Antifibrin antibody for use as antithrombotic tools

 

(57) Abstract:

The invention relates to medicine and related methods of use antifibrin antibodies for inhibition of in vivo blood clots, as well as pharmaceutical compositions and a kit containing a pharmaceutical composition for use in such methods. The essence of the invention lies in the fact that as antifibrin antibodies used monoclonal MN-1 antibody produced by hybridomas ADS 9739. The invention allows to inhibit the formation of thrombus at the injury site of blood vessel, 9 C. and 15 C.p. f-crystals, 2 Il.

1. The technical field to which the invention relates

The invention relates to methods of applying antifibrin antibodies for inhibition of in vivo blood clots. It also relates to pharmaceutical compositions for use in such methods.

2. Background of the invention

2.1. Blood clots during illness or surgery

Blood clots, or thrombi formed in injured blood vessels. Clinical manifestations of a pathological thrombosis or thrombotic disorders are extremely diverse is inania other vascular diseases (for example, of arteriosclerosis) can result in the blockage of the main arteries, which leads to ischemia of organs and associated life-threatening condition, such as stroke (stroke), myocardial infarction, etc.

In addition, invasive surgical procedures, including but not limited to it, balloon angioplasty, and organ transplants (both natural and artificial) can cause the formation of blood clots. For example, balloon angioplasty is a procedure used to clear clogged arteries can actually be damaged artery wall, causing reocclusion by deposition of a new blood clot. It is reported that percutaneous transluminal coronary angioplasty leaves the vessel affected due to reocclusion with a frequency of 25-35%. (Gimple, L. w.et al., Circulation, 86: 1536-46 (1992). Cm. also Sarembock, I. I. et al., Circulation, 80: 1029-40 (1989); and Ip. , I. H. al. , IACC 17: V-V (1991). Indeed, in one report claims that "the mechanism of angioplasty including, in most cases, endothelial damage and destruction of platelets or bundle [link] very similar to the process that leads to acute coronary syndromes. "Tenaglia, A. N., Ann.Rev. Med., 44: 465-79, 466 (1993).

As it was shown that systemic treatment Antico: 77B-88B (1991)), has a small effect or no is, and as such treatment anticoagulants often carries the risk of total bleeding in patients (Physicians' Desk Reference, 47 th Ed., Medical Economics Data (1993)), would be a useful new site-specific methods of treatment, when blood clots inhibited or their education is an obstacle, as in the case of preclinical vascular disease, and invasive surgical procedures.

2.2. The hemostatic system

The mechanism of hemostasis, through which is formed a blood clot, is a complex physiological reactive mechanism that is activated when the restoration of the damage to the injured blood vessel. Cm. Harber. L. A., and Mann, K. G."Thrombosis and Eibrinolysis in Thrombosis in Cardiovascular Disorders Euster, V., and verstraete, M. (eds), W. B. Saunders Co. (1992), pp.1-16.

Hemostasis is achieved with joint cooperation of the walls of damaged blood vessel, platelets and the coagulation system. Cm. Furie, B. and Furie, B. C., Cell, 53: 505-18 (1988).

The role of the coagulation system is to provide an insoluble fibrin matrix to stabilize and consolidate trombozitemii tube, which is going to subendothelial the structure of the damaged vessel in place powerdevelopment (factors of the blood coagulation system) sequentially activated with the formation of active enzyme. The formation of the fibrin matrix of circulating fibrinogen is a result of this cascading sequence of enzymatic reactions that result in the desired place quickly formed the Enzyme thrombin, which is the conversion of fibrinogen to fibrin, and fibrin stitched factor of HSA, which forms the clot.

The sequence of reactions can simply be represented by using the following three-stage process.

Thrombin

Stage 1 - Proteolysis: Fibrinogen -->

The fibrin monomers (A A DOS Des AABB)

+ FPA and F.

Stage 2 - Polymerization: Monomers of fibrin

Soluble fibrin polymers (unstitched and stitched).

Stage 3 - Coagulation: Soluble fibrin polymers -->

Insoluble fibrin clot.

Fibrinogen consists of three pairs of nonidentical polypeptide chains A, B and . Cm. L. Stryer, Biochemistry, 3 rd Ed., W. H. Freeman and Company, New Iork (1988) p. 249. At the initial stage, when fibrinogen is converted into fibrin, which is shown above in stage 1, fibrinogen is cleaved by thrombin to release fibrinopeptide A (FPA) from the amino-terminal endings of the two fibrinogenic A-chains. The remaining portion of the molecule fibrinogen is a "monomial is returned fibrinopeptide b (FPB) from the amino-terminal endings of the two fibrinogenic B-chains. The remaining part of the molecule of fibrinogen after the second cleavage is also a fibrin monomer, denoted Des AABB allocation FPA and FPB - and-chains of fibrin monomer identification of new amino-terminal end. Cm. W. Nieuwenhuiren, Blood Coagulation and Fibrinolysis 4:93-96 (1993) In stage 2 of the fibrin monomers spontaneously begin to form limonaire non-covalent linkages (unstitched) with the formation of soluble polymer. Factor He acts on the polymer by enzymatic addition of covalent cross-links between chains of fibrin monomer. Cross-linked polymer may be soluble, but at a certain moment in the process of polymerization and crosslinking of fibrin polymer becomes insoluble, forming a fibrin clot, as indicated at stage 3.

Soluble fibrin polymers are intermediate precursor of fibrin clot. As a result, I strongly believe that individuals with impending or existing thrombosis level of soluble fibrin polymers in the plasma increases. It is shown that the detection and measurement of the amount of these polymers in the blood, in particular, soluble fibrin polymers Des AABB useful as guidance on starting obrazovanii system

As found in nature, and expressed in the laboratory antibodies play a role in the characterization of the components of the hemostatic system and explain their functions.

Marciniak, E. , and Greenwood, MF, Blood, 53; 81-82 (1979) describe an example of an inhibitor of blood clotting in the serum of 14-year-old patient with down syndrome, who was present in the IgG-fraction, and which inhibited enzyme selection of fibrinopeptide A from fibrinogen. Hoots, W. K., et al, New End. I. Med., 304: 857-61 (1981) describe the case of a 13-year-old patient suffering from active hepatitis, as well as coagulation disorders, in which the violation was detected by the presence of antibodies in the blood of the patient, reflecting a high affinity to fibrinogen and fibrin, and which inhibit the polymerization of fibrin monomers, preventing thereby the formation of a fibrin gel.

Sola, B, et al, Thromb Res, 29: 643-53 (1983), describe the creation of lines of hybridoma cells secreting monoclonal antibodies specific for human fibrinogen and fibrin. Elms, M. I. et al., Thromb Haemostas, 50; 591-94 (1983), describe the receiving line hybridoma cells secreting monoclonal antibodies that recognize antigenic determinant in D-dimer - specific al., Proc. Natl Acad Sci. USA., 82: 7091-95 (1985), describe the use of synthetic antigens hexapeptides representing the amino-ends - and-chain, respectively, of human fibrin, to obtain monoclonal antibodies that bind to fibrin, even in the presence of fibrinogen. Kudryk, B, et al, Mol Immunol., 21" 89-94 (1984), describe the receiving line hybridoma cells secreting monoclonal antibodies to N-DSK-part fibrinogen. Sobel, I. H.,et al., Thromb and Haem., 60: 153-59 (1988), describe the use of two different monoclonal antibodies that are bound to CNBr fragments A-chain of fibrinogen, to research one of the first events of cross-linking between neighboring molecules of fibrin. Mirshahi, et al., Fibrinogen, 4: 49-54 (1990), describe the receiving line hybridoma cells which secrete monoclonal antibodies to fibrinogen, which are able to inhibit the polymerization of fibrin. Cierniewski, C. S. , and Budzynski, A. Z. Biochemistry, 31: 4248-53 (1992), describe the preparation and use of polyclonal and monoclonal antibodies directed against purified human fibrinogen, which inhibit the intensity of the polymerization of fibrin monomers, and also prevent the effects of thrombin on fibrinogen. Tymkewycz, P. H., et al., Blood Coag and Fibrinol. ,4: 211-21 (1993), described the om. Gargan, P. E., et al., Fibrinolysis, 7: 275-83 (1993), describe the creation of the line hybridoma cell MN-1, which produces monoclonal antibodies specific to both stitched and unstitched polymeric structure of fibrin, but not detect immunoreactivity neither fibrinogen nor to any degradation products of fibrin or fibrinogen. In addition, the antibody MN-1 does not react with the monomers of fibrin Des AA or Des AA BB, and is not any reactive in relation to individual -, - or-chains of fibrinogen

For further discussion of the use of monoclonal antibodies specific for components of the hemostatic system, see Kudryk, B. I., et al, "Monoclonal Antibodies as Probes for Fibrin (ogen) Proteolysis" Monoclonal Antibodies in Immunoscintigraphy, Chatal, I-F (ed), CRC Press, Boca Raton, Et (1989), pp. 365-398.

Developed modified antibodies for hemostatic components for clinical use as a means for scintigraphy of blood clots in situ. Cm. for example, Lian, C-S, and Su, C-T, I Form Med Assoc, 88; 209-12 (1989); Wasser, M. N. I. M. et al., Blood, 74: 708-14 (1989); Walker, K. Z. et al., Eur. I. Nucl. Med. 16: 787-94 (1990); Alavi, A., et al., Radiology, 175; 79-85 (1990); Wasser, M,N,I. M., et al., Thromb. Res., Supp.x; 91-104 (1990); and Kanke, M., et al., I. Nucl. Med., 32:1254-60 (1991).

Designed for therapeutic use of antibodies to fibrin, anywherefrom with thrombolytic enzymes the ski tools has greatly increased efficiency means the destruction of clots.

Finally, published several U.S. patents relating to the receipt and/or methods using monoclonal antibodies specific for fibrinogen, or to the products of its destruction. Cm. U.S. patents NN 4722903, 4758524 and 4916070 - list, not exhaustive.

2.4. Ways to prevent blood clots

Antithrombotic treatment usually involves the introduction of one or more anticoagulants, such as heparin or coumarin. Physicians' Desk Reference, 47 th Ed., Medical Economies Data (1993); The Merk Manual of Diagnostics and Therapy, 15 th Ed., Merk Sharpe & Dohme Rescarch Labs (1987). These anticoagulants are often used in an attempt to prevent recurrent thrombosis in patients suffering from vascular disease, and when you try to warn the acute thrombotic reocclusion after angioplasty.

The main disadvantage of the use of such a system of anticoagulants is the risk of systemic bleeding. Doctors warn that "in patients receiving heparin, bleeding can occur virtually anywhere". Physicians' Desk Reference, 47 th Ed. , Medical Economics Data (1993), p. 2568.

In addition, it is shown that the effectiveness of these anticoagulants in the prevention of reocclusion after angioplasty or not atleastone, advantages in the use of these anticoagulants are neutralized as the risk of bleeding, and their questionable efficacy in the prevention of reocclusion.

Many biological and mechanical approaches were used in attempts to reduce the degree of reocclusion after angioplasty, but the treatment did not show positive results. Accordingly, there is a growing interest in "site-specific" or "targeted" delivery of antithrombotic funds to areas of vascular damage. Gimple, L. W., et al, Curculation 86: 1536-46 (1992).

Site-specific delivery of antithrombotic means using antibodies to the corresponding components of the hemostatic system should serve as a localization of such funds in the damaged vessel. One of the advantages of this localization is that you will need less input dose. Another advantage of localization should be reducing the risk of systemic bleeding.

In some cases it was shown that antibodies to specific components of the hemostatic system themselves inhibit the polymerization of fibrin. Francis, S. E., et al, Am, I Hem., 18: 111-19 (1985) describe studies of monoclonal antibodies, founded the 90), describe the receiving line hybridoma cells secreting monoclonal antibody in which the antigen was a fragment of the D-product plasminogen destruction of fibrinogen. These monoclonal antibodies reacted vigorously with fibrinogen and inhibited the polymerization of fibrin. Ciernewski, CS, and Budrynski, A. Z. Biochemistry, 31: 4248-53 (1992), describe the three different lines of hybridoma cells in which the antigen is a natural human fibrinogen, and which secrete monoclonal antibodies that suppress the intensity of the polymerization of fibrin.

However, the development of site-specific therapeutic or prophylactic antithrombotic agents all these three messages are of limited use or no use. This is true, because in all these cases, the antibodies are cross-interact with either fibrinogen or degradation products of fibrinogen, which are all ubiquitary for the hemostatic system. Any such antibody administered to the patient, should closely contact with fibrinogen or with the products of destruction of fibrinogen and will not be able to inhibit the polymerization of fibrin or blood clots in the place in which the problem of inhibiting the formation of blood clots, using monoclonal antibodies, which are fibrin-specific (antifibrinolytic) (i.e., no significant cross-reaction with fibrinogen or products formed during plasmino the destruction of fibrinogen or fibrin). The present invention relates to method of application mentioned antifibrin monoclonal antibodies, which, surprisingly, is inhibited thrombus formation at the injury site of blood vessel.

3. Summary of the invention

The present invention relates to a method of inhibiting the formation of thrombus at the injury site of blood vessel of a person in need of such inhibition, through the introduction of antifibrin monoclonal antibodies. The present invention also relates to fragments or derivatives antifibrin monoclonal antibodies, and the above-mentioned fragments and derivatives containing the binding domain of the antibody. The present invention also relates to pharmaceutical compositions containing antifibrin monoclonal antibody or its fragment, or its derivative, and a pharmaceutically effective carrier, which, in sufficient quantities, is effective in the IAM, contains one or more ingredients of the pharmaceutical compositions of the invention.

4. Brief description of drawings

Fig. 1. The inclusion of monoclonal antibodies to MN-1 in the reaction mixture of the fibrinogen-trombino coagulation inhibits both the extent and the absolute value of coagulation. Control reaction mixtures are the reaction mixture "fibrinogen" and "45I".

Fig. 2. The inclusion of an increasing number of monoclonal antibodies to MN-1 in the reaction mixture of the fibrinogen-trombino coagulation results in increasing the level of inhibition of coagulation. The control reaction mixture is "fibrinogen".

5. Detailed description of the invention

5. 1. The usefulness of the present invention

Surprisingly discovered that antifibrin monoclonal antibody can inhibit the polymerization of fibrin and blood clots. Thus, antifibrin monoclonal antibodies can be used to prevent or treat any condition in humans, which is amenable to prevention or treatment through inhibition of polymerization of fibrin or blood clots. For example, antifibrin monoclonal antibodies is the danger of pathological thrombosis. Examples, which is not limited to the list of such vascular diseases are deep vein thrombosis (DVT), arterial to venous thrombosis, stroke, thromboembolism, pulmonary embolism and thromboembolic complications of arteriosclerosis.

Antifibrin monoclonal antibodies such useful, for example, in the treatment of people who are trained to invasive surgical procedures, during, or after recovering from these procedures. In describing the present invention, the term "invasive surgical procedure" refers to any surgical procedure that can result in damage to the arterial or venous blood vessel, which can lead to thrombotic complications. Examples, which are not limited to, such procedures are balloon angioplasty and organ transplants (both natural and artificial).

In addition antifibrin monoclonal antibodies, can be created by well-known techniques of antibody fragments which contain the idiotype (binding domain) antifibrin antibodies. For example, such fragments include, but are not limited to, (1) F (ab')2-fragments, which can be obtained pepsinogen perevarivanii the SUB>2fragments; and (3) Fab-fragments, which can be obtained by treating the antibody molecules with papain and a reducing agent. Such antibody fragments are included in the scope of the present invention. To get familiar with other examples of the production and use of fragments of antibodies, see Parham. P., et al., I. Immunol Meth. , 53: 133 (1982), and Khaw, B-A et al., I. Nucl. Med., 34: 2264-68 (1993)

In addition antifibrin monoclonal antibodies and fragments of antibodies can be obtained by known techniques other derivatives of antibodies, which contain the idiotype (binding domain) antifibrin antibodies. For example, can be synthesized by recombinant and synthetic oligopeptides and their analogues, which have the same inhibiting effect on the formation of blood clots, as antifibrin monoclonal antibody or its fragments. Such derivatives are included in the scope of the present invention. Other examples of synthetic oligopeptides with sequences based on the initial binding of monoclonal antibodies, see Knight, L., et al, I. Nucl. Med, 35: 282-88 (1994).

Finally, the scope of the present invention includes any functionally equivalent antifibrin antibody, fragment or derivative. Teroson, which is capable of sufficiently inhibiting the formation of blood clots in humans in need of such effects by binding to the same epitope to which the antibody binds MN-1.

5.2. Obtaining and characterization of monoclonali antibody MN-1

Many of the previous approaches taken to growing antifibrin antibodies were focused on immunization of animals soluble fragments of fibrin and synthetic peptides that mimic available neoantigen sites on fibrin. Cm. Hui, K. Y., et al., Science, 222: 1129-32 (1983); Shecfers-Borchel, V, et al, Proc Natl. Acad. Sci USA, 82-7091-95(1985); Elms, M. I, et al., Thromb. Haemostas, 50: 591-94(1983) and Kudryk, B., et al, Mol Immunol., 21: 89-94 (1984)

However, it is assumed that the binding site of these antibodies is maintained during the process of destruction of fibrin, and therefore, such antibodies can also contact the products of destruction of fibrin.

U.S. patent N 5120834 (hereinafter referred to here as the "patent '834"), Gargan et al., issued June 9, 1992, which is included in the present reference, relates mainly to a method of obtaining monoclonal antibodies from sterile animals by immunization mentioned animals selected antigen. I believe that the benefits use which can be very enhanced immune response to the antigen, increasing the likelihood of locating lymphocyte that produces an antibody capable of binding with a specific epitope of the antigen. It is established that this system is particularly suitable for the creation of highly specific antibodies to fibrin, which has a small cross-interaction with fibrinogen or does not have it. The cultivation of such selecting antibodies was problematic, as there is an estimation that the structural and conformational similarity between fibrin and fibrinogen exceed 98% (Plow, EF., et al., Semin Thramb Haemostas, 8: 36 (1982). B a result, only a small percentage of epitopes on the molecule of fibrin are actually neoantigens (i.e., for the sole purpose of fibrin).

Patent '834 refers to the line of hybridoma cells was ATSS N HB 9739, secreting monoclonal antibody MN-1. Monoclonal antibody MN-1 is specific to the stitched and unstitched polymers of fibrin, but does not cross react with fibrinogen or products plasminogen destruction of fibrinogen or fibrin.

MN-1 is cleaned in accordance with the procedures described in the patent '834. MN-1 specifically binds to fibrin and does not cross react with fibrinogen at konkurentini fibrinogen, nor with any of the products formed when plasmino the destruction of fibrin. As a result, we can conclude that (1) MN-1 recognizes an epitope of intact fibrin molecules, which is not present or does not open on the surface of the molecule predecessor - fibrinogen; and (2) the epitope is explicitly destroyed by digestion with plasmin cross-linked fibrin.

In addition, MN-1, to determine the affinity of MN-1 to fibrin, is described by Scatchard analysis (Prankel, et al. Mol Immunol 16:101-6 (1979)) using labeled1251 antibody MN-1. The value obtained for the dissociation constants KDthat is 6.7 10-10that corresponds to the affinity of 5000 times from the affinity of tissue plasma activator for fibrin.

Western Western blot turns-analysis shows that MN-1 does not cross react with A- , B - or-chains of fibrinogen. The same method shows that MN-1 reacts with cross-linked fibrin, and with unstitched fibrin, and the affinity to cross-link fibrin is the larger of these two values.

5.3. The routes of administration

According to the present invention antifibrin monoclonal antibodies or fragments thereof, or derivatives thereof is administered to a human in need of treatment with inhibition ttora is unconjugated to any agent tagging radioactive isotope, thrombolytic or other agent, or any other molecule, or a part of it. However, the methods of the invention can also be carried out using antibodies or their derivatives, which konjugierte with radioisotope labels, thrombolytic or other agents, or to any molecule or part of it, and covered by the scope of the present invention.

The term "carrier" refers to a diluent, excipient or filler that is injected antifibrin antibody or its fragment, or a subclass thereof.

The term "pharmaceutically acceptable carrier" means a carrier approved by the Federal government or the state, or included in the list of United States Pharmacopeia or other generally recognized Pharmacopoeia for use in animals and, more specifically, to the people.

The term "thromboembolic composition" used in the present invention refers to compositions containing antifibrin monoclonal antibody or its fragment, or its derivative, and a pharmaceutically acceptable carrier, which, in sufficient quantities can inhibit the formation of blood clots in humans, in need of the above-mentioned inhibition.

5.4. The routes of administration

Antifibrin monoclonal antibodies or their fragments or derivatives thereof can be administered to the person by any suitable means, including parenteral, in the form of a single injection loading dose of a substance, continuous infusion or by a combination of the intracoronary introduction, when this is shown.

Preparation of drugs for parenteral administration includes, for example, restoring liofilizovannyh antifibrin monoclonal antibodies or their fragments, or derivatives thereof in a sterile, free of endotoxins, physiological solution or, more generally, may include restoring or dispersion antifibrin monoclonal antibodies or their fragments, or derivatives thereof in a sterile aqueous or nonaqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable organic esters, such as etiloleat. Aqueous carriers include water, spirtovanie solutions, emulsions or suspensions, including saline and buffered environment. Parenteral carriers include sodium chloride, dextrose and ringer, dextrose and sodium chloride, lakirovannuyu dextrose ringer's or fixed oils. Intravenous carriers include liquid and additional nutritional substances, such as on the basis of dextrose ringer, and the like. Can also contain preservatives and other additives, such as, for example, antimicrobial amateurindia compositions of the present invention were sterile form, which meets the standards of sterility, established by the Department of food and drugs of the United States.

The present invention also relates to pharmaceutical kits containing one or more ingredients of the pharmaceutical compositions of the invention, stored in the same or in different containers. May be accompanied by a leaflet, not necessarily in that container (containers), in the form prescribed by a governmental Agency controlling the manufacture, use or sale of pharmaceuticals or biological products, and this leaflet reflects the Agency's permission to manufacture, use or sale for human use. Can be accompanied by instructions, not necessarily tied to sets that contain only liofilizovane antibody, or fragments, or derivatives, of the media that are suitable for administration to a human in need of treatment.

The amount, duration, timing and method of treatment will vary depending on age, weight, status, and gender of the person in need of treatment, and depending on the type and severity of the disease, which requires lechitsa immediately before, during or after any surgical procedure, or both before and during the procedure, or both during and after the procedure, depending on the specific set of conditions in this case.

For example, during a surgical procedure, such as balloon angioplasty, it often happens that a sick person already has a large number of significantly scattered blood clots. In such a situation, in General, be advantageous to introduce antithrombotic composition directly after the procedure angioplasty, using, preferably, the catheter while it is still in place, in order to directly wash the damaged portion of the blood vessel antithrombotic composition, focusing, by means of this treatment in one place. This prevents loss of antithrombotic composition, which may result from, for example, binding of an antibody to a blood clot in the unaffected areas.

In situations involving invasive surgical procedures for patients not suffering from pathological thrombosis in General, it would be preferable to introduce antithrombotic composition either directly before or during the procedure, so C.

In addition, in most cases, including surgical procedures, the time during which there is an increased risk of blood clots, tends to be limited to the completion of the procedure. As a result, sufficient for the effective inhibition of blood clots may be only a limited number of treatments or even a single treatment of sufficient duration, whereby significantly reduces the danger of coagulation induced by surgical intervention, with all its inherent dangers for the patient.

In the case of chronic thrombotic disease timing, duration, and number of antithrombotic treatments depend largely on the diagnosis of the patient's condition before and after treatment and its response to the preliminary antithrombotic treatment that should be defined, monitored and evaluated by the treating physician.

5.5. To determine the effectiveness of treatment

The effectiveness of antithrombotic treatment with monoclonal antibody MN-1 or a fragment, or derivative may be determined by standard methods. Examples of such methods include, but are not limited the tion in the artery or Vienna, when the appearance of obstacles in the way of contrast indicates the need for further treatment; (2) the use of scintigraphy using antithrombotic antibody conjugated with a radioisotope label, when it can be tracked the size and location of the thrombus in order to establish the need for further treatment; (3) control over the appearance and degree of clinical symptoms, when the increase in the number or severity of symptoms may indicate the need for further treatment; (4) the measurement of thrombin in the blood of the patient, as there is an inverse correlation between the levels of fibrinogen and fibrin, when the fall in the level of fibrinogen may indicate possible a corresponding increase in the level of fibrin and polymerization of fibrin, pointing to the need for further treatment.

Now, with a General description of the present invention, it will be easier to understand, appealing to a characteristic example included in this description only for the purpose of illustration, and not intended for any restrictions.

5.6. Example: inhibition of coagulation in vitro

Conduct a number of in vitro experiments to determine the influence of MN-1 on coagulation. Purified human fibrin pH, concentration 1 mg/ml, and placed approximately 100 ál in each well tiralongo microplate (Costar). In each well to form a reaction mixture, add approximately 50 μl of bovine thrombin to a final concentration of 0.5 NIH units on the ml To the reaction mixture, in zero time, add approximately 50 μl of either (1) a monoclonal antibody MN-1, to obtain a final concentration of antibody 100 µg/ml; or (2) monoclonal antibodies 45 I (monoclonal antibody 45 I, which cross react with fibrin and fibrinogen, is secreted by the hybridoma line cells of ATSS N HB 9740, which receive conventional techniques, using traditional mice Ba 1b/c, when mice are subjected to immunization fibrin), to obtain a final concentration of 200 µg antibody/ml; or (3) saline solution. Allow reaction to proceed for 60 minutes at 37oC. Coagulation determine spectrometrically at 340 nm every 2 minutes. The increase of absorption indicates that coagulation progresses. The end of the clotting process can be observed as a plateau on the curve "absorption time".

Fig. 1 shows that treatment of MN-1 inhibits the coagulation rate, which is obvious from Myeong the structure or control of the reaction mixture with 45 I, or with a control mixture with salt solution (fibrinogen). In addition, MN-1 reduces the absolute value of coagulation that occurs that is defined at a lower level gently sloping section of the curve for MN-1 when compared with the curves for the control reaction mixtures. The control reaction mixture containing the antibody 45 I (at twice the concentration against MN-1), does not detect any inhibition of coagulation when compared with the control reaction mixture with brine. These data support the assumption that the monoclonal antibody MN-1 is able to inhibit the formation of blood clots.

In the second experiment, examine the effect of MN-1 at multiple concentrations in clotting time compared with the control reaction mixture with fibrinogen. In Fig. 2 shows the profiles of the coagulation reaction (absorption time) when increasing the concentration of MN-1 in the reaction mixture. (1:1000 = 1 mg/ml; 1:100 = 10 µg/ml; 1:50 = 20 μg/ml 1:20 = 50 µg/ml; 1: 10 = 100 µg/ml; fibrinogen = control)

5.7. The depositing of hybridoma

Line hybridoma cell MN-1 and 45 I deposited in the American type culture collection (ATSS) June 9, 1988 under inventory numbers HB 9739 and NEITHER 9740, meet the PA-chain, and it has been observed that MN-1 cross-reacts with human fibrin and rabbit fibrin.

The present invention is not limited in the amount of the deposited line of hybridoma cells was ATSS N HB 9739 or a monoclonal antibody MN-1, but assume that they are separate illustrations antifibrin monoclonal antibody, which inhibits the formation of blood clots. Any functionally equivalent antifibrin monoclonal antibody, or its fragment, or derivative, are included in the scope of the present invention. The term "functionally equivalent" indicates that the monoclonal antibody, or fragment, or its derivative capable of inhibiting the formation of blood clots by binding to the same epitope to which the antibody binds MN-1.

The present invention can be implemented in other specific forms without departing from its essence or its essential characteristics, and, therefore, should rather refer to the attached claims, than to the above description), as a statement about the scope of the invention.

All publications and patents cited above are included in the present as references.

2. Method of inhibiting the formation of blood clots in humans, comprising the administration to a person in need of the above-mentioned inhibition, an effective amount of a fragment or derivative antifibrin monoclonal antibody MN-1, produced by hybridomas ADS 9739, or antibody, which binds to the same epitope as monoclonal antibody MN-1, a fragment or derivative which contains a binding domain indicated antibodies.

3. The method of treatment of a person to reduce human risk surgical thrombotic complications, including the introduction of a seeker in the above treatment, an effective amount antifibrin monoclonal antibody MN-1, produced by hybridomas ADS 9739, or antibody, which binds to the same epitope as monoclonal antibody MN-1.

4. The method according to p. 3, characterized in that the surgical thrombotic complications are the result of angioplasty.

5. The method according to p. 3, Otley is

6. The method of treatment of a person to reduce human risk surgical thrombotic complications, including the introduction of a seeker in the above treatment, an effective amount of a fragment or derivative antifibrin monoclonal antibody MN-1, produced by hybridomas ADS 9739, or antibody, which binds to the same epitope as monoclonal antibody MN-1, a fragment or derivative containing the binding domain of the specified antibodies.

7. The method according to p. 6, characterized in that the surgical thrombotic complications are the result of angioplasty.

8. The method according to p. 6, characterized in that the surgical thrombotic complications are the result of organ transplantation.

9. A method of treating thrombotic vascular disease in humans, comprising the administration to a person in need of the above-mentioned treatment, an effective amount antifibrin monoclonal antibody MN-1, produced by hybridomas ADS 9739, or antibody, which binds to the same epitope as monoclonal antibody MN-1.

10. The method according to p. 9, wherein the thrombotic vascular disease is of is a lung embolism.

12. The method according to p. 9, wherein the thrombotic vascular disease is a deep vein thrombosis.

13. The method according to p. 9, wherein the thrombotic vascular disease is an arterial or venous thrombosis.

14. The method according to p. 9, wherein the thrombotic vascular disease represents atherosclerosis.

15. A method of treating thrombotic vascular disease in humans, comprising the administration to a person in need of the above-mentioned treatment, an effective amount of a fragment or derivative antifibrin monoclonal antibody MN-1, produced by hybridomas ADS 9739, or antibody, which binds to the same epitope as monoclonal antibody MN-1, a fragment or derivative which contains a binding domain indicated antibodies.

16. The method according to p. 15, wherein the thrombotic vascular disease is a kick.

17. The method according to p. 15, wherein the thrombotic vascular disease is a lung embolism.

18. The method according to p. 15, wherein the thrombotic vascular disease represents the e represents the arterial or venous thrombosis.

20. The method according to p. 15, wherein the thrombotic vascular disease represents atherosclerosis.

21. Pharmaceutical composition containing unconjugated antifibrin monoclonal antibody MN-1, produced by hybridomas ADS 9739, or antibody that binds the same epitope as monoclonal antibody MN-1, and a pharmaceutically acceptable carrier, while said composition is in sterile form.

22. Pharmaceutical composition containing unconjugated fragment or derivative antifibrin monoclonal antibody MN-1, produced by hybridomas ADS 9739, or antibody that binds the same epitope as monoclonal antibody MN-1, a fragment or derivative which contains a binding domain indicated antibodies, and a pharmaceutically acceptable carrier, while said composition is in sterile form.

23. Set to treat the person in need of the inhibition of the formation of a blood clot, while the set includes unconjugated antifibrin monoclonal antibody MN-1, produced by hybridomas ADS 9739, or antibody that binds the same epitope h antibody and pharmaceutically acceptable carrier, while the set is in sterile form.

24. Set on p. 23, characterized in that it also contains a pharmaceutically acceptable carrier, which is in sterile form.

 

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The invention relates to a new compound is a sodium salt of 6-nitro-3H-gynazole-4-yl-3-acetic acid of formula (I), which is antioxidant and anti-ischemic activity and may find application in medicine

The invention relates to new derivatives of oxazolidinones General formula (I) listed in the description, as well as their salt

The invention relates to a new compound - monoethanolammonium salt of 6-nitro-3H-gynazole-4-yl-3-acetic acid f-crystals (I), which is antioxidant and anti-ischemic activity and may find application in medicine

The invention relates to medicine, the inhibitor of intimal hypertrophy, containing as the active ingredient oxindole derivative represented by formula I, or its salt, where R represents a hydrogen atom, phenyl group which may be substituted by a lower alkyl group, lower alkoxygroup, lower alkylaminocarbonyl, hydroxyl group, amino group, lower alkylamino or halogen atom, or pyridyloxy group which may be substituted by a lower alkyl group, lower alkoxygroup, lower alkylaminocarbonyl, hydroxyl group, amino group, lower alkylamino, a halogen atom, a lower alkoxycarbonyl group or a carboxyl group, a represents a phenyl group, which may be substituted, or pyridyloxy group which may be substituted by a lower alkyl group, lower alkoxygroup, lower alkylaminocarbonyl, hydroxy-group, amino group, lower alkylamino, a halogen atom, a lower alkoxycarbonyl group or a carboxyl group, a represents a hydrogen atom, a lower alkyl group, benzyl group or benzosulfimide group which may be substituted, or acyl group,axially group, lower alkoxycarbonyl group, phenylcarbamoyl group which may be substituted, or triptorelin group represents CH or N; n represents an integer from 0 to 4, inclusive, that indicates the number of substituents and double dotted/solid line represents a simple bond or double bond, which has an excellent inhibitory action against intimal hypertrophy, and used as an agent for prevention (treatment) attenuation of proliferative vascular diseases such as restenosis after RTSA, arteriosclerosis, peripheral embolism and angia, use oxendolone derived to obtain an inhibitor of intimal hypertrophy, compositions for inhibiting intimal hypertrophy and method of prevention and treatment of intimal hypertrophy

The invention relates to medicine, particularly cardiology, and for slowing the progression of corneoscleral

The invention relates to medicine, namely to vertebrology in the treatment of myofascial pain of the spine

The invention relates to biotechnology and concerns gumanitarnogo immunoglobulin specific for the protein L-selectin person

The invention relates to medicine, namely to Oncology, and relates to a method of obtaining specific antisera to universal tumor antigen and method for the diagnosis of malignant tumors using this antisera
The invention relates to the field of veterinary Microbiology and biotechnology, in particular the production of vaccines or sera against viral and bacterial diseases of cattle

The invention relates to medicine, in particular to pharmaceutical compositions for the treatment of chronic rheumatoid arthritis and inhibitor of the growth of synovial cells

The invention relates to the field of veterinary Virology, Microbiology and biotechnology, in particular to the production of a biological product intended for specific immunotherapy and passive prophylaxis of mixed forms of infectious enteritis and enterocolitis of newborn calves
The invention relates to veterinary medicine, in particular to the examination of animal products
The invention relates to chemical-pharmaceutical industry, medicine and relates to antiviral, antimicrobial and protivokariosnoe tools
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