -D is 4,7,10,13-tetraoxahexadecanedioic acid bis-({1- [1- (1-chloroacetyl- 4-guanidino-butylcarbamoyl) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide).

  In a further embodiment of the invention, the LM of the compound having the formula A- (LM) -D is ethylene glycol-bisbissuccinate-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl)- 2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide).

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is N- [3- (4- {3- [3- (1- {1- [1- (2-chloroacetyl) ) -4-Guanidinobutylcarbamoyl] -2-phenylethylcarbamoyl} -2-phenylethylcarbamoyl) propionylamino] propyl} piperazin-1-yl) -propyl] -succinic acid-1- [1- (1-chloroacetyl) -4-guanidino-butylcarbamoyl) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide.

  In a further embodiment of the invention, the LM of the compound having the formula A- (LM) -D is bis-[(1- {1- [1- (2-chloro-acetyl) -4-guanidino-butyl] eicosanedioate. Rucarbamoyl] -2-phenyl-ethylcarbamoyl} -2-phenyl-ethyl) -amide].

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is bis-pentanedioate-[(1- {1- [1- (2-chloro-acetyl) -4-guanidino-butyl]. Rucarbamoyl] -2-phenyl-ethylcarbamoyl} -2-phenyl-ethyl) -amide].

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is N, N′-bis- (1- {1- [1- (2-chloro-acetyl) -4-guanidino- Butylcarbamoyl] -2-phenyl-ethylcarbamoyl} -2-phenylethyl) -oxalamide).

  In a further embodiment, a pharmaceutical composition comprising an amount of a compound having the formula A- (LM) -D, wherein A and D are FVII polypeptides that bind TF; and less than 30,000 Daltons A pharmaceutical composition that is a linker moiety having a molecular weight of

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight of less than 20,000 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight of less than 10,000 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight of less than 5,000 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight of less than 2000 daltons.

  In a further embodiment of the invention the LM of the compound having the formula A- (LM) -D is a linker moiety having a molecular weight of less than 1000 daltons.

  In a further embodiment, LM is [bis- (2-carboxymethyl-[(1- {1- [1- (2-chloro-acetyl) -4-guanidino-butylcarbamoyl] -2-phenyl-ethylcarbamoyl}- Not 2-phenyl-ethylcarbamoyl) -methyl] -amino} -ethyl) -amino] acetic acid.

ではない。 In a further aspect, LM is

is not.

ではない。 In a further embodiment, the LM is

is not.

  In a further embodiment of the invention, the disease or disorder associated with TF is deep vein thrombosis, arterial thrombosis, postoperative thrombosis, coronary artery bypass graft (CABG), percutaneous percutaneous coronary reconstruction ( PTCA), stroke, cancer, tumor metastasis, angiogenesis, ischemia / reperfusion, arthritis including rheumatoid arthritis, thrombolysis, arteriosclerosis and restenosis after angioplasty, acute and chronic signs such as inflammation, sepsis Atherosclerosis at risk for septic chock, sepsis, hypotension, adult respiratory distress syndrome (ARDS), disseminated intravascular coagulation syndrome (DIC), pulmonary embolism, platelet deposition, myocardial infarction, or thrombosis Preventive treatment of mammals with multiple blood vessels.

  The FVII polypeptide of the TF dimer antagonist is chemically linked to the molecular linker moiety. LM can bind two FVII polypeptides simultaneously to the TF site. To accomplish this, the backbone must be long enough to span the distance between the TF sites, but flexible so that it can bind well to the second TF site. In other words, a suitable LM can be assumed to be a stable, extended secondary structure arrangement, but remain flexible and sufficiently soluble in water or physiological systems.

  “Linker moiety” (LM) or “backbone” refers to any biocompatible molecule that functions as a means to join two FVII polypeptides together. Each FVII polypeptide, or its equivalent of the FVII polypeptide, is bound to the molecule LM via a chemical bond, for example via an amide or peptide bond between the amino and carboxyl groups of LM, or vice versa. is there. It should be understood that LM may include covalent and non-covalent chemical bonds or a mixture thereof. “Flexible” refers to the multiple carbon-carbon bonds with free rotation about these axes so that two FVII polypeptides can be separated by a suitable distance for the LM to bind the two TF sites. means.

  Suitable LM or backbones include peptides; polynucleotides; monosaccharides, disaccharides and oligosaccharides, and sugars including cyclodextrins and dextrans; polyethylene glycol, polypropylene glycol, polyvinyl alcohol, hydrocarbons, polyacrylates, and amino- , Hydroxy-, thio-, or carboxy-functionalized silicones, polymers including other biomaterial units; and combinations thereof, including but not limited to. LM materials such as those described above are widely commercially available or are available via synthetic organic methods generally known to those skilled in the art.

LM, for example, has the following structure: linear or branched C _1-50 -alkyl, linear or branched C _2-50 -alkenyl, linear or branched C _2-50 -alkynyl, to chain 1 to 50 membered linear or branched, C _3-8 cycloalkyl, containing carbon and at least one N, O, or S atom, 3 to 3 containing carbon and at least one N, O, or S atom in the ring Selected from among 8-membered cyclic rings, aryl, heteroaryl, amino acids, the structure may optionally be substituted with one or more of the following groups: H, hydroxy, phenyl, phenoxy, benzyl , Thienyl, oxo, amino, C _1-4 -alkyl, CONH ₂ , CSNH ₂ , C _1-4 monoalkylamino, C _1-4 dialkylamino, acylamino, sulfonyl, carboxy, carboxyamide, halogeno, C _1-6 Alkoxy, C _1-6 alkylthio, trifluoroalkoxy Si, alkoxycarbonyl, haloalkyl. The LM may be linear or branched and may contain one or more double bonds or triple bonds. The LM may contain one or more heteroatom-like N, O, or S. It should be understood that an LM can include multiple classes of the group described above, as well as multiple members within a class. Where the LM comprises multiple classes of groups, such LMs are preferably obtained by connecting different units via these functional groups. Methods for forming such bonds include standard organic synthesis and are well known to those skilled in the art.

  Peptides, proteins, and amino acids as used herein include or refer to “natural”, ie, naturally occurring amino acids, as well as “non-classical” D-amino acids, D-isomer of amino acids, α-isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, β-methylamino acid , Designer amino acids such as Cα-methyl amino acid, Nα-methyl amino acid, and general amino acid analogs. In addition, the amino acids are Abu, 2-aminobutyric acid; γ-Abu, 4-aminobutyric acid; ε-Ahx, 6 aminohexanoic acid; Aib, 2-aminoisobutyric acid; β-Ala, 3-aminopropionic acid; Orn, Ornithine; Hyp, trans-hydroxyproline; Nle, norleucine; Nva, norvaline.

  The three-letter designation “GLA” as used herein means 4-carboxyglutamic acid (field γ-carboxyglutamic acid).

  “Combination of these” means that the LM can contain multiple classes of groups as described above, as well as multiple members within a class. Where an LM contains multiple classes of groups, such LMs are preferably obtained by connecting different units via these functional groups. Methods for forming such bonds include standard organic synthesis and are well known to those skilled in the art.

  In order to select an appropriate linker moiety, FVII polypeptides are bound to different LMs and screened in a TF activity assay.

LM is for example hydroxy, oxo, amino, C _1-4 monoalkylamino, acylamino, sulfonyl, carboxy, carboxyamide, halogeno, C _1-6 alkoxy, C _1-6 alkylthio, trifluoroalkoxy, alkoxycarbonyl, or haloalkyl Functional groups such as groups can be included. The LM can also include a functional group having a charge, such as an ammonium group or a carboxylate group.

  The charged functional group can provide the TF dimer antagonist with sufficient solubility in water or physiological systems, providing a reactive site for ionic bond formation with other species, TF / FVIIa Enhance their binding activity to other members of the / FXa complex. It is within the purview of those skilled in the art to select specific acids and their concentrations to provide optimal solubility and binding activity properties for the TF dimer antagonist. Preferably, the total amount of charged functional groups is minimized so as to maximize the specificity of the TF dimer antagonist for the TF site but not significantly reduce solubility.

  The FVII polypeptide is located on the LM so that the distance between them is sufficient to bind the FVII polypeptide to two TF molecules. Since the LM of a TF dimer antagonist is flexible, the FVII polypeptide can exhibit a conformation that allows the FVII polypeptide to bind to two TF molecules. Such distance between FVII polypeptides can be measured by any method known in the art, such as molecular modeling, or can be predicted theoretically. Molecular modeling programs that can be used are commonly known and available in the art.

  It should be understood that the two FVII polypeptides of a TF dimer antagonist can be the same or different.

  The LM can further comprise an FVIIa inhibitor.

  “FVIIa inhibitor” means a substance that binds to FVIIa and reduces or prevents the conversion of FX to FVIIa catalyzed by FXa. FVIIa inhibitors are at least 50% at a concentration of 400 μM substance A in the FVIIa amidolytic assay described by Persson et al. (Persson et al., J. Biol. Chem. 272: 19919-19924, 1997). May be identified as Substance A that reduces the amidolytic activity. Substances that reduce amidolytic activity to at least 50% at a concentration of 300 M substance A are preferred; substances that reduce amidolytic activity to at least 50% at a concentration of 200 M substance A are more preferred.

  The “FVIIa inhibitor” may be selected from any one of several groups of FVIIa directed inhibitors. Such inhibitors are broadly classified for the purposes of the present invention: i) inhibitors that reversibly bind to FVIIa and are cleavable by FVIIa, ii) reversibly bind to FVIIa, Inhibitors that cannot be cleaved, and iii) inhibitors that bind irreversibly to FVIIa. For a review of serine protease inhibitors, see Proteinase inhibitors (Research Monographs in cell and Tissue Physiology; v. 12) Elsevier Science Publishing Co., Inc., New York (1990).

  The FVIIa inhibitor moiety may also be an irreversible FVIIa serine protease inhibitor. Such irreversible active site inhibitors generally form a covalent bond with the protease active site. Such irreversible inhibitors include common serine protease inhibitors such as the peptide chloromethyl keto (see Williams et al., J. Biol. Chem. 264: 7536-7540 (1989)) or peptidyl chloromethane. Azapeptides; various guanidinobenzoate derivatives and acylating agents such as 3-alkoxy-4-chloroisocoumarin; sulfonyl fluorides such as phenylmethylsulfonyl fluoride (PMSF); diisopropylfluorophosphate (DFP); Tosylpropyl chloromethyl ketone (TPCK); tosyl lysyl chloromethyl ketone (TLCK); nitrophenyl sulfonate and related compounds; including but not limited to heterocyclic protease inhibitors such as isocoumarin and coumarin.

  Examples of peptidic irreversible FVIIa inhibitors are Phe-Phe-Arg chloromethyl ketone, Phe-Phe-Arg chloromethyl ketone, D-Phe-Phe-Arg chloromethyl ketone, D-Phe-Phe-Arg chloromethyl Ketone, Phe-Pro-Arg chloromethyl ketone, D-Phe-Pro-Arg chloromethyl ketone, Phe-Pro-Arg chloromethyl ketone, D-Phe-Pro-Arg chloromethyl ketone, L-Glu-Gly-Arg chloro Including but not limited to methyl ketone and D-Glu-Gly-Arg chloromethyl ketone.

  Examples of FVIIa inhibitors also include benzoxazinones such as those described in PCT / DK99 / 00138 or their heterocyclic analogs.

Examples of other FVIIa inhibitors include, for example, Phe-Phe-Arg, D-Phe-Phe-Arg, Phe-Phe-Arg, D-Phe-Phe-Arg, Phe-Pro-Arg, D-Phe-Pro- Small peptides such as Arg, Phe-Pro-Arg, D-Phe-Pro-Arg, L- and D-Glu-Gly-Arg; peptidomimetics; benzamidine systems; heterocycles substituted by one or more amidino groups Aromatic substituted by one or more C (= NH) NHR groups, wherein R is H, C _1-3 alkyl, OH, or a group that is easily cleaved in vivo The term “C _1-50 -alkyl” or “C _1-50 -alkanediyl” as used herein includes _1-50 carbon atoms, including but not limited to aromatic or heteroaromatic systems. A straight or branched, saturated or unsaturated hydrocarbon chain having.

"C _2-50 - alkenyl" or - The term "C _2-50 alkenediyl" is as used herein, 2-50 carbon atoms and at least one double bond, the unsaturated A branched or straight hydrocarbon chain.

The term “C _2-50 -alkynyl” or “C _2-50 -alkyndiyl” as used herein is an unsaturated moiety having 2 to 50 carbon atoms and at least one triple bond. A branched or straight hydrocarbon chain. C _1-50 -alkyl residues include aliphatic hydrocarbon residues, unsaturated aliphatic hydrocarbon residues, and alicyclic hydrocarbon residues. Examples of C _1-50 -alkyl within this definition are decanyl, hexadecanyl, octadecanyl, nonadecanyl, icosanyl, docosanyl, tetracosanyl, triacotanyl, decandiyl, hexadecandiyl, octadecandiyl, nonadecandiyl, icosandiyl, docosanediyl, tetracosanediyl, triacantanediyl Including but not limited to diyl.

The term C _3-8 -cycloalkyl is an alicyclic hydrocarbon residue, including saturated alicyclic hydrocarbon residues having 3 to 8 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc .; And C _5-6 unsaturated alicyclic rings having 5 to 6 carbon atoms such as 1-cyclopentenyl, 2-cyclopentenyl, 3-cyclopentenyl, 1-cyclohexenyl, 2-cyclohexenyl, 3-cyclohexenyl, etc. Means a hydrocarbon residue of the formula

The term “C _1-6 -alkoxy” as used herein, alone or in combination, is attached via an ether oxygen and has its free valence bond derived from the ether oxygen. And straight or branched monovalent substituents, including C _1-6 -alkyl groups having 1 to 6 carbon atoms such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, pentoxy.

The term “C _1-6 -alkylthio” as used herein, alone or in combination, is attached via a thioether sulfur atom and has its free valence bond from the thioether sulfur. And a linear or branched monovalent substituent containing a C _1-6 -alkyl group having 1 to 6 carbon atoms.

  The terms “aryl” and “heteroaryl” as used herein refer to optionally substituted aryl or optionally substituted, including: heteroaryl, Phenyl, biphenyl, indene, fluorene, naphthyl (1-naphthyl, 2-naphthyl), anthracene (1-anthracenyl, 2-anthracenyl, 3-anthracenyl), thiophene (2-thienyl, 3-thienyl), furyl (2-furyl) , 3-furyl), indolyl, oxadiazolyl, isoxazolyl, quinazoline, fluorenyl, xanthenyl, isoindanyl, benzhydryl, acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl , Triazoyl (1,2,3-triazol-1-yl, 1,2,3-triazol-2-yl 1,2,3-triazol-4-yl, 1,2,4-triazol-3-yl) , Oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2-thiazolyl, 4-thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2- Pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl, 7-quinolyl, 8-quinolyl), isoquinolyl (1-isoquinolyl, 3-isoquinolyl, 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl, 8-isoquinolyl), benzo [b] furanyl ( 2-Benzo [b] furani , 3-benzo [b] furanyl, 4-benzo [b] furanyl, 5-benzo [b] furanyl, 6-benzo [b] furanyl, 7-benzo [b] furanyl), 2,3-dihydrobenzo [ b] furanyl (2- (2,3-dihydro-benzo [b] furanyl), 3- (2,3-dihydro-benzo [b] furanyl), 4- (2,3-dihydro-benzo [b] furanyl) ), 5- (2,3-dihydro-benzo [b] furanyl), 6- (2,3-dihydro-benzo [b] furanyl), 7- (2,3-dihydro-benzo [b] furanyl), Benzo [b] thiophenyl (2-benzo [b] thiophenyl, 3-benzo [b] thiophenyl, 4-benzo [b] thiophenyl, 5-benzo [b] thiophenyl, 6-benzo [b] thiophenyl, 7-benzo [ b] thiophenyl), 2,3-dihydrobenzo [b] thiophenyl (2- (2,3-dihydro-benzo [b] thiophenyl), 3- (2,3-dihydro-benzo [b] thiophenyl), 4- (2,3-dihydro-benzo [b] thiophenyl), 5- (2,3-dihydro- Benzo [b] thiophenyl), 6- (2,3-dihydro-benzo [b] thiophenyl), 7- (2,3-dihydro-benzo [b] thiophenyl)), indolyl (1-indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl) , Benzimidazolyl (1-benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5-benzimidazolyl, 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl) , 2-benzoxazolyl), benzothiazolyl (1-benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzo Thiazolyl, 7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz [b, f] azepine (5H-dibenz [b, f] azepin-1-yl, 5H-Dibenz [b, f] azepin-2-yl, 5H-Dibenz [b, f] azepin-3-yl, 5H-Dibenz [b, f] azepin-4-yl, 5H-Dibenz [b, f] Azepine-5-yl), 10,11-dihydro-5H-dibenz [b, f] azepine (10,11-dihydro-5H-dibenz [b, f] azepin-1-yl, 10,11-dihydro-5H -Dibenz [b, f] azepin-2-yl, 10,11-dihydro-5H-dibenz [b, f] azepin-3-yl, 10,11-dihydro-5H-dibenz [b, f] azepine-4 -Yl, 10,11-dihydro-5H-dibenz [b, f] azepin-5-yl).

  The invention also relates to partially or fully saturated analogs of the aforementioned ring structures.

The term "C _1-4 monoalkylamino" and "C _1-4 dialkylamino", methylamino, dimethylamino, N- ethyl -N- methylamino, ethylamino, diethylamino, propylamino, dipropylamino, N One or both of the hydrogens, such as-(n-butyl) -N-methylamino, n-butylamino, di (n-butyl) amino, sec-butylamino, t-butylamino, are 1-4 carbon atoms An amino group having an alkyl group independently substituted with alkyl as defined above.

The term “acyl” or “carboxy” refers to a monovalent substituent containing a C _1-6 -alkyl group attached through a carbonyl group, such as acetyl, propionyl, butyryl, isobutyryl, pivaloyl, valeryl, and the like. .

The term “acylamino” refers to a C _1-n C (═O) NH— group.

The term “carboxamide” refers to the group —C (═O) NHC _1-n .

The term “trifluoroalkoxy” means that _{three of} its hydrogen atoms bonded to one or more of the carbon atoms, such as (CF ₃ ) O—, (CF ₃ ) CH ₂ O—, are replaced by fluorine atoms Refers to a C _1-6 alkoxy group as defined above.

The term “alkoxycarbonyl” refers to a —C (═O) (R) group, wherein R is a C _1-6 alkoxy group as described above.

The term “C _1-6 -alkoxycarbonyl” as used herein includes, for example, carbomethoxyl, carboethoxy, propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl, sec-butoxycarbonyl, tert-butoxy. Carbonyl, 3-methylbutoxycarbonyl, n-hexoxycarbonyl, etc .; refers to a monovalent substituent containing a C _1-6 -alkoxy group bonded via a carbonyl group.

  The term “leaving group” as used herein includes, but is not limited to, a halogen, sulfonate, or acyl group. Suitable leaving groups are known to those skilled in the art.

  “Halogen” refers to fluorine, chlorine, bromine, and iodine.

  “Halo” refers to fluoro, chloro, bromo, and iodo.

  “Any” or “optionally” means that the event or situation described thereafter may or may not occur, as well as that the event or situation occurs, and It means to include a case that does not happen. For example, “… optionally substituted aryl” means that aryl may or may not be substituted, and that the description includes both unsubstituted and substituted aryl. means.

LM containing FVIIa inhibitors for use in the preparation of TF dimer antagonists may be prepared by the following method. In the following method, the FVIIa inhibitor is referred to as the letter F _n and when n is 1 or 2, it indicates that the LM may comprise two different FVIIa inhibitors.

Method 1
The LM containing the FVIIa inhibitor is F ₁ -BX, wherein X is a functional group capable of reacting with the structure F ₂ -Y where Y is a functional group F ₁ -BX By reacting by the usual coupling reaction methods using known coupling reagents.

Method 2
LM containing FVIIa inhibitors may be prepared by a reaction between F ₁ -BZ where Z is a leaving group and F2W where W is a nucleofiles. Examples of leaving groups are halogen, sulfonate, phosphonate. Examples of nucleofiles are hydroxy, amino, N-substituted amino, and carbanions.

Method 3
LM containing FVIIa inhibitors may be prepared by a reaction between F ₂ -BZ where Z is a leaving group and F ₁ -W where W is a nucleofile. Examples of leaving groups are halogen, sulfonate, phosphonate. Examples of nucleofiles are hydroxy, amino, N-substituted amino, and carbanions.

Method 4
Linker B can be reacted with structures F ₁ and F ₂ bound to the solid surface using methods well known in the art. This approach is particularly valuable when the structures F ₁ and F ₂ are identical, and this method allows the convergence here by the optimal distance between the reagent bound to the solid phase and the linker size. A significant increase in rate occurs.

Method 5
An LM containing an FVIIa inhibitor first reacts F ₁ or F ₂ with an activated linker moiety that forms the F ₁ -B and F ₂ -B moieties, respectively, and then converts the products formed to F ₂ , respectively. It may be prepared by the order of the reaction by reacting with the F ₁ moiety. Actual bond formation occurs upon reaction to functional groups or derivatives or leaving groups / nucleofiles, as described in Methods 1-3.

  The reaction can be performed in solution phase using procedures known by those skilled in the art or on a solid support.

Formation of FVII dimers by DNA technology
Although the use of reagents directed to the active site represents one approach to making TF dimer antagonists, this also involves more than one non-covalent interaction. This can be accomplished by adding a peptide tag to the FVII polypeptide that has the ability to specifically bind the FVII polypeptide. Perhaps most suitable tags to accomplish this are via the leucine zipper (LZ) domain, a naturally occurring element that can mediate firm protein-protein interactions.

LZ is present in many gene regulatory proteins, including: CCATT-box and enhancer binding proteins (C / EBP), cAMP response element (CRE) binding proteins (CREB, CRE-BP1, ATFs), transcription Jun / AP1 family of regulators, yeast common regulator protein GCN4, fos oncogene and fos-related proteins fra-1 and fosB, C-myc, L-myc, and N-myc oncogenes, octamer-binding transcription Factor 2 (Oct-2 / OTF-2).

おそらく、最も研究されたLZは、酵母の一般的な制御タンパク質GCN4のものであるが、免疫原性が懸念されうるので、ヒト化産物またはヒト相当物のうちの1つを使用してもよい。したがって、好ましい態様において、ヒトATF7またはATF4由来のLZおよびこれらの誘導体が使用される。実際の融合タンパク質は、PCRまたはプライマ-二量体カセットを含む、当業者に基地の従来の分子生物学的方法を使用して作製してもよい。タグは、FVIIポリペプチドのコード配列に対してC末端に導入され、ある程度の高次構造上の柔軟性を提供するために役立つ短いスペーサ部分（グリシン-セリン-アラニン）の後にあり、したがって、所望のオリゴマーの形成を支持する（その他のリンカー部分を想像することもでき、実際により長いスペーサが、オリゴマー形成による構造上の衝突を避けるために必要にされるであろう）。したがって、本発明にしたがったTF二量体アンタゴニストの１つの例は、天然に形成されたFVII-Gly-Ser-Ala-LZ構築物の二量体であることができる。FVII-LZ融合タンパク質は、その他のFVIIポリペプチドについて記載されているように産生される。 Structurally, LZ is typically a pattern ^{(H 1 · P · P ·} H 2 · P · P · P) n, wherein H is selected Leu, Ile, Val, or Asn, And consisting of periodic repeats of leucine or other hydrophobic residues every 7 positions over a distance covering 8 helix turns according to a pattern where n ≧ 2, typically n ≧ 4. Segments containing these periodic arrays of leucine residues are present in the higher order structure of the amphipathic α-helix. A leucine side chain extending from one α helix tagged on one FVII polypeptide interacts with that from a similar α helix tagged on a second FVII polypeptide, dimer The structure formed by the cooperation of these two areas forms a coiled coil. The structural data available for LZ indicate that the dimeric morphology present in parallel conformations, ie the N-terminus of both helices is at the same end of the dimer. This is important because the TF binding domain may provide for the presentation of dimerized FVII polypeptides, both of which are oriented to facilitate binding. An additional feature of the LZ motif is that specific mutations in the two hydrophobic positions of the peptide allow the user to switch between dimeric, trimeric, or tetrameric conformations of the target protein. It is. More complex oligomeric forms of LZ provide individual elements with different orientations depending on the composition of the individual binding partners (Harbury, PB et al., Science (1993) 262: 1401-1407).

Perhaps the most studied LZ is that of the yeast general control protein GCN4, but one of humanized products or human equivalents may be used as immunogenicity may be a concern . Accordingly, in a preferred embodiment, LZ derived from human ATF7 or ATF4 and derivatives thereof are used. The actual fusion protein may be made using conventional molecular biology methods based on those skilled in the art, including PCR or primer-dimer cassettes. The tag is introduced at the C-terminus relative to the coding sequence of the FVII polypeptide and is after a short spacer portion (glycine-serine-alanine) that serves to provide some degree of conformational flexibility and is therefore desired (Other linker moieties can also be envisioned, and indeed longer spacers would be needed to avoid structural collisions due to oligomer formation). Thus, one example of a TF dimer antagonist according to the present invention can be a dimer of a naturally formed FVII-Gly-Ser-Ala-LZ construct. FVII-LZ fusion proteins are produced as described for other FVII polypeptides.

  As used herein, amino acids are represented using the abbreviations shown in Table 1 approved by the Biochemical Nomenclature Committee (CBN) of IUPAC-IUB. Those having amino acids and isomers represented by names or the following abbreviations are natural L forms unless otherwise specified. Furthermore, the left and right ends of the amino acid sequence of the peptide are the N and C ends, respectively, unless otherwise specified.

Table 1: Abbreviations for amino acids:
Amino acid 3 letter code 1 letter code Glycine
Proline Pro P
Alanine Ala A
Valin
Leu L
Isoleucine Ile I
Methionine Met M
Cystine Cys C
Phenylalanine Phe F
Tyrosine Tyr Y
Tryptophan Trp W
Histidine His H
Lysine Lys K
Argin R
Glutamine Gln Q
Asparagine Asn N
Glutamate Glu E
Aspartic acid Asp D
Serine Ser S
Threonine Thr T
The invention also relates to a method for preparing a human FVII polypeptide as described above. Suitable human FVII polypeptides are desirably produced by recombinant DNA technology. To this end, the DNA sequence encoding human FVII is a DNA sequence that encodes all or part of the protein by preparing a genomic or cDNA library, and hybridization using synthetic oligonucleotide probes according to standard techniques. (See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989). For this purpose, the DNA sequence encoding the protein is preferably of human origin, ie derived from a human genomic DNA or cDNA library.

  Alternatively, a DNA sequence encoding a human FVII polypeptide can be obtained by established standard methods, such as the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or (It may be prepared synthetically by the method described in Matthes et al., EMBO Journal 3 (1984), 801-805. According to the phosphoamidite method, oligonucleotides are synthesized on an automated DNA synthesizer. Purified, annealed, ligated and cloned into an appropriate vector.

  DNA sequences are also prepared by polymerase chain reaction using specific primers, as described, for example, in US Pat. No. 4,683,202, Saiki et al., Science 239 (1988), 487-491 or Sambrook et al. May be.

  The DNA sequence encoding the human FVII polypeptide is usually inserted into a recombinant vector, which may be any vector, which will conveniently be subjected to recombinant DNA procedures, and vector selection may be Often depends on the host cell into which the is to be introduced. Thus, the vector may be a vector that replicates independently, ie, a vector that exists as an extrachromosomal entity, such as a plasmid, whose replication is independent of chromosomal replication. Alternatively, the vector may be one that integrates into the host cell genome when introduced into the host cell, and may replicate with the chromosome into which it is integrated.

  The vector is preferably an expression vector in which a DNA sequence encoding a human FVII polypeptide is operably linked to an additional segment that is required for transcription of the DNA. In general, expression vectors are derived from plasmid or viral DNA, or may contain both elements. The term “operably linked” refers to a DNA sequence that encodes a polypeptide, eg, transcription is initiated at a promoter, so that the segment functions with emphasis for these intended purposes. It is shown to be arranged so as to progress through.

  A promoter may be any DNA sequence that exhibits transcriptional activity in a selected host cell, and may be derived from a gene encoding a protein that is homologous or heterologous to the host cell.

  Examples of suitable promoters for directing transcription of DNA encoding human FVII polypeptide in mammalian cells include the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854-864), MT-1 ( Metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814), CMV promoter (Boshart et al., Cell 41: 521-530, 1985), or adenovirus 2 major late promoter (Kaufman and Sharp, Mol. Cell Biol, 2: 1304-1319, 1982).

  Examples of suitable promoters used in insect cells include the polyhedrin promoter (US Pat. No. 4,745,051; Vasuvedan et al., FEBS Lett. 311, (1992) 7-11), the P10 promoter (JM Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), Autographa californica polynuclear disease virus basic protein promoter (EP397485), baculovirus immediate early gene 1 promoter (US Pat. No. 5,155,037; US Pat. No. 5,162,222) Or the baculovirus 39K delayed-early gene promoter (US Pat. No. 5,155,037; US Pat. No. 5,162,222).

  Examples of suitable promoters used in yeast host cells include promoters derived from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419-434) or alcohol dehydrogenase gene (Young et al., In Genetic Engineering of Microorganisms for Chemicals (Hollaender et al. Eds.), Plenum Press, New York, 1982 et al.) Or TPI1 (US 4,599,311) or Contains the ADH2-4c (Russell et al., Nature 304 (1983), 652-654) promoter.

  Examples of suitable promoters used in filamentous fungal host cells are, for example, the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093-2099) or the tpiA promoter. Examples of other useful promoters include A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral alpha amylase, A. niger acid stable amylase, A. niger or A. awamori glucoamylase (gluA), Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, or A. niger It is derived from the gene encoding nidulans acetamidase. This is preferably the TAKA-amylase and gluA promoter. Suitable promoters are mentioned for example in EP238023 and EP383779.

  In addition, the DNA sequence encoding the human FVII polypeptide is operably connected to human growth hormone terminator (Palmiter et al., Science 222, 1983, pp. 809-814) or TPI1 as necessary. Appropriate (Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 419-434) or ADH3 (McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099) as appropriate terminators Any terminator may be optionally attached. The vector may also include a set of RNA splice sites located downstream of the promoter or upstream from the insertion site for the FVII sequence itself. Preferred RNA splice sites may be obtained from adenovirus and / or immunoglobulin genes. A polyadenylation signal located downstream of the insertion site is also included in the expression vector. Particularly preferred polyadenylation signals are the early or residual polyadenylation signal from SV40 (Kaufman and Sharp, ibid), the polyadenylation signal from the adenovirus 5Elb region, the human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res. 9 : 3719-3730, 1981), or a polyadenylation signal derived from the human FVII gene or the bovine FVII gene. The expression vector also includes an untranslated viral leader sequence such as a tripartite leader of adenovirus 2 located between the promoter and the RNA splice site; and an enhancer sequence such as the SV40 enhancer.

  The recombinant vector may further comprise a DNA sequence that allows the vector to replicate in the host cell in question. An example of such a sequence (when the host cell is a mammalian cell) is the SV40 origin of replication.

  Suitable sequences that allow the vector to replicate when the host cell is a yeast cell are the yeast plasmid 2μ replication gene REP1-3 and the origin of replication.

  Vectors also include selectable markers, such as genes encoding dihydrofolate reductase (DHFR), such as genes for products that complement host cell defects, or the TSI gene (PR Russell, Gene, Schizosaccharomyces pombe). 40, 1985, pp. 125-130), or drugs that confer resistance to ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin, or methotrexate. For filamentous fungi, selectable markers include amdS, pyrG, argB, niaD, or sC.

  In order to direct the human FVII polypeptide of the invention into the secretory pathway of the host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence, or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is linked to the DNA sequence encoding the human FVII polypeptide in the appropriate reading frame. The secretory signal sequence is generally placed 5 'to the DNA sequence encoding the peptide. The secretory signal sequence may be usually a protein or may be derived from a gene encoding another secreted protein.

  For secretion from yeast cells, the secretory signal sequence may encode any signal peptide that ensures efficient orientation of the human FVII polypeptide expressed in the cell's secretory pathway. The signal peptide. It may be a naturally occurring signal peptide or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides include the α-factor signal peptide (see US Pat. No. 4,870,008), the mouse peptide peptide signal (see O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), Modified carboxypeptidase signal peptide (see LA Valls et al., Cell 48, 1987, pp. 887-897), yeast BAR1 signal peptide (see WO 87/02670), or yeast It is known to be an aspartic protease 3 (YAP3) signal peptide (see M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137).

  For efficient secretion in yeast, a sequence encoding a leader peptide may be inserted downstream of the signal sequence and upstream of the DNA sequence encoding the human FVII polypeptide. The function of the leader peptide allows the expressed peptide to be directed from the endoplasmic reticulum to the Golgi and further to the secretory vesicle for secretion into the medium (ie, across the cell wall or at least around the cell membrane of the yeast cell) Export of human FVII polypeptide across the cell membrane into the cavity). Leader peptides are described in yeast alpha factor leaders (eg, US Pat. No. 4,546,082, US Pat. No. 4,870,008, European Patent No. 16201, European Patent No. 123294, European Patent No. 123544, and European Patent No. 163529). Use). Alternatively, the leader peptide may be a synthetic leader peptide, ie the leader peptide is not found in nature. Synthetic leader peptides may be constructed, for example, as described in WO 89/02463 or WO 92/11378.

  For use in filamentous fungi, the signal peptide is conveniently a gene encoding Aspergillus sp. Amylase or glucoamylase, a gene encoding Rhizomucor miehei lipase or protease, or Humicola It may be derived from Humicola lanuginosa lipase. The signal peptide is preferably a gene encoding A. oryzae TAKA amylase, A. niger neutral alpha amylase, A. niger acid stable amylase, or A. niger glucoamylase. Derived from. Suitable signal peptides are disclosed, for example, in EP 238023 and EP215594.

  For use in insect cells, the signal peptide may be an insect gene (WO 90/05783), such as the Lepidoptera Tobacco Manduca sexta lipokinetic hormone precursor signal peptide (see US Pat. No. 5,023,328). For convenience).

Used to bind human FVII polypeptides, promoters, and optionally DNA sequences encoding terminator and / or secretory signal sequences, respectively, and to insert them into appropriate vectors containing information necessary for replication The procedures performed are well known to those skilled in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).

  Methods for transfecting mammalian cells and expressing DNA sequences introduced into the cells are described, for example, by Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb, Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.

  The selectable marker may be introduced into the cell simultaneously with the gene of interest on separate plasmids, or they may be introduced on the same plasmid. In the case of the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, with the latter arrangement producing a dicistronic message. This type of construct is known in the art (eg, Levinson and Simonsen, US Pat. No. 4,713,339). It may also be advantageous to add additional DNA, known as “carrier DNA”, to the mixture that is introduced into the cell.

  After the cells have taken up the DNA, they are cultured in an appropriate cell growth medium, typically 1-2 days, to initiate expression of the gene of interest. As used herein, the term “appropriate cell growth medium” refers to a medium containing nutrients and other components required for cell growth and expression of the human FVII polypeptide of interest. Means. The medium generally contains a carbon source, a nitrogen source, essential amino acids, essential sugars, vitamins, salts, phospholipids, proteins, and growth factors. For production of gamma-carboxylated proteins, the medium preferably contains vitamin K at a concentration of about 0.1 g / ml per about 5 g / ml. Drug selection is then applied to the selection of cell growth that expresses the selectable marker in an appropriate manner. For cells transfected with a selectable marker that can be amplified, cells with increased expression levels may be selected by increasing the drug concentration and increasing the copy number of the cloned sequence. The stably transfected cell clones are then screened for expression of the human FVII polypeptide of interest.

  A host cell into which a DNA sequence encoding a human FVII polypeptide has been introduced can produce a post-translationally modified human FVII polypeptide and can be used in any cell, including yeast, fungi, and higher eukaryotic cells. There may be.

Examples of mammalian cell lines used in the present invention are COS-1 (ATCCCRL1650), baby hamster kidney (BHK), and 293 (ATCCCRL1573; Graham et al., J. Gen. Virol. 36: 59-72 , 1977) cell lines. A preferred BHK cell line is the tk ^- ts13 BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79: 1106-1110, 1982, incorporated herein by reference), below BHK570 Called cells. The BHK570 cell line has been deposited under the ATCC accession number CRL10314 by American Type Culture Collection 12301 Parklawn Dr., Rockville, Maryland 20852. The tk ^- ts13BHK cell line is also available from ATCC under accession number CRL1632. In addition, RatHepI (rat hepatocytoma; ATCCCRL1600), rat HepII (rat hepatocytoma; ATCCCRL1548), TCMK (ATCCCCL139), human lung (ATCCHB8065), NCTC1469 (ATCCCCL9.1), CHO (ATCCCCL61), and DUKX cells Many other cell lines including (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216-4220, 1980) may be used within the scope of the present invention.

  Examples of suitable yeast cells include cells of yeast spp. (Saccaromyces spp.) Or fission yeast spp. (Schizosavvaromyces spp.), In particular Saccharomyces cerevisiae or Saccaromyces kluyveri strains. Methods for transforming yeast cells with heterologous DNA and methods for producing heterologous polypeptides therefrom include, for example, U.S. Pat.No. 4,599,311, U.S. Pat.No. 4,931,373, U.S. Pat.Nos. 4,870,008, 5,037,743, And U.S. Pat. No. 4,845,075, which are incorporated herein by reference. Transformed cells are selected by phenotype determined by a selectable marker, commonly drug resistance, or the ability to grow in the absence of a particular nutrient (eg, leucine). A preferred vector for use in yeast is the POT1 vector disclosed in US Pat. No. 4,931,373. The DNA sequence encoding the human FVII polypeptide may be after the signal sequence and optionally the leader sequence, eg, as described above. Further examples of suitable yeast cells are K. lactis, Kluyveromyces such as Hansenula, eg H. polymorpha, or Pichia, eg Pichi. P. pastoris strain (see Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465).

  Examples of other fungal cells are filamentous fungi, such as Aspergillus spp. (Aspergillus spp.), Panchobi spp. (Neurospora spp.), Fusarium spp. (Fusarium spp.), Or Trichoderma spp. (Trichoderma spp.), In particular, cells of A. oryzae, A. nidulans or A. niger strains. The use of Aspergillus spp. For the expression of proteins is described, for example, in European Patent No. 272 277, European Patent No. 238 023, European Patent No. 184 438. Transformation of F. oxysporum may be performed, for example, as described by Malardier et al., 1989, Gene 78: 147-156. The transformation of Trichoderma spp. May be performed, for example, as described in EP 244 234.

  When filamentous fungi are used as host cells, they may be transformed with the DNA constructs of the invention by conveniently integrating the DNA construct into the host chromosome in order to obtain recombinant host cells. This integration is generally regarded as advantageous to make it more likely that the DNA sequence is stably maintained in the cell. Integration of the DNA construct into the host chromosome may be performed according to conventional methods, for example by homologous recombination or heterologous recombination. Transformation of insect cells and production of heterologous polypeptides therein are described in US Pat. No. 4,745,051; US Pat. No. 4,879,236; US Pat. No. 5,155,037; US Pat. No. 5,162,222; European Patent No. 397,485 (all of which are , Which is incorporated herein by reference). Insect cell lines used as hosts are optimally Lapidoptera cell lines such as Spodoptera frugiperda cells or Trichoplusia ni cells (see US Pat. No. 5,077,214). Also good. The culture conditions may optimally be as described, for example, in WO 89/01029 or WO 89/01028 or any of the references mentioned above.

  The transformed or transfected host cells described above are then cultured in an appropriate nutrient medium under conditions that allow for culturing for expression of the human FVII polypeptide, after which all or part of the resulting peptide is removed from the culture. It may be recovered. The medium used to culture the cells may be any conventional medium suitable for culturing host cells, such as minimal medium or complex medium with appropriate supplementation. Appropriate media are available from commercial sources or may be prepared according to published prescriptions (eg, catalogs from the American Type Culture Collection). The human FVII polypeptide produced by the cell is then separated from the medium by centrifugation or filtration, precipitated the protein water-soluble component of the supernatant, or depending on the type of polypeptide in question, or It may be recovered from the medium by conventional procedures including filtration by salt, eg ammonium sulfate procedures, purification by various chromatographic procedures, eg ion exchange chromatography, gel filtration chromatography, affinity chromatography, and the like.

  For the preparation of recombinant human FVII polypeptides, cloned wild type FVII DNA sequences are used. This sequence may be modified to encode the desired FVII variant. The complete nucleotide and amino acid sequence of human FVII is known. See US Pat. No. 4,784,950, which describes the cloning and expression of recombinant human FVII, which is incorporated herein by reference. The bovine FVII sequence is described in Takeya et al., J. Biol. Chem, 263: 14868-14872 (1988), which is incorporated herein by reference.

  Changes in amino acid sequence may be achieved by various techniques. Modification of the DNA sequence may be by site-directed mutagenesis. Techniques for site-directed mutagenesis are well known in the art and are described, for example, in Zoller and Smith (DNA 3: 479-488, 1984). Thus, FVII nucleotide and amino acid sequences may be used to introduce selection changes.

  The DNA sequences used in the present invention are typically pre-processed at the amino terminus of the FVII protein so that appropriate post-translational processing (eg, gamma-carboxylation of glutamic acid residues) and secretion from the host cell can be obtained. Encodes the peptide. The pre-propeptide may be that of FVII or another plasma protein that is dependent on vitamin K, such as Factor IX, Factor X, prothrombin, protein C, or protein S. As is well known to those skilled in the art, further modifications can be made to the amino acid sequence of FVII if these modifications do not significantly impair the ability of the protein to act as a clotting factor. For example, as generally described in US Pat. No. 5,288,629 (incorporated herein by reference), catalytic triad FVII is converted to a double-stranded form of its activation of the enzyme precursor FVII. Modifications can be made to the activated cleavage site to inhibit conversion.

  Within the scope of the present invention, transgenic animal technology may be used to produce human FVII polypeptides. It is preferred to produce the protein in the mammary gland of the host female mammal. Expression of the protein of interest in the mammary gland and subsequent secretion into milk overcomes many of the problems encountered in isolating proteins from other sources. Milk is easily recovered, available in large quantities, and is well characterized biochemically. In addition, major milk proteins are present in milk at high concentrations (typically about 1-15 grams / liter). From a commercial point of view, it is preferable to use as the host a species with a clearly large milk yield. Small animals such as mice and rats can be used, but livestock mammals such as pigs, goats, sheep and cows are preferably used. Sheep are particularly preferred due to factors such as the previous history of this type of transgene, milk yield, cost, and the availability of equipment for collecting sheep milk. For a comparison of factors affecting host species selection, see WIPO Publication WO 88/00239. It is generally desirable to select a host animal that has been bred for dairy use, such as East Friesland sheep, or to introduce a milk strain at a later date by genetically modified breeding.

  In any case, animals of known good health should be used. To obtain expression in the mammary gland, a transcription promoter derived from a milk protein gene is used. Milk protein genes include genes encoding casein (see US Pat. No. 5,304,489, incorporated herein by reference), beta-lactoglobulin, alpha-lactalbumin, and whey acidic protein. The β-lactoglobulin (BLG) promoter is preferred. In the case of the ovine β-lactoglobulin gene, a region of at least 406 base pairs near the 5 ′ flanking sequence of the gene is generally used, but approximately 4.25 kbp including the 5 ′ flanking promoter and the non-coding portion of the β-lactoglobulin gene. Most sequences of 5 ′ flanking up to about 5 kbp, such as the DNA portion, are preferred. See Whitelaw et al., Biochem J. 286: 31-39 (1992). Similar promoter DNA fragments from other species are also suitable.

  In addition, other regions of the β-lactoglobulin gene may be incorporated into the construct, or may be regions of the genome of the gene to be expressed. For example, it is generally accepted in the art that constructs lacking introns are not well expressed compared to those containing such DNA sequences (Brinster et al., Proc. Natl. Acad. Sci. USA 85 : 836-840 (1988); Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478-482 (1991); Whitelaw et al., Transgenic Res. 1: 3-13 (1991), International Publication No.89 / And International Publication No. WO 91/02318, each incorporated herein by reference, in this regard, if possible, the natural intron of the gene encoding the protein or polypeptide of interest. It is generally preferred to use a genomic sequence that includes all or part of, and thus further contain at least some introns, eg, derived from the β-lactoglobulin gene. A portion of DNA that provides intron splicing and RNA polyadenylation derived from the 3 'non-coding region of the toglobulin gene, when replaced with the 3' non-coding sequence of the native gene. Can enhance and stabilize the expression level of the protein or polypeptide of interest Within other embodiments, the region surrounding the starting ATG of the sequence encoding the human FVII polypeptide is milk specific. Substitution with corresponding sequences from typical protein genes, such substitutions provide a putative tissue-specific initiation environment to enhance expression.All pre-pro sequences of the human FVII polypeptide It is convenient to replace the 5 ′ non-coding sequence with, for example, that of the BLG gene, but a narrower region may be replaced.

  For expression of a human FVII polypeptide in a transgenic animal, a DNA segment encoding the human FVII polypeptide is operably linked to an additional DNA segment required for its expression and an expression unit is combined. Make it. Such additional segments include the promoters described above and sequences that provide for transcription termination and polyadenylation of mRNA. The expression unit further comprises a DNA segment encoding a secretory signal sequence operably linked to the segment encoding human FVII polypeptide. The secretory signal sequence may be the natural secretory signal sequence of the human FVII polypeptide or may be that of another protein such as milk protein. See, for example, von Heinje, Nuc. Acids Res. 14: 4683-4690 (1986); and Meade et al., US Pat. No. 4,873,316, which are incorporated herein by reference.

  The construction of an expression unit for use in a transgenic animal is conveniently performed by inserting a sequence encoding a phage vector containing a human FVII polypeptide or additional DNA segment into a plasmid, but the expression unit consists essentially of It may be constructed by any ligation sequence. It is particularly convenient to provide vectors containing DNA segments encoding milk proteins, and to replace milk protein coding sequences with those of human FVII polypeptides, thereby including expression control sequences for milk protein genes Make a gene fusion. In any event, cloning of the expression unit in a plasmid or other vector facilitates amplification of the human FVII polypeptide. Amplification is conveniently performed in bacterial (eg, E. coli) host cells, and thus vectors typically include an origin of replication and a selectable marker that is functional in the bacterial host cell.

  The expression unit is then introduced into fertilized eggs (including early fetuses) of the selected host species. The introduction of heterologous DNA is site-specific using microinjection (eg, US Pat. No. 4,873,191), retroviral infection (Jaenisch, Science 240: 1468-1474 (1988)), or embryonic stem (ES) cells. Incorporation (Bradley et al., Bio / Technology 10: 534-539 (1992) can be achieved by one of several routes. The ovum is then transplanted into a pseudopregnant female fallopian tube or uterus. Progeny with DNA introduced into these germ lines can normally pass DNA to these offspring in a Mendelian fashion and can generate transgenic groups.

  General procedures for producing transgenic animals are known in the art. For example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al., Bio / Technology 6: 179-183 (1988); Wall et al., Biol. Reprod. 32: 645-651 (1985) Buhler et al., Bio / Technology 8: 140-143 (1990); Ebert et al., Bio / Technology 9: 835-838 (1991); Krimpenfort et al., Bio / Technology 9: 844-847 (1991); Wall et al., J. Cell. Biochem. 49: 113-120 (1992), U.S. Pat. Nos. 4,873,191 and 4,873,316; WIPO publications WO 88/00239, WO 90/05188, WO 92/11757; as well as GB87 / 00458, which are hereby incorporated by reference. Techniques for introducing foreign DNA sequences into mammals and these embryonic cells were originally developed in mice. For example, Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380-7384 (1980); Gordon and Ruddle, Science 214: 1244-1246 (1981); Palmiter and Brinster, Cell 41: 343-345 (1985) and Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442 (1985). These techniques were subsequently applied to larger animal uses, including livestock species (eg, WIPO publications WO 88/00239, WO 90/05188, and WO 92). / 11757; see Simons et al., Bio / Technology 6: 179-183 (1988)). In summary, the most efficient route used to date in the production of transgenic mice or livestock is the production of hundreds of linear molecules of DNA of interest in the pronucleus of fertilized eggs according to established techniques. Inject into one. Alternatively, DNA injection into the cytoplasm of the zygote can be used. Alternatively, the production of genetically modified plants may be used. Expression may be generalized or directed to a specific organ such as a tuber. Hiatt, Nature 344: 469-479 (1990); Edelbaum et al., J. Interferon Res. 12: 449-453 (1992); Sijmons et al., Bio / Technology 8: 217-221 (1990), and European Patent Office publications See European Patent No. 255,378. FVII made according to the present invention may be purified by affinity chromatography against an anti-FVII antibody column. The immunosorbent column preferably contains a highly specific monoclonal antibody. As described in Wakabayashi et al., J. Biol. Chem, 261: 11097-11108, (1986) and Thim et al., Biochem. 27: 7785-7793, (1988), incorporated herein by reference, It is particularly preferred to use a calcium-dependent monoclonal antibody. Further generation may be performed by conventional chemical purification means such as high performance liquid chromatography. Other purification methods, including barium citrate precipitation, are known in the art and may be applied to the purification of FVII described herein (generally, Scopes, R., Protein Purification, (See Springer-Verlag, NY, 1982). For pharmaceutical applications, substantially pure FVII that is at least about 90-95% uniform is preferred, and most preferably 98-99% or more uniform. Once purified partially or as homogeneous as required, FVII may then be used for therapy.

  Using Factor XIIa as described by Hedner and Kisiel (1983, J. Clin. Invest. 71: 1836-1841) or by other proteases with trypsin-like specificity (Kisiel and Fujikawa, Behring Inst. Mitt. 73: 29-42, 1983), conversion of single chain FVII to active double chain FVIIa may be achieved. Alternatively, FVII may be self-activated by passage through an ion exchange chromatography column such as Mono Q.RTM (Pharmacia Fire Chemicals) or the like (Bjoern et al., 1986, Research Disclosures 269: 564-565). The FVII molecules of the invention and their pharmaceutical compositions are useful for administration to humans to treat various conditions, particularly including intravascular coagulation.

  The compounds of the present invention may have one or more asymmetric centers and are stereoisomers (optical isomers) as separated, pure or partially purified stereoisomers, Or racemic mixtures are also contemplated to be within the scope of the present invention.

  Within the scope of the present invention, TF dimer antagonists may be prepared in the form of pharmaceutically acceptable salts, particularly acid addition salts, including salts of organic and mineral acids. Examples of such salts are salts of organic acids such as formic acid, fumaric acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid including. Suitable inorganic acid addition salts include salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like. Further examples of pharmaceutically acceptable inorganic or organic acid addition salts include the pharmaceutically acceptable salts listed in the Journal of Pharmaceutical Science, 66, 2 (1977) known to those skilled in the art.

  Hydrates that can form the present compounds are also contemplated as pharmaceutically acceptable acid addition salts.

  Acid addition salts may be obtained as direct products of compound synthesis. Alternatively, the salt may be isolated by dissolving the free base in a suitable solvent containing the appropriate acid and evaporating the solvent or otherwise separating the salt and solvent.

  The compounds of the present invention may form solvates with standard low molecular weight solvents using methods known to those skilled in the art. The TF dimer antagonists of the present invention are useful for preparing pharmaceutical compositions for the treatment or prevention of thrombotic or coagulopathy related diseases or disorders including vascular diseases and inflammatory reactions. This includes deep vein thrombosis, arterial thrombosis, postoperative thrombosis, coronary artery bypass graft (CABG), percutaneous percutaneous coronary artery reconstruction (PTCA), stroke, cancer, tumor metastasis, angiogenesis , Ischemia / reperfusion, arthritis including rheumatoid arthritis, thrombolysis, arteriosclerosis and restenosis after angioplasty, acute and chronic signs such as inflammation, septic shock, sepsis, hypotension, adult Blood vessels such as respiratory distress syndrome (ARDS), disseminated intravascular coagulation syndrome (DIC), pulmonary embolism, platelet deposition, myocardial infarction, or prophylactic treatment of mammals with atherosclerotic blood vessels at risk for thrombosis Diseases or disorders related to TF-mediated clotting activity, including disorders, thrombotic or clotting-related diseases or disorders, or chronic thromboembolic diseases related to inflammatory reactions and fibrin formation Or disorder, and including other diseases, but not limited to. Diseases or disorders associated with TF are not limited to in vivo coagulopathy disorders such as those named above, but in-line from the patient in processes such as dialysis procedures, hemofiltration, or blood bypass during surgery. Includes in vitro TF / FVIIa related processes, such as clotting that would be caused by extracorporeal circulation of blood, including removed blood.

  The TF dimer antagonist may be administered in the form of a pharmaceutically acceptable acid addition salt or, where appropriate, as an alkali metal or alkaline earth metal or lower alkyl ammonium salt. Good. Such salt forms are believed to exhibit approximately the same order of activity as the free base system.

  Apart from the pharmaceutical use of the compounds, these may also be useful in vitro tools for investigating inhibition of FVIIa, FXa, or TF / FVIIa / FXa activity.

Pharmaceutical composition
In another aspect, the invention provides a pharmaceutically acceptable range pharmaceutical composition, or a pharmaceutically acceptable salt thereof, which includes within its scope a TF dimer antagonist as an active ingredient. Contains with carrier or diluent.

  Optionally, the pharmaceutical composition of the invention comprises a TF dimer antagonist in combination with one or more other compounds exhibiting anticoagulant activity, such as a platelet aggregation inhibitor.

  The compounds of the present invention may be formulated in a pharmaceutical composition or diluent that includes the compound and a pharmaceutically acceptable carrier. Such carriers include water, saline, ethanol, polyols such as glycerin or propylene glycol, or vegetable oils. Also, as used herein, “pharmaceutically acceptable carrier” includes any and all of solvents, dispersion media, coatings, antifungal agents, preservatives, isotonic agents, and the like. . Except for which any conventional media is incompatible with the active ingredient and its intended use, its use of the composition of the present invention is envisioned.

  The composition may be prepared in conventional form by conventional techniques, such as a capsule, tablet, solution, or suspension. The pharmaceutical carrier used may be a conventional solid or liquid carrier. Examples of solid carriers are lactose, clay, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are syrup, peanut oil, olive oil, and water. Similarly, the carrier or diluent may include any time delay material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. The formulations may also contain wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents. The formulations of the present invention may be formulated to provide simple, sustained or delayed release of the active ingredient after administration to a patient by using procedures well known in the art.

  The pharmaceutical composition can be sterilized and optionally, supplementary drugs, emulsifiers, salts for affecting osmotic pressure, buffers, and / or non-hazardous without reacting with the active compound, and / or Or a coloring substance etc. can be mixed.

  The route of administration is active at the appropriate or desired site of action, such as oral or parenteral, e.g. rectal, transdermal, subcutaneous, intranasal, intramuscular, topical, intravenous, intraurethral, ophthalmic solution or ointment. While any route that efficiently transports the compound may be used, the oral route is preferred.

  When using a solid carrier for oral administration, the preparation can be tableted and placed in a hard gelatin capsule in powder or pellet form, or in the form of a troche or lozenge. The amount of solid carrier may vary widely but will usually be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation may be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous suspension or solution.

  For nasal administration, a preparation may contain a compound of formula (I) dissolved or suspended in a liquid carrier, particularly an aqueous carrier, for aerosol application. The carrier may contain solubilizers such as parabens, propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrins, or additives such as preservatives.

  For parenteral application, particularly suitable are solutions or suspensions for injection, preferably aqueous solutions containing the active compound dissolved in polyhydroxylated castor oil.

  Tablets, dragees or capsules with talc and / or carbohydrate carriers or binders etc are particularly suitable for oral application.

  Preferred carriers for tablets, dragees, or capsules include lactose, corn starch, and / or potato starch. If a sweetened medium can be used, syrup or elixir can be used.

A typical tablet may be prepared by conventional tableting techniques and includes the following:
core:
Active compound (as free compound or salt thereof) 10mg
Colloidal silicon dioxide (Areosil®) 1.5mg
Cellulose, microcrystals (Avicel®) 70mg
Modified cellulose gum (Ac-Di-Sol (registered trademark)) 7.5mg
Magnesium stearate
Coating :
HPMC about 9mg
* Mywacett (registered trademark) 9-40T 0.9 mg
* Acylated monoglyceride used as plasticizer for film coating.

  The compounds of the present invention may be administered to mammals, particularly humans, in need of treatment, prevention, elimination, alleviation, or recovery of various thrombolytic or coagulopathy disorders or disorders as described above. Also good. Such mammals also include animals such as wildlife, livestock such as domestic pets and non-domestic animals.

  Usually, a suitable dosage form for oral, nasal, pulmonary or transdermal administration is about 0.001 mg to about 100 mg, preferably about 0.01 mg to about 50 mg of a compound of formula I or a pharmaceutically acceptable carrier or Contains mixed with diluent.

  The compounds may be administered by the oral, rectal, or parenteral (including subcutaneous) route in parallel with, simultaneously with, or together with the pharmaceutically acceptable carrier or diluent. The compound is often and preferably in the form of its alkali metal or alkaline earth metal salt.

  The appropriate dosage range depends on the exact mode of administration, the form of administration, the indications to be administered, the subject and the subject's body weight, and the choice or experience of the physician or veterinarian. Different as shown.

  The compounds of the present invention have interesting pharmacological properties. For example, the compounds of this invention can be used to modulate and normalize impaired hemostatic balance in mammals caused by deficiency or dysfunction of blood clotting factors or their inhibitors. The activity of FVIIa and in particular TF / FVIIa plays an important role in the control of the coagulation cascade, and such key modulatory modulators such as the present invention are thrombotic or coagulant, including vascular diseases and inflammatory responses. It can be used for the treatment and prevention of disorder-related diseases or disorders. Accordingly, the pharmaceutical compositions of the present invention will be useful for adjusting and normalizing impaired hemostatic balance in mammals. In particular, the pharmaceutical composition will be useful for the treatment and prevention of thrombotic or coagulopathy related diseases or disorders including vascular diseases and inflammatory reactions.

  “Adjusting and normalizing impaired hemostatic balance” means achieving an effect on the clotting system and / or animal model that reduces the risk of thrombosis or bleeding as measured by in vitro assays.

  More particularly, the pharmaceutical composition may be used as an inhibitor of mammalian blood coagulation, as an inhibitor of mammalian clotting activity, as an inhibitor of mammalian fibrin deposition, as an inhibitor of mammalian platelet deposition, as a deep vein thrombosis , Arterial thrombosis, postoperative thrombosis, coronary artery bypass graft (CABG), percutaneous percutaneous coronary reconstruction (PTCA), stroke, cancer, tumor metastasis, angiogenesis, ischemia / reperfusion, rheumatism Arthritis, including arthritis, thrombolysis, arteriosclerosis and restenosis after angioplasty, acute and chronic signs such as inflammation, septic shock, sepsis, hypotension, adult respiratory distress syndrome (ARDS), dissemination Useful for the treatment of mammals suffering from vascular blood coagulation syndrome (DIC), pulmonary embolism, platelet deposition, myocardial infarction, or for the prophylactic treatment of mammals with atherosclerotic blood vessels at risk for thrombosis It will allo. The compositions of the present invention may also be used as an adjunct to thrombolytic therapy.

  Furthermore, the present invention provides a method for inhibiting TF-inducing activity in a mammal, wherein a pharmaceutically acceptable amount of at least one compound of the present invention is administered to a mammal in need of such treatment. In combination with the excipients and / or carriers to be administered.

Assay Method Inhibition of FX-FVIIa / phospholipid-embedded TF-catalyzed activation by dimer TF antagonist FXa production assay (assay method 1):
In the following examples, all concentrations are final concentrations. Lipidated TF (10 pM), FVIIa (100 pM), and dimeric TF antagonist or FFR-rFVIIa (0-50 nM) HBS / BSA (50 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM CaCl ₂ , 1 mg / ml BSA) solution is incubated for 60 minutes at room temperature before adding FX (50 nM). After 10 minutes, the reaction is stopped by the addition of 1/2 volume of stop buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 20 mM EDTA). The amount of FXa produced is determined by adding substrate S2765 (0.6 mM, Chromogenix) and measuring absorbance at 405 nm continuously for 10 minutes. IC ₅₀ values of TF antagonist inhibition of FVIIa / lipidated TF mediated FX activation may be calculated. The IC ₅₀ value for FFR-rFVIIa is 51 +/− 26 pM in this assay.

Inhibition of FVIIa / cell surface TF-catalyzed FX activation by dimeric TF antagonist (assay method 2):
In the following examples, all concentrations are final concentrations. Human lung fibroblast WI-38 (ATCC number CCL-75) or human bladder carcinoma cell line J82 (ATCC number HTB-1) monolayer or human keratinocyte cell line CCD1102KerTr (ATCC) constitutively expressing TF No. CRL-2310) is used as a TF source for the activation of FX catalyzed by FVIIa / TF. Confluent cell monolayers in 96-well plates once with buffer A (10 mM Hepes, pH 7.45, 150 mM NaCl, 4 mM KCl, and 11 mM glucose) and buffer B (1 mg / ml BSA and 5 mM) Wash once with buffer A) supplemented with Ca ²⁺ . FVIIa (1 nM), FX (135 nM), and various concentrations of dimeric TF antagonist or FFR-rFVIIa in buffer B solution are added simultaneously to the cells. Allow FXa to form for 15 minutes at 37 ° C. An aliquot of 50 μl is removed from each well and stop buffer (Buffer A supplemented with 10 mM EDTA and 1 mg / ml BSA) is added to 50 μl. The amount of FXa produced is determined by transferring 50 μl of the above mixture to a microtiter plate well and adding 25 μl ChromozymX (final concentration 0.6 mM) to the well. Absorbance at 405 nm is measured continuously and the initial rate of color generation is converted to FXa concentration using the FXa calibration curve. The IC ₅₀ value for FFR-rFVIIa is 1.5 nM in this assay.

Inhibition of 125I-FVIIa binding to cell surface TF by a dimeric TF antagonist (assay method 3):
In the following examples, all concentrations are final concentrations. Binding studies are all constitutively expressing TF, human bladder carcinoma cell line J82 (ATTC number HTB-1) or human keratinocyte cell line (CCD1102KerTr ATCC number CRL-2310) or NHEKP166 (Clonetics number CC-2507) It was used. Wash confluent monolayers of 24-well tissue culture plates with Buffer A supplemented with 5 mM EDTA (see Assay Method 8), then once with Buffer A and Buffer B (see Assay Method 8) Washed once. Preincubate monolayer with 100 μl of cooling buffer B for 2 minutes. Various concentrations of Mabs (or FFR-FVIIa) and radiolabeled FVIIa (0.5 nM ¹²⁵ I-FVIIa) are added simultaneously to the cells (final volume 200 μl). Incubate the plate at 4 ° C. for 2 hours. At the end of the incubation, unbound material is removed and the cells are washed 4 times with ice cold buffer B and lysed in 300 μl lysis buffer (200 mM NaOH, 1% SDS, and 10 mM EDTA). Radioactivity is measured with a gamma counter (Cobra, Packard Instruments). Using GraFit4, binding data were analyzed and fitted to the curve (Erithacus Software, Ltd., (UK). The FFR-rFVIIa IC ₅₀ value is 4 nM in this assay.

  The invention is further illustrated by the following examples.

  The present invention is not limited in scope by the specific embodiments disclosed in the examples, but is illustrative of many aspects of the present invention, and any embodiment that is functionally equivalent is within the scope of the present invention. It is contemplated. Those skilled in the art will be able to know or ascertain many equivalents to the specific embodiments of the invention described herein using only routine testing. These and all other equivalents are intended to be included within the scope of the claims.

オクタン二酸ビス-（{1-[1-（1-クロロアセチル-4-グアニジノ-ブチルカルバモイル）-2-フェニル-エチルカルバモイル]-2-フェニル-エチル}-アミド）（実施例1）

N、N’ビス-（1-{1-[1-（2-クロロ-アセチル）-4-グアニジノ-ブチルカルバモイル]-2-フェニル-エチルカルバモイル}-2-フェニルエチル）-オキサルアミド（実施例2）

N,N’-ビス-（1-{1-[1-（2-クロロ-アセチル）-4-グアニジノ-ブチルカルバモイル]-2-フェニル-エチルカルバモイル}-2-フェニルエチル-2,3-ジヒドロキシスクシンアミド（実施例3）

ペンタン二酸ビス-[（1-{1-[1-（2-クロロ-アセチル）-4-グアニジノ-ブチルカルバモイル]-2-フェニル-エチルカルバモイル}-2-フェニル-エチル）-アミド]（実施例4）

ドコサ-10,12ジエン二酸ビス-[（1-{1-[1-（2-クロロ-アセチル）-4-グアニジノ-ブチルカルバモイル]-2-フェニル-エチルカルバモイル}-2-フェニル-エチル）-アミド]（実施例5）

オクタデカン二酸ビス-[（1-{1-[1-（2-クロロ-アセチル）-4-グアニジノ-ブチルカルバモイル]-2-フェニル-エチルカルバモイル}-2-フェニル-エチル）-アミド]（実施例8）

エイコサン二酸ビス-[（1-{1-[1-（2-クロロ-アセチル）-4-グアニジノ-ブチルカルバモイル]-2-フェニル-エチルカルバモイル}-2-フェニル-エチル）-アミド]（実施例7）

ドコサン二酸ビス-[（1-{1-[1-（2-クロロ-アセチル）-4-グアニジノ-ブチルカルバモイル]-2-フェニル-エチルカルバモイル}-2-フェニル-エチル）-アミド]（実施例6）
実施例1
オクタン二酸ビス-（{1-[1-（1-クロロアセチル-4-グアニジノ-ブチルカルバモイル）-2-フェニル-エチルカルバモイル]-2-フェニル-エチル}-アミド）（1）
ジスクシンイミジルスベラート（Pierce #21555）（5.86mg）を1.5mlのFFR-CMK（25mg）のリン酸緩衝液pH7.4溶液と混合し、2滴のDMFを添加後、混合物をN₂雰囲気下で1日間閉じた容器中で撹拌し、引き続く蒸発により、黄色の油生の生成物物（35mg）を得て、これを1ml/分の一定の流れでHPLC（逆相カラム（Symmetry Shield,C₈、Waters、Part no. WAT200655））で精製した。溶出は、水相（0.1%のTFA水溶液）に関して有機相（0.1%のトリフルオロ酢酸（TFA）を含有するアセトニトリル）の割合を増大することによって達成した。FFR-CMKの二量体の形態が約28分で溶出される場合、35分にわたる14%から50%の有機相の直線濃度勾配を使用した。 The following LMs containing FVIIa inhibitors were synthesized:

Octanedioic acid bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide) (Example 1)

N, N′bis- (1- {1- [1- (2-chloro-acetyl) -4-guanidino-butylcarbamoyl] -2-phenyl-ethylcarbamoyl} -2-phenylethyl) -oxalamide (Example 2) )

N, N'-bis- (1- {1- [1- (2-chloro-acetyl) -4-guanidino-butylcarbamoyl] -2-phenyl-ethylcarbamoyl} -2-phenylethyl-2,3-dihydroxy Succinamide (Example 3)

Pentanedioic acid bis-[(1- {1- [1- (2-chloro-acetyl) -4-guanidino-butylcarbamoyl] -2-phenyl-ethylcarbamoyl} -2-phenyl-ethyl) -amide] Example 4)

Docosa-10,12 dienedioic acid bis-[(1- {1- [1- (2-chloro-acetyl) -4-guanidino-butylcarbamoyl] -2-phenyl-ethylcarbamoyl} -2-phenyl-ethyl) -Amide] (Example 5)

Octadecanedioic acid bis-[(1- {1- [1- (2-chloro-acetyl) -4-guanidino-butylcarbamoyl] -2-phenyl-ethylcarbamoyl} -2-phenyl-ethyl) -amide] Example 8)

Eicosanedioic acid bis-[(1- {1- [1- (2-chloro-acetyl) -4-guanidino-butylcarbamoyl] -2-phenyl-ethylcarbamoyl} -2-phenyl-ethyl) -amide] Example 7)

Docosanedioic acid bis-[(1- {1- [1- (2-chloro-acetyl) -4-guanidino-butylcarbamoyl] -2-phenyl-ethylcarbamoyl} -2-phenyl-ethyl) -amide] Example 6)
Example 1
Octanedioic acid bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide) (1)
Disuccinimidyl suberate (Pierce # 21555) (5.86 mg) is mixed with 1.5 ml FFR-CMK (25 mg) in phosphate buffer pH 7.4, 2 drops of DMF are added, and the mixture is then N ₂ atmosphere Stirring in a closed vessel for 1 day and subsequent evaporation yields a yellow oily product (35 mg) which is HPLC (Symmetry Shield, C ₈ , Waters, Part no. WAT200655)). Elution was achieved by increasing the proportion of the organic phase (acetonitrile containing 0.1% trifluoroacetic acid (TFA)) relative to the aqueous phase (0.1% aqueous TFA). If the dimeric form of FFR-CMK elutes at about 28 minutes, a linear gradient of 14% to 50% organic phase over 35 minutes was used.

  A fraction with an rt (retention time) of 30.94 minutes was isolated. MS (M + 1) 1141, yield 12%, oil.

Example 2
N, N'-bis- (1- {1- [1- (2-chloro-acetyl) -4-guanidino-butylcarbamoyl] -2-phenyl-ethylcarbamoyl} -2-phenylethyl) -oxalamide (2)
Disuccinimidyl suberate (4.2 mg) and FFR-CMK (25 mg) were mixed in 1.5 ml phosphate buffer pH 7.4, 2 drops of DMF were added and the mixture was closed under N ₂ atmosphere for 1 day Stirring in a vessel and subsequent evaporation gave a yellow oily crude product (35 mg) that was HPLC (reverse phase column (Symmetry Shield, C ₈ , Waters, Part no. WAT200655)). Elution was achieved by increasing the proportion of the organic phase (acetonitrile containing 0.1% trifluoroacetic acid (TFA)) relative to the aqueous phase (0.1% aqueous TFA). If the dimeric form of FFR-CMK elutes at about 28 minutes, a linear gradient of 14% to 50% organic phase over 35 minutes was used.

  The rt fraction of 31.08 minutes was isolated. MS (M + 1) 1057, 8% yield, yellow oil.

Example 3
N, N'-bis- (1- {1- [1- (2-chloro-acetyl) -4-guanidino-butylcarbamoyl] -2-phenyl-ethylcarbamoyl} -2-phenylethyl-2,3-dihydroxy succin amide (3)
Disuccinimidyl tartrate (Pierce # 20589) (5.16 mg) and FFR-CMK (25 mg) were mixed in 1.5 ml of phosphate buffer pH 7.4, 4 drops of DMF were added, and the mixture was placed under N ₂ atmosphere. Stirred in a closed vessel overnight and subsequent evaporation gave a yellow oily crude product (35 mg) which was HPLC (Symmetry Shield, C ₈ , Waters, Part no. WAT200655)). Elution was achieved by increasing the proportion of the organic phase (acetonitrile containing 0.1% trifluoroacetic acid (TFA)) relative to the aqueous phase (0.1% aqueous TFA). If the dimeric form of FFR-CMK elutes at about 28 minutes, a linear gradient of 14% to 50% organic phase over 35 minutes was used.

  The rt fraction of 29.20 minutes was isolated. MS (M + 1) 1117, 4% yield, yellow oil.

Example 4
Pentanedioic acid bis- (1- {1- [1- (2-chloro-acetyl) -4-guanidino-butylcarbamoyl] -2-phenyl-ethylcarbamoyl} -2-phenyl-ethyl) -amide] (4)
Disuccinimidyl glutarate (Pierce # 20593) (4.89 mg) and FFR-CMK (25 mg) are mixed in 1.5 ml phosphate buffer pH 7.4, 1 ml DMF is added, and the mixture is placed under N ₂ atmosphere Stirring in a closed vessel for 3 days and subsequent evaporation gave a yellow oily crude product (35 mg) which was HPLC (Symmetry Shield, C) with constant flow of 1 ml / min. ₈ , Waters, Part no. WAT200655)). Elution was achieved by increasing the proportion of the organic phase (acetonitrile containing 0.1% trifluoroacetic acid (TFA)) relative to the aqueous phase (0.1% aqueous TFA). If the dimeric form of FFR-CMK elutes at about 28 minutes, a linear gradient of 14% to 50% organic phase over 35 minutes was used.

  The rtrt fraction of 29.20 minutes was isolated. MS (M + 1) 1099, 18% yield, yellow oil.

Example 5
10,12-docosadiynedioic acid bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide)
Docosa-10,12-diindioic acid bis- (2,5-dioxo-pyrrolidin-1-yl) ester (9.4 mg) mixed with 1.0 mL of phosphate buffer pH 7.4 solution of FFR-CMK (25 mg) After adding 1.0 ml of DMF, the mixture was stirred in a closed vessel under N ₂ atmosphere for 4 days and subsequent evaporation gave a yellow oily product (35 mg).

Example 6
Docosanedioic acid bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide)
Docosanedioic acid bis- (2,5-dioxo-pyrrolidin-1-yl) ester (9.5 mg) is mixed with 1.0 mL phosphate buffer pH 7.4 solution of FFR-CMK (25 mg) and 1.0 ml DMF The mixture was stirred in a closed vessel under N ₂ atmosphere for 4 days and subsequent evaporation gave a yellow oily crude product.

Example 7
Icosanedioic acid bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide)
Eicosanedioic acid bis- (2,5-dioxo-pyrrolidin-1-yl) ester (9.1 mg) mixed with 1.0 mL phosphate buffer pH 7.4 solution of FFR-CMK (25 mg) and 1.0 mL DMF The mixture was stirred in a closed vessel under N ₂ atmosphere for 4 days and subsequent evaporation gave a yellow oily crude product.

Example 8
Octadecanedioic acid bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide)
Octadecanedioic acid bis- (2,5-dioxo-pyrrolidin-1-yl) ester (8.6 mg) mixed with 1.0 mL phosphate buffer pH 7.4 solution of FFR-CMK (25 mg) and 1.0 mL DMF The mixture was stirred in a closed vessel under N ₂ atmosphere for 4 days and subsequent evaporation gave a yellow oily crude product.

Example 9:
Octanedioic acid bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide, 150 mM NaCl and 10 mM Mixed with rFVIIa in 10 mM glycylglycine pH 7.5 containing calcium chloride in a 1: 2 molar ratio and incubated for 6 days at 4 ° C. Samples were 20 mM Tris Cl, 250 mM NaCl, 5 mM calcium chloride. And loaded into HiLoad 16/60 Superdex 75 (Parmacia) equilibrated by pH 8.0.Fractions were analyzed by SDS-PAGE (Novex) and fractions 16-19 showing a band with a molecular weight of 100,000 were pooled. The concentrated sample was loaded onto a HiLoad 16/60 Superdex 200 (Parmacia) equilibrated with 20 mM Tris Cl, 250 mM NaCl, 5 mM calcium chloride, pH 8.0, and SDS. -Fractions containing a band with a molecular weight of 100,000 were pooled and concentrated.

  Size exclusion chromatography of rFVIIa reacted with octanedioic acid bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide (Figure 1a) and SDS-PAGE of fractions (Figure 1b) Samples for SDS-PAGE were not reduced, MARK 12 (Invitrogen) was used as the standard molecular weight (left lane). In 19, fractions were pooled, concentrated and loaded onto a Superdex200 column.

Example 10:
Production and expression of FVII-LZ.

The plasmid vector pFVII-fus for expressing human FVII fusion protein in mammalian cells is based on the pCI-neo plasmid vector (Promega, Madison, Wis.) And the existing BamHI site is deleted by BamHI digestion followed by T4DNA. A plasmid vector called pCI-neo (ΔBamHI) was obtained by ligation with polymerase. Briefly, the pFVII-fus vector is a propeptide under the control of a strong CMV promoter for transcription of the inserted cDNA and a neomycin phosphotransferase cDNA under the control of the SV40 early promoter for use as a selectable marker. Having a cDNA nucleotide sequence encoding human FVII. The FVII insert is made from IMAGE clone ID: 1848877, but any full length FVII cDNA can also be used by PCR using Expand High Fidelity (Roche). PCR products are generated by procedures well known to those skilled in the art for preparing DNA constructs using polymerase chain reaction with specific primers (see PCR Protocols, 1990, Academic Press, San Diego, California, USA). Be made) using oligonucleotide primers 1 and 2:
Primer 1. 5'-AAATAG GCTAGC ATGGTCTCCCAGGCCCTCAGG-3 '(SEQ ID NO: 1)
Primer 2. 5'-ACGCGT GAATTC TCAGGCGGATCCGGGAAATGGGGCTCG-3 '(SEQ ID NO: 2)
The resulting product contains the complete FVII coding sequence followed by an in-frame portion encoding Gly-Ser-Ala containing the BamHI site used for frame insertion of the C-terminal tag. The fragment is digested with NheI and EcoRI (primer sequences are underlined) and introduced into the NheI and EcoRI sites of pCI-neo (BamHI) to obtain a plasmid vector called pFVII-fus.

  Different leucine zipper insertions are made as primer dimers using overlapping oligonucleotides (italic overlapping regions), which are joined using T4 or Taq DNA polymerase, and a BamHI site encoding Gly-Ser-Ala A double-stranded DNA fragment encoding the selected LZ was obtained after the stop codon and EcoRI site before. (Primer sequences are underlined). Thus, an example of a design that yields a fusion protein containing ATF4-derived LZ is shown below.

Primer 3 (ATF4LZ forward):
5'-AT GGGATCC GCCAAGGAGCTGGAGAAGAAGAACGAGGCCCTGAAGGAGCGCGCCGACAGCCTGGC-3 '(SEQ ID NO: 3)
Primer-4 (ATF4LZ recerse):
5'-CAT GAATTC TCACTCCTCGATCAGGTCCTTCAGGTACTGGATCTCCTTGGCCAGGCTGTCGGCGC-3 '(SEQ ID NO: 4)
Following the reaction ligated by T4 polymerase, the DNA polymerase is removed, the double-stranded DNA product is digested with BamHI and EcoRI, and the resulting insert is purified using conventional methods. The purified insert is ligated to the BamHI and EcoRI sites of the pFVII-fus vector using T4 DNA ligase and transformed into an appropriate E. coli strain, such as DH5α or XL1 Blue; a plasmid vector containing the desired LZ tag ( pFVII-LZ) is identified, isolated using standard techniques, and sequenced by DNA sequencing. The resulting expression vector encodes the following proteins:
FVII-GSA- KELEKKNEALKERADSLAKEIQYLKDLIEE
The pFVII-LZ vector is transfected into CHO cells using lipofectamine or similar techniques, and stable clones are isolated following neomycin selection. Clones that secrete FVII were identified using FVII ELISA, and high-producing clones were further subcloned to produce clones with very specific FVII expression in Dulbecco's modified Eagle medium with 10% fetal bovine serum. obtain. The clones are then adapted for serum-free suspension culture using commercially available CHO medium (JRH Bioscience). The resulting recombinant FVII-LZ material can then be purified from the culture medium using conventional methods (Thim, L. et al., Biochemistry (1988) 27: 7785-93).

Example 11
Expression of FVII-LZ.

BHK cells were transfected with the pFVII-LZ vector essentially as described above (Thim et al. (1988) Biochemistry 27, 7785-7793; Persson and Nielsen (1996) FEBS Lett. 385, 241-243)) Obtain expression of FVII-LZ. The FVII-LZ protein is purified as follows:
The conditioned medium is 5 mM EDTA, 0.1% Triton X-100, 10 mM Tris, pH is adjusted to 8.0, and water is added to adjust the conductivity to 10-11 mS / cm, followed by Q Sepharose. Fill a 25 ml column of Fast Flow (Pharmacia Biotech).

  Protein elution was performed using 10 mM Tris, 50 mM NaCl, 0.1% Triton X-100, pH 8.0-10 mM Tris, 50 mM NaCl, 25 mM Calcium Chloride, 0.1% Triton X-100, pH 8.0. Achieved by stages. Fractions containing S314E / K316H-FVII are pooled and loaded onto a 25 ml column containing monoclonal antibody F1A2 (NovoNordisk, Bagsvard, Denmark) conjugated to CNBr active Sepharose 4B (Pharmacia Biotech).

Equilibrated with Hepes (pH 7.5) of 50mM containing 10mM of CaCl _2, 100 mM NaCl and 0.02% Triton X-100 column.

  After washing with equilibration buffer and equilibration buffer containing 2M NaCl, the bound material is eluted with equilibration buffer containing 10 mM EDTA instead of calcium chloride. Prior to use or storage, excess calcium chloride above EDTA is added or FVII-LZ is transferred to a buffer containing Ca2 +. The yield of each step was performed by factor VII ELISA measurement, and the purified protein was analyzed by SDS-PAGE. Subsequent inactivation of the FVII portion of the FVII-LZ molecule, for example by FFR-CMK, is known to those skilled in the art.

Example 12
FFR-FVIIa dimer and FFR.FVIIa were analyzed by competitive clotting assay as follows: bis-octanedioate ({1- [1- (1-chloroacetyl-4-guanidino-butyrate) as a linker FFR-FVIIa dimer or FFR-FVIIa monomer prepared using (rucarbamoyl) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide, 20 nM FVIIa, 10 nM calcium chloride, and Incubate with 0.1% BSA for 60 min at 37 ° C at the indicated concentration with 200 mM diluted 50 mM imidazole, 0.1 M NaCl, pH 7.4 solution of lipidated recombinant TF (Innovin, Dade Behring) A clotting time of 30 seconds was given in the sample without antagonist, which was measured on an ACL300 Research instrument (Instrumentation Laboratories) after adding 0.5 volume of FVII deficient plasma (Helena BioSciences). FFR-FVIIa monomer concentration required to double Was 0.23 μg / ml, but the concentration of FFR-FVIIa dimer required to double the clotting time was 0.002 μg / ml, ie the dimer had a clotting time It was almost 100 times more effective in stretching.

Size exclusion chromatography of rFVIIa was performed using octanedioic acid bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide. Reacted with (fraction (b) a) and SDS-PAGE. Effect of FFR-FVIIa active site dimer and FFR-FVIIa monomer in competitive coagulation assay using lipidated recombinant TF (Innovin) as TF source. Sequence used in the present invention. Sequence used in the present invention. Sequence used in the present invention.

Claims (25)

  A compound having the formula A- (LM) -D, wherein A and D are FVII polypeptides that bind to TF; and LM is a linker moiety having a molecular weight of less than 30,000 Daltons; and The compound is provided that the compound is not a compound having the formula wtFVIIai- (DPTA-dim-FFR-cmk) -wtFVIIai or FVIIai (Q10E32)-(DPTA-dim-FFR-cmk) -FVIIai (Q10E32) A compound that inhibits TF activity.   2. The compound of claim 1, wherein the affinity of the compound binding to TF is higher than the affinity of either A or D alone binding to TF.   3. A compound according to any one of claims 1-2, wherein A and D are human recombinant FVIIa.   3. The compound according to any one of claims 1-2, wherein A and D are human recombinant FVIIai.   The compound according to any one of claims 1 to 4, wherein LM comprises an amino acid sequence.   6. The compound according to any one of claims 1 to 5, wherein LM is a leucine zipper.   The compound according to any one of claims 1 to 6, wherein LM is an amino acid sequence independently selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 6.   The compound according to any one of claims 1 to 7, wherein LM is an amino acid sequence (Gly-Gly-Gly-Gly-Ser) n, and n is an arbitrary integer of 1 to 10. The compound according to any one of claims 1 to 8, wherein LM is a molecule in which the molecule is linear or branched C _1-50 -alkyl, linear or branched C _2-50 -alkenyl, Chain or branched C _2-50 -alkynyl, 1-50 member straight or branched chain containing carbon and at least one N, O, or S atom in the chain, C _3-8 cycloalkyl, carbon in the ring and Selected from the group consisting of a 3-8 membered cyclic ring containing at least one N, O, or S atom, aryl, heteroaryl, amino acid, wherein the structure is optionally in one or more of the following groups Substituted compounds: H, hydroxy, phenyl, phenoxy, benzyl, thienyl, oxo, amino, C _1-4 -alkyl, CONH ₂ , CSNH ₂ , C _1-4 monoalkylamino, C _1-4 dialkylamino, acylamino Sulfonyl, carboxy, carboxyamide, halogeno, C _1-6 alkoxy, C _1-6 alkylthio, trifluoroalkoxy, alkoxycarbonyl, haloalkyl.   10. The compound according to any one of claims 1 to 9, wherein LM is independently Phe-Phe-Arg chloromethyl ketone, Phe-Phe-Arg chloromethyl ketone, D-Phe-Phe-Arg chloro. Methyl ketone, D-Phe-Phe-Arg chloromethyl ketone, Phe-Pro-Arg chloromethyl ketone, D-Phe-Pro-Arg chloromethyl ketone, Phe-Pro-Arg chloromethyl ketone, D-Phe-Pro-Arg Contains a divalent chloromethyl ketone inhibitor consisting of two monomers selected from the group comprising chloromethyl ketone, L-Glu-Gly-Arg chloromethyl ketone, and D-Glu-Gly-Arg chloromethyl ketone Compound.   11. A compound according to any one of claims 1 to 10, wherein LM comprises two FVIIa inhibitors.   12. The compound according to any one of claims 1 to 11, wherein LM is bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-phenyl octadecadioate]. -Ethylcarbamoyl] -2-phenyl-ethyl} -amide), icosanedioic acid bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-phenyl-ethylcarbamoyl] -2 -Phenyl-ethyl} -amide), bis-docosanedioic acid bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl}- Amide), 10,12-docosadiynedioic acid bis-({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-phenyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide) , And bis-octanedioate ({1- [1- (1-chloroacetyl-4-guanidino-butylcarbamoyl) -2-phen Nyl-ethylcarbamoyl] -2-phenyl-ethyl} -amide).   A method for reducing TF activity, comprising contacting a compound having the formula A- (LM) -D with a TF expressing cell, wherein A and D bind to TF And LM is a linker moiety having a molecular weight of less than 30,000 daltons; and said compound is a compound having the compound wtFVIIai- (DPTA-dim-FFR-cmk) -wtFVIIai or FVIIai (Q10E32)-(DPTA -dim-FFR-cmk)-a method of inhibiting TF activity on condition that it is not a compound having the formula of FVIIai (Q10E32).   14. The method of claim 13, wherein the compound having the formula A- (LM) -D is described in any one of claims 1-12.   A pharmaceutical composition comprising an amount of a compound having the formula A- (LM) -D and a pharmaceutically acceptable carrier or excipient, wherein A and D are FVII polyamines that bind to TF. And LM is a linker moiety having a molecular weight of less than 30,000 daltons; and said compound has the compound wtFVIIai- (DPTA-dim-FFR-cmk) -wtFVIIai or FVIIai (Q10E32)-(DPTA-dim -FFR-cmk) -FVIIai (Q10E32) A composition that inhibits TF activity provided that it is not a compound having the formula.   16. The pharmaceutical composition according to claim 15, wherein the compound having the formula A- (LM) -D is described in any one of claims 1-12.   The pharmaceutical composition according to any one of claims 15 to 16, further comprising a platelet aggregation inhibitor.   A compound for use as a medicament having the formula A- (LM) -D, wherein A and D are FVII polypeptides that bind to TF; and LM has a molecular weight of less than 30,000 Daltons And the compound is not a compound having the compound wtFVIIai- (DPTA-dim-FFR-cmk) -wtFVIIai or FVIIai (Q10E32)-(DPTA-dim-FFR-cmk) -FVIIai (Q10E32) A compound that inhibits TF activity on the condition. Formula of   19. A compound according to claim 18, wherein the compound having the formula A- (LM) -D is described in any one of claims 1-12.   Use of a compound having the formula A- (LM) -D, wherein A and D are FVII polypeptides that bind to TF; and LM is a linker moiety having a molecular weight of less than 30,000 Daltons And said compound is not a compound having the formula wtFVIIai- (DPTA-dim-FFR-cmk) -wtFVIIai or FVIIai (Q10E32)-(DPTA-dim-FFR-cmk) -FVIIai (Q10E32); Use to inhibit TF activity as a condition.   21. Use according to claim 20, wherein the compound having the formula A- (LM) -D is described in any one of claims 1-12.   The use according to any one of claims 20 to 21, wherein the TF-related disease or disorder is deep vein thrombosis, arterial thrombosis, postoperative thrombosis, coronary artery bypass graft (CABG), transdermal Percutaneous coronary reconstruction (PTCA), stroke, cancer, tumor metastasis, angiogenesis, ischemia / reperfusion, arthritis including rheumatoid arthritis, thrombolysis, arteriosclerosis and restenosis after angioplasty, inflammation Acute and chronic signs such as septic chock, sepsis, hypotension, adult respiratory distress syndrome (ARDS), disseminated intravascular coagulation syndrome (DIC), pulmonary embolism, platelet deposition, myocardial infarction, or Use for preventive treatment of mammals with atherosclerotic blood vessels at risk for thrombosis.   A method for the prevention or treatment of a TF related disease or disorder in a mammal comprising administering to the mammal an effective amount of at least one compound having the formula A- (LM) -D, Where A and D are FVII polypeptides that bind to TF; and LM is a linker moiety having a molecular weight of less than 30,000 daltons; and said compound is a compound of wtFVIIai- (DPTA-dim-FFR- cmk) -wtFVIIai or FVIIai (Q10E32)-(DPTA-dim-FFR-cmk) -A method for inhibiting TF activity, provided that it is not a compound having the formula of FVIIai (Q10E32).   24. The method of claim 23, wherein the compound having the formula A- (LM) -D is described in any one of claims 1-12.   25. The method according to any one of claims 23 to 24, wherein the TF-related disease or disorder is deep vein thrombosis, arterial thrombosis, postoperative thrombosis, coronary artery bypass graft (CABG), transdermal Percutaneous coronary reconstruction (PTCA), stroke, cancer, tumor metastasis, angiogenesis, ischemia / reperfusion, arthritis including rheumatoid arthritis, thrombolysis, arteriosclerosis and restenosis after angioplasty, inflammation Acute and chronic signs such as septic chock, sepsis, hypotension, adult respiratory distress syndrome (ARDS), disseminated intravascular coagulation syndrome (DIC), pulmonary embolism, platelet deposition, myocardial infarction, or 13.Prophylactic treatment of mammals having atherosclerotic blood vessels at risk for thrombosis, the method according to any one of claims 1 to 12 for mammals in need of such treatment. Therapeutic treatment of at least one compound Comprising administering an effective amount, with pharmaceutically acceptable excipients and / or carriers.

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