Trombotsitopeiiya chimeric immunoglobulin and methods of its use

 

(57) Abstract:

The invention relates to medicine, namely to trombozitopatiam the chimeric immunoglobulins and their application. The inventive patient for inhibiting stenosis and/or restenosis prescribe an effective amount of a chimeric immunoglobulin or its fragment having specificity for glycoprotein IIb/IIIA, with the specified immunoglobulin or fragment contains the region of binding antigen, not owned by the person and specific for the glycoprotein IIb/IIIA, and constant region of human immunoglobulin. The invention allows to reduce the number of allergic reactions. 8 C. and 25 C. p. F.-ly, 26 tab., 15 Il.

Platelet aggregation plays an important role in the formation of blood clots. Under normal circumstances, blood clots are used to prevent loss of blood from the vascular system. However, under certain conditions, a blood clot can block blood flow or completely overlap, which leads to cell necrosis.

For example, platelet aggregation and subsequent thrombosis at the site of atherosclerotic plaques is an important causal factor in the Genesis of conditions, such as the banking operations on vessels. Patients affected by strokes, are usually treated with the use of thrombolytic agents such as tissue plasminogen activator or streptokinase, which is dissolved component fibrin clots. The main complication associated with fibrinolysis, is re-blockage due to platelet aggregation, which leads to further heart damage. Because, as you know, the receptor glycoprotein (GP)IIb/IIIa responsible for platelet aggregation, it can be expected that the reagents that block these receptors will reduce or prevent re-occlusion after application of thrombolytic therapy and to increase the speed of thrombolite. It can be expected that such reagents are useful in the treatment of other diseases associated with thrombosis of blood vessels, and therapy of thromboembolic diseases.

In one of the approaches to blocking of platelet aggregation using monoclonal antibodies specific for receptor GPIIb/IIIa. Murinova monoclonal antibodies, which are given in European patent applications numbers 205207 and 206532, designated as ES, inhibit platelet aggregation and probably useful in the treatment of thrombotic diseases man Yeni limit their use in human therapy. As foreign proteins, murinova antibodies are able to induce immune responses that reduce or negate their therapeutic effectiveness and/or cause allergic reactions or hypersensitivity patients. The need for repeated administration of these therapeutic procedures in thromboembolic diseases raises the possibility that the immune reactions of the specified type.

Chimeric antibodies with binding regions that do not belong to the person who is connected with a constant regions belonging to the person proposed as a means to circumvent the problems caused by immunoactivity marinovich antibodies (see Proc. Natl. Acad. Sci. USA, 81: 6851 (1984) and International application N PCT/GB85 00392). Because a constant region is largely responsible for immunoactivity molecule antibodies, chimeric antibodies with constant regions of human origin should be less cause antimurine response in humans. But beforehand, it is impossible to determine whether the accession of the constant region of a person to murinova binding region having the desired specificity, to reduce immunoactive is giving refers to trombozitopatiam the chimeric immunoglobulins, containing the variable region or antigen binding, not owned by the person, and a constant region that belongs to the person. Chimeric antibodies can be specific for receptor GPIIb/IIIa, or other components of the platelet. These antibodies bind to platelets and can block platelet aggregation and, therefore, are useful as antithrombotic means to prevent or reduce the likelihood of blockage or re-blockage of blood vessels in various clinical situations (in particular, after thrombolytic therapy, which is held in conjunction with plastic surgery on the blood vessels) or to prevent stenosis or restenosis. Antiplatelet antibodies of the present invention is also useful in obtaining images of a blood clot.

In Fig. 1 shows the result of analysis by the method of Naser blot heavy chain and light chain mRNA for monoclonal antibody 7E3 using as probes cloned variable regions.

In Fig. 2A and 2B is a schematic representation of the plasmids p7E3VKCK(Fig.2A) and p7E3VHh C4(Fig. 2B), which contain the chimeric gene engineering design, the t binding of chimeric immunoglobulin 7E3, encoded vectors p7E3VKhCKand p7E3VHh C4c platelets.

In Fig. 4 shows the inhibition of platelet aggregation chimeric immunoglobulin 7E3 (c7E3).

In Fig. 5 shows a graph of the concentration of antibodies in plasma (ng/ml) depending on time (in days), which shows a rapid initial removal of c7E3 Fab (1K) from the plasma of patients suffering from chronic diseases of the coronary vessels after injection of a dose of 0.20 mg/kg c7E3 Fab in the form of intravenous infusion for five minutes.

Fig. 6 (A-C) explains generalized effect on the activity of platelets dose of chimeric 7E3 Fab in a single bolus (0.15 mg/kg, 0.20 mg/kg or 0.25 mg/kg) every 2 hours after the appointment antibodies (1K) dose-Dependent response was observed when studying the activity of platelets from the point of view of the blockade of the receptor (Fig. 6A, top), platelet aggregation (Fig. 6B, middle) and bleeding time (Fig. C, below). Lines correspond to the average values.

Fig. 7 (A-C) illustrates the duration of antiplatelet effects of chimeric 7E3 Fab (1K) appointed prior to having any plastic surgery on the blood vessels in the form of a single bolus dose of 0.25 mg/kg Line SDA), platelet aggregation (Fig. 7B, middle) and bleeding time (Fig. 7C, bottom).

Fig. 8 (A-C) illustrates the total antiplatelet activity of a single bolus dose of 0.25 mg/kg followed by continuous infusion for 12 hours (10 µg/min) chimeric 7E3 Fab (1K) in eleven patients. Lines correspond to the average values determined for the percentage of blockade of the receptor (Fig. 8A, top), expressed in percentage of the degree of platelet aggregation for pre-dose (baseline at time zero) (Fig. 8B, middle) and bleeding time (Fig. 8C, below).

Fig. 9 illustrates the absolute change in hematocrite from baseline values within 24 h after injection for 47 patients described in Example 4.

Fig. 10 is a graph showing Kaplan-Meier estimates, which illustrates the dependence of the probability is not caused by repeated percutaneous repair of vessels from the time of randomization to the three groups of subjects.

In Fig. 11 chart shows the probability coefficients and 95% confidence intervals for key subgroups (listed to the right), which is subjected to testing. Data are shown for the Skye operation on vessels or the placement of a coronary stent or intra-aortic balloon pump in stand ischemia). In addition, the left table shows the absolute values for the frequency of observations the main event of termination of the test in each subgroup (Frequency of event (%)).

In Fig. 12 is a graph showing the proportion of all patients whose cases were not identified during the six-month observation period.

In Fig. 13 is a graph showing the proportion of those patients whose cases were not identified during the six-month observation period and have successful surgery, which had no consequences after 30 days.

In Fig. 14 is a graph showing the proportion of those patients whose cases are not marked during the six-month observation period, and taking into account those patients whose cases are detected at 48 hours after initially successful surgery.

In Fig. 15 is a graph showing the proportion of all patients who did not show attempts to restore blood vessels subjected to the operation of the artery during the six-month observation period.

Chimeric immunoglobulins of the present invention consist of individual himel, derived from the heavy chain not owned by the person antigen specific for platelets (in particular, specific receptor GPIIb/IIIa), which is bound to the constant region of the heavy chain of a human. Chimeric light chain includes antigennegative region derived from the heavy chain not owned by the person antigen, which is bound to the constant region of the light chain of a human.

The immunoglobulins of the present invention can be monovalent, bivalent or polyvalent. Monovalent immunoglobulin (HL) represents a dimer formed chimeric heavy chain, linked by disulfide bridges with the chimeric light chain. Bivalent antibodies are tetramera (H2L2) formed by two dimers associated at least with one disulfide bridge. Can be also obtained polyvalent immunoglobulins, for example, due to the aggregation constant region heavy chain (in particular, the constant regions of the heavy chain). Can be also obtained fragments, chimeric antibodies, such as Fab, Fab' or F(ab')2.

Not belonging to a man of antig the Preferred antibodies are immunoglobulins, specific receptor GPIIb/IIIa platelet count, which can block the binding of ligand to the receptor complex of glycoprotein IIb/IIIa.

Thrombosis begins with the adhesion of platelets in the defeat of the vessel walls. The process of attachment of platelets takes place under the control of the surface receptors of platelets that bind proteins of the extracellular matrix of the exposed surface of the subendothelial layer, such as factor von Willebrand's disease, collagen, fibronectin, vitronectin and laminin. Adhesion of platelets leads to the formation of compiled monolayer. Further, in response to agonists such as epinephrine, ADP, collagen and thrombin, is the activation of platelets. Activation leads to the exposure of the receptor glycoprotein IIb/IIIa (GPIIb/IIIa) on the surface of platelets. After that GPIIb/IIIa on activated platelets becomes available for binding to fibrinogen, which stimulates platelet aggregation. The binding of GPIIb/IIIa with other adhesive proteins, such as factor von Willebrand's disease, also causes cross-linking and aggregation of platelets. Thus, the binding of adhesion molecules, such as fibrinogen or factor a background of Villebranda to GPIIb/IIIa, causing Ib/IIIa attractive target for therapeutics which may interfere with the interaction of GPIIb/IIIa with these molecules. Moreover, it can be expected that when using anti-GPIIb/IIIa chimeric antibodies will be suppressed aggregation of activated platelets, and the initial adhesion of platelets is not disturbed. Selective inhibition of platelet aggregation may be desirable, since the adhesion of platelets, not accompanied by aggregation, can help stop bleeding.

Examples of suitable antibodies specific for platelets, are 7E3 and 10E5. Cm. European patent application numbers 205207 and 206532, descriptions of which are given here for reference. Antibody 7E3 (or interacts with the antibody or functionally equivalent epitope) is the most preferred because it is specific for the complex forms of the receptor GPIIb/IIIa. Can also be used for other antibodies specific for receptor GPIIb/IIIa (antigen, which recognizes 7E3), such as antibodies specific for any of the components IIb or IIIa. Can be used antibodies specific for other antigens platelets. For example, can be used antibodies capable of interacting with others who CLASS="ptx2">

Antigennegative region of the chimeric antibody can be obtained from immunoglobulin that is not owned by the person. Antigennegative region is preferably murinova origin, because murinova antibodies against platelets and, in particular, receptor GPIIb/IIIa, is available or can be obtained in marinovich systems. Alternative sources antigenspecific areas are other species of animals or rodents (see, e.g., Newman et al. , Bio/Technology, 10: 1455-1460 (1992). In one of the embodiments of the present invention antigennegative region of chimeric immunoglobulin contains at least a portion of trombozitarnogo immunoglobulin which does not belong to man sufficient for specific or selective binding of the gene, such as one or more hypervariable sites or portions thereof, selected from does not belong to human immunoglobulin (see, in particular, Winter, U.S. Patent 5225539, European patent application 0239400, the United Kingdom Patent 2188638; Adair et al., International patent application WO 91/09967; Jolliffe et al., International patent application WO 91/09966). In another embodiment, the present invention chimeric immunoglobulin comprises at least one human immunoglobulin, which is connected with at least part of a constant region of heavy chain of a human, and at least one chimeric light chain containing the variable area shaded from the light chain does not belong to human immunoglobulin, which is connected with at least part of a constant region of the light chain of a human. There are also other combinations of variable and constant regions (see, particularly, U.S. Patent 5169939).

The constant region of the chimeric antibody derived from human immunoglobulins. The constant region of the heavy chain, you can choose from one of the five isotypes alpha, Delta, Epsilon, gamma or mu. Moreover, the heavy chains of different subclasses (such as the IgG subclasses of heavy chains) are responsible for different functions of effector and, thus, choosing the constant region of the desired heavy chain, you can get a chimeric antibody with a desired effector function. Preferred constant regions are gamma 1 (IgG1), gamma 3 (IgG3) and gamma 4 (IgG4). The constant region of the light chain may be of the type Kappa or lambda.

In the General case, chimeric antibodies get ready for each of the components of the light or heavy chain chimeric part thrombocytopathies variable regions, not owned by the person (for example, functionally reconstructed variable plot together with neighboring segments) and associated with the second DNA segment that encodes at least a portion of a constant region of human immunoglobulin. Each gene merge collected or injected into the expression vector, obtaining the expression vector that contains a gene mergers able to be expressed in the form. In one of the embodiments of the present invention DNA, including the region of binding of antigen covalently connects to the constant region by an intron. In another embodiment, the present invention can be created or received genetic engineering designs that are missing one or more introns. Then subjected to transfection with genes cells of the recipient is able to Express the gene products. Subjected to transfection of the recipient cells are grown under conditions that permit expression of the introduced genes, and then expressed immunoglobulins or chain immunoglobulin allocate.

The genes encoding the variable regions of light and heavy chains of the immunoglobulin may be obtained from the lymphoid cells that produce traymanager for chimeric antibodies of the present invention. Available also other rodent cell lines. Cell lines can be obtained by introducing the rodent provocative test of human platelets, or the component containing the receptor GPIIb/IIIa, or fractions of platelets, forming a fused hybrid cells between the cells that produce antibodies, and cell myeloma line, exposing the hybrid cloning and selecting clones which produce antibodies against platelets or receptor glycoprotein IIb/IIIa.

The constant region can be obtained from producing antibodies in human cells, using standard cloning techniques. Otherwise, because subjected to cloning genes representing two classes of light chains and five classes of heavy chains of these clones easily get constant region belonging to the person linking the fragments of chimeric antibodies, such as fragments F(ab')2or Fab can be obtained by constructing a chimeric gene of the heavy chain in a truncated form. For example, a chimeric gene encoding a portion of the F(ab')2heavy chain would include DNA sequences encoding region CH and the area of the "waist" of the heavy chain. Alternatively, such fragments can be obtained by enzymatic rasdale the AMB, accordingly, the Fab fragments or F(ab')2.

Gene fusion encoding the chimeric light and heavy chains (or their portions) mainly collected in two different expression vectors that can be used for katrushenko transfer into cells of the recipient. Each vector contains two breeding gene for selection in bacterial system, and the second selection in eukaryotic system, where each vector has a different pair of genes. These vectors allow the production and amplification of the gene merge in a bacterial system and subsequent contrasenya transfer in eukaryotic cells and the selection of cells subjected kurashina transfer. Examples of breeding genes for use in bacterial system are genes that give resistance to ampicillin, and genes that give resistance against the action of chloramphenicol. Preferred two breeding gene: (i) gene xanthan gum-guaninephosphoribosyltransferase (gpt) and (ii) gene phosphotransferase from Tn5 (known as neo). Breeding with gtp based on the ability of the enzyme encoded by the specified genome, using xanthine as a substrate in the synthesis of purine nucleotide acid, which blocks the conversion of monophosphate inosine monophosphate in xanthine, can survive only in cells expressing the gpt gene. The product of the neo gene blocks the inhibition of protein synthesis in eukaryotic systems, caused by the antibiotic G418 and other antibiotics in this class. Two procedures of selection can be used simultaneously or sequentially for the selection of gene expression immunoglobulin chain is introduced into a eukaryotic cell in two different DNA vectors.

The preferred cell line recipient is cell myeloma. Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded subjected to transfection immunoglobulin genes. Moreover, they know the mechanism of glycosylation of immunoglobulin. Especially preferred recipient cell is neproducyruth line of myeloma cells, such as Sp2/0. These cell lines produce only immunoglobulin encoded subjected to transfection immunoglobulin genes. Myeloma cells can be grown in the medium for growing crops or in the peritoneum of a mouse and secretory immunoglobulin can be isolated from ascitic fluid. Suitable cells restful several ways transfection of lymphoid cells by vectors, containing the genes encoding the immunoglobulin. The preferred method of introducing DNA into lymphoid cells is electroporation. When performing this technique, the recipient cells exposed to electric pulses in the presence of DNA, which must be entered (see, for example, Potter et al. , Proc. Natl. Acad. Sci. USA, 81: 7161 (1984)). Another method of introducing DNA is fusion of protoplasts. In this method, removal of the cell walls of bacteria that hides the recombinant plasmid containing the chimeric gene of the immunoglobulin, using lysozyme. The resulting spheroplast merge with the myeloma cells in the presence of polyethylene glycol. After fusion of protoplasts, the transfectants are selected and isolated. Another method that can be used to introduce DNA into many types of cells is the deposition of calcium phosphate.

Chimeric genes of the immunoglobulin can be expressed in non-lymphoid cells, such as bacteria or yeast. When expression in bacteria heavy chain and light chain become part of the Taurus inclusion. Thus, the chain should be isolated and purified, and then assembled into functional molecules of the immunoglobulin. There are other ways to li in the form of a fused protein, containing a signal sequence.

Use trombotsitopeiiya chimeric immunoglobulins

Chimeric thrombocytopaenia antibodies of the present invention are useful as antithrombotic therapeutics. For example, chimeric antibodies (or fragments thereof) can be used to suppress platelet aggregation and the formation of thrombus in patients with thrombus or at risk of clotting. Antibodies can also be used to suppress variance in the menstrual cycle caused by platelet aggregation, which may precede the formation or transformation of blood clots. Antibodies can be used in various situations where it is necessary to prevent the formation or transformation (occlusion) of a blood clot. Moreover, antibodies can be used in different situations, when you want to suppress (reduce, delay or prevent stenosis or restenosis.

For example, patients at risk or suffering from coronary artery disease, you may feel relief when assigning an effective amount of the chimeric fragments antithrombolytic antibodies of the present invention (in particular, the chimeras the Oia occlusion, reocclusion, stenosis and/or restenosis of blood vessels.

For example, antibodies can be administered to an individual (in particular, a mammal such as a human) to prevent thrombosis and pulmonary embolism, transient ischemic disorders of cerebral circulation, deep venous thrombosis, while conducting operations on coronary artery surgery for installing artificial heart valve or the vessel (in particular, native, non-native or synthetic graft vessel). Antibodies of the present invention can also be assigned to the individual, to prevent platelet aggregation and thrombosis before, during and after plastic surgery on vessels, which are methods of balloon coronary atherectomy, laser plastic surgery on vessels, or other suitable means. The antibody can be administered before performing plastic surgery on vessels, during the plastic surgery on the blood vessels and after plastic surgery on the blood vessels. Such treatment can prevent thrombosis and, thus, reduce the likelihood of thrombotic complications after carrying out plastic surgery on the percutaneous cresolate reconstruction of the coronary artery or bypass operations coronary artery (in cases of acute ischemia). In addition, such treatment may lead to a more lasting impact by reducing the likelihood of ischemia or complications of surgical intervention on the coronary arteries (in particular, when performing plastic surgery on vessels, such as death, myocardial infarction or recurrent ischemia leading to the need for percutaneous cresolate reconstruction of the coronary artery or bypass operations coronary artery (operations revascularization), as evidenced by the reduction of the probability of occurrence, delay or prevent stenosis or restenosis of the vessel lumen.

For example, as shown in Example 4, the purpose of chimeric antitromboticos antibody (fragment, chimeric 7E3 Fab) as adjunctive therapy before performing plastic surgery on blood vessels (percutaneous cresolate reconstruction of the coronary artery) effectively increases the bleeding time and reduces induced by an agonist of platelet aggregation, which is confirmed by ex vivo analysis of platelet aggregation. The results of the studies described in Examples 4 and 5 also show that the Blo is Boticelli effective in the treatment of people.

The results of a randomized impersonal analysis using placebo as a control in the appointment of a fragment of the chimeric antibody 7E3 shown in Examples 6 and 7. The data presented in Example 6, indicate that the purpose of the fragment of the chimeric antibody 7E3 patients undergoing plastic surgery on the blood vessels and are subject to significant risk of sudden blockage (occlusion) of blood vessels, can prevent sudden blockage (occlusion) of a blood vessels and reduce the likelihood of acute ischemia. As further shown in Example 7, the purpose of the fragment of the chimeric antibodies in patients undergoing plastic surgery on the blood vessels and are subject to significant risk of sudden blockage (occlusion) of vessels that can later be reduced to postpone and/or prevent the development of restenosis.

Long-term beneficial effects are evident in the reduction of the probability of occurrence of mild ischemic complications in the appointment of a fragment of the chimeric antibody anti-GPIIb/IIIa (see Example 7) is unparalleled. To prevent or reduce the likelihood of stenosis or restenosis can be used for more connections, colorshine probability of mild ischemic complications. Once appointed, these compounds can prevent the blockage or re-blockage of blood vessels. These compounds include, for example, antagonists of GPIIb/IIIa, the immunoglobulin or nimmanahaeminda peptides or proteins (in particular, synthetic, recombinant), their analogs, and nucleic acids and analogs of nucleic acids. Patients at risk or suffering from coronary artery disease may experience beneficial effects when prescribing an effective amount of a compound that selectively binds to the receptor GPIIb/IIIa, thereby inhibiting thrombosis, recurrent thrombosis, stenosis and/or restenosis of blood vessels.

Platelet aggregation activates the blood clotting system and leads to the formation of more stable fibrin mesh structure and obstructive clot, which can be subjected to lysis under the action of thrombolytic agents. Antibodies of the present invention or compounds that selectively bind to the receptor GPIIb/IIIa, can be assigned to an individual (particularly a human) alone or in combination with thrombolytic agent, such as plasminogen activator (in particular, cell activator platinette, antithrombin means), or antiplatelet agent such as aspirin, heparin, hirulog, hirudin or coumarin anticoagulants (including warfarin), for example, to prevent and reduce the likelihood of re-blockage of blood vessels, which may occur after thrombolyse, and to accelerate the lysis of the clot. Connection, the antibody or fragment can be administered before, together or after the appointment of thrombolytic funds antiterminator means of anticoagulant or antiplatelet funds in amounts sufficient to prevent aggregation of platelets, which can cause blockage or re-occlusion of the vessel, and/or to delay or prevent the development of stenosis or restenosis.

An effective amount (i.e. an amount sufficient to achieve the desired therapeutic effect, such as an amount sufficient for inhibition of platelet aggregation and thereby to suppress the formation or transformation of a blood clot, or an amount sufficient to prevent or delay the development of stenosis or restenosis, or ischemia) compounds or antibodies, or antibody fragment, may appoint the physiological salt solution. You can enter the buffer additives. In the composition comprising the antibody, and may include additional additives, such as stabilizers (in particular, Polysorbate 80, in accordance with USP and Additions to the US Pharmacopoeia). The antibody may be administered in a single dose, continuously or by using multiple injections (in particular, by injection of a bolus followed by continuous infusion), otherwise the connection or the antibody may be administered with the use of controlled selection (in particular, by using a polymer or delivery system "foci") or other suitable method. Assigned number depends on various factors, such as clinical symptoms, the weight of the individual, and whether the assignment of other drugs (in particular, thrombolytic agents).

When conducting repeated therapy with antiplatelet antibodies may occur thrombocytopenia caused by drugs; this phenomenon may be due to the fact that the body takes antibody-coated platelets for foreign proteins, mobilizes antibodies against them, and then outputs them to the outside through the reticuloendothelial system faster than usual. Vsledstvii one platelet) and a large number of platelets in circulating blood (approximately (0.25 to 0.5)1061 μl), thrombocytopenia can be a serious complication in the treatment of antiplatelet antibodies, the use of chimeric antiplatelet (in particular, anti-GPIIb/IIIa) antibodies allows you to bypass this problem. Chimeric antiplatelet antibodies of the present invention can minimize thrombocytopenia (reduce or prevent), which otherwise might occur when the use of antiplatelet antibodies. For example, the minimum reduction in the platelet count in the blood is observed in the appointment of chimeric 7E3 Fab (see, in particular, Examples 6 and 7).

Secondly, when appointing people to the fragment of the chimeric antibody 7E3 Fab shows a surprisingly low incidence of induced immunogenicity compared to its murinova double (see, in particular, Examples 4 and 7). In the process of binding of platelets large part of murinova antiplatelet components of the chimeric antibody binds to the surface, in particular through receptor GPIIb/IIIa, and, thus, inaccessible to the immune system, making chimeric antibodies are functionally indistinguishable from human antibodies directed against the same epitope. This in and despite their connecting region murinova antigen.

Thrombocytopaenia chimeric immunoglobulins of the present invention is also useful for visualization of blood clots. To this end, in the General case is more preferable to use fragments of antibodies. As indicated earlier, the chimeric gene of the heavy chain can be obtained in a truncated form, in order to obtain fragments of chimeric immunoglobulin (in particular, Fab, Fab' or F(ab')2for immunoscintigraphy. In these molecules, you can enter a label directly or by using a chelating agent such as DTPA, with radioisotopes, such as131iodine,125iodine,99mtechnetium or111indium and get agents to radioimmunoscintigraphy. Otherwise, by means of genetic engineering in the site of the chimeric antibody can be maintained domain binding (chelating) radioactive metals, to obtain a site for insertion of the label. Thus, chimeric immunoglobulin can be created in the form of protein, which contains not owned by the person trombozitopenicescoy variable region, constant region of a human (preferably truncated) domain and binding of the metal derived from linking the metal of the protein, such as metallothionein.

Trombotsitopeiiya chimeric immunoglobulin or its FR is required for to containing the label immunoglobulin was localized at the location of the thrombus, find the generated tag signal using fotoscandela device, such as a gamma camera. The detected signal is then converted into an image of a blood clot. The image allows to localize the thrombus in vivo and to develop an appropriate strategy of therapy.

The invention is further illustrated by the following examples, which in no case limit.

Example 1

Obtaining chimeric trombozitarnogo IgG4

A. General strategy

The strategy for cloning the variable regions for genes with light and heavy chain of hybridoma E based on the bond formation in the genome between the variable region and the corresponding J-fragment binding fragment) functionally rearranging (and expressed) genes Ig. Probes J-DNA fragment can be used for screening genomic libraries to isolate DNA that is connected with the J-segment; DNA in embryonic configuration (prearrangement) also hybridized in J-probes, however, is not connected with the variable region of the sequence and can be identified by analysis of the selected clones using enzyme rest is th of rearranging genes of heavy and light chain when using the JHand JK. These clones are subjected to examination by the method nazem blot, in order to determine, whether expressed their sequence in hybridoma E. Clones that contain expressed sequences, introducing the expression vector containing the constant region of a human, and placed in myeloma cells of the mouse in order to determine if the antibody. The antibody of producing its cells then have to determine the binding specificity and function in comparison with murinova antibody 7E3.

Depositing derived cell lines murinova hybridoma I spend American collection tissue cultures (American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852) may 30, 1985. Registration number NW assigned after successful testing viability.

B. Materials and methods

Unless specified, all parts and percentages are given by weight and temperatures are indicated in degrees Celsius.

Construction of genomic library of heavy chain

To highlight gene variable regions of the heavy chain of hybridoma 7E3 construct a genomic library of separation by size, using phage lambda vector gt10 strip size 3, 5 thousand base pairs, corresponding transformed the locus of the heavy chain. Probably, this fragment contains a gene variable regions of the heavy chain. From hybridoma cells 7E3 produce DNA with high molecular weight and is subjected to full fermentation using the restriction endonuclease EcoRI. Then DNA fractionary 0.7% agarose gel and directly from the gel allocate DNA size 3-4 thousand base pairs. After extraction with a mixture of phenol/chloroform and gel filtration through a column of Sephadex G-50 fragments of size 3-4 thousand base pairs are ligated to the shoulders Lamba gt10 (from Promega Biotech, Inc. ") and packaged in vitro into phage particles using Packagene from Promega Biotech". This library is subjected to direct screening with a density of approximately 30,000 platelets per Petri dish 150 mm, using JH-a sample that contains the tag32P. Hybridization of platelets is carried out in 5X SSC, 50% formamide, 2X denhardt's reagent, 200 μg/ml denatured DNA salmon sperm 42oC for 18-20 hours. Finish washing is carried out in 0.5 X SSC, 0,1% sodium dodecyl sulfate at 65oC. Positive clones identified after autoradiography.

Construction of genomic library of light chain

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DNA with high molecular weight are subjected to partial fermentation with restriction endonuclease Sau3A and fractionary size in the density gradient 10-40% sucrose solution. The DNA fragments by size 18-23 thousand base pairs are ligated to the shoulders EMBL-3 and packaged in vitro into phage particles using Packagene. This library is subjected to screening with a density of approximately 30,000 platelets per Petri dish 150 mm, using JK-test. Conditions for hybridization and washing are identical to the conditions used for the library of heavy chain.

DNA probes

JH-probe heavy chain of the mouse is a fragment of BamHI/EcoRI size 2 thousand base pairs, which contains the segment J3, and the segment J4. JKthe probe light chain mouse is a Hindlll fragment size 2.7 thousand base pairs, which contains all five segments of Jx. Probes containing the label P, receive enzymatic legirovaniem single-stranded gap in double-stranded DNA ("nick"-broadcast) using a kit from Amersham, Inc. Free nucleotides are centrifugation through a column of Sephadex G-50. The specific activity of the probes approximately 109counts per minute per microgram the 1% gel agarose/formaldehyde (Maniatis et al., Molecular Cloning) and transferred to a pulp. Spot hybridized using DNA probes subjected to "nick"is broadcast in 50% formamide, 2X denhardt's solution, 5X SSC, and 200 μg/ml denatured DNA salmon sperm 42oC for 10 hours. Finish washing is carried out in 0.5 X SSC, 0,1% sodium dodecyl sulfate at 65oC.

Transfection of DNA using electroporation

Plasmid DNA that must be subjected to transfection, twice purified by centrifugation until the balance in the gradient of solutions of ethidium bromide/chloride cesium. 10-50 μg DNA plasmid type 8106SP2/0 cells in phosphate buffered saline physiological solution when cooled in a bath of ice and the resulting mixture is placed in the apparatus for carrying out electroporation firm "Biorad". Electroporation is carried out at a voltage of 200 volts, and the cells placed on the tablets for micrometrology with 96 cells. Selection using appropriate medications carried out after 48 hours and after 1-2 weeks identify colonies that are resistant to the drug.

Quantitative determination of reproduction antibodies

The liquid above the sediment tissue culture to analyze the protein content of IgG immunoassay method conciencia for purified IgG. The concentration of the chimeric antibody 7E3 with constant regions of a human is determined using beads made of polystyrene, coated with the obtained from goat antibody anti-human IgG Fc and obtained from goat conjugated with fluorescein antibody anti-human IgG Fc. The analysis is performed on the automated installation (from "Pandex Laboratories, Inc.").

Cleaning trombotsitopeiiya chimeric antibodies IgG4

The liquid above the sediment tissue culture concentrate using Diaflo ultrafiltration membrane YMIOO ("Amicon") and placed on a column filled with A protein - separate. Chimeric antibody elute from the column with protein A solution of sodium citrate in a gradient of pH from 6.5 to 3.5. The purified antibody concentrate using Diaflo ultrafiltration membrane YM100. The concentration of antibody is determined by measuring the absorption at a wavelength of 280 nm.

Analysis of inhibition of binding

Purified antibody (as murinova 7E3 and chimeric 7E3) use in competitive analysis with antibody 7E3, containing the label of radioactive iodine in the study of the binding of human platelets. Platelet-rich plasma obtained by centrifugation containing citrate buffer of whole blood with the speed of 1875 the exchange to the desired concentration of the solution of the test antibodies and initiate the reaction by adding 150 μl of platelet-rich plasma. Spend incubation for 1-2 hours at room temperature and platelets with bound antibody was separated from free antibody by centrifugation through a 30% sucrose with acceleration 12000 g for 4 minutes in a tube for centrifugation capacity of 0.4 ml Cap tube containing pill platelet/antibody, cut and define the number of samples in the Geiger counter. Competition binding to platelets between containing iodine 7E3 and chimeric 7E3 compare with competitor containing iodine 7E3 and not containing the label 7E3 IgG.

Inhibition of platelet aggregation

The purified antibody 7E3 or chimeric antibody 7E3 add to containing citrate buffer of whole blood and incubated in the incubator at 37oC for 10 minutes. The rate of platelet aggregation was determined after activation by collagen or ADP with the help of aggregometry whole blood ("Chronolog Corp.").

C. Results

Cloning trombotsitopeiiya variable regions of the gene

Several positive clones secrete after screening of a library of heavy chain and the library of light chain and approximately one million platelets, respectively using probes JHand JK. After approximately three emission with either EcoRI (clone heavy chain), with either HindIII (clones light chain) and fractionary 1-percentage agarose gels.

DNA transferred to nitrocellulose and samples hybridized with DNA probes JH(heavy chain) or JK(light chain), containing the label P. For heavy chain allocate 2 clone containing the EcoRI DNA fragments with a mass of 3.5 thousand base pairs, which was hybridisable with probe JH. Two classes of HindIII fragments with sizes of 3.0 and 6.0 thousand base pairs identify with probe JK.

Cloned DNA corresponding to the authentic regions of the heavy and light chains from hybridoma 7E3 must gibridizatsiya in mRNA isolated from hybridoma. Non-functional rearrangement DNA loci in both heavy and light chain should not be expressed. In Fig. 1 shows the results of analysis by the method of Naser blot showing that the expected EcoRI fragment of the heavy chain (3,5 thousand base pairs and estimated HindIII fragment light chain size 6.0 thousand base pairs both hybridize to the corresponding size of mRNA from hybridoma 7E3. In the subcloned fragments enter the label P method "nick"-broadcast and hybridized them in Naser-blotty containing total RNA isolated from SP2/0 (merge partner g is implemented with mRNA size 2 thousand base pairs in RNA 7E3, but not in RNA SP2/0. Similarly, the fragment of the light chain HindIII size 6.0 thousand base pairs hybridizes with mRNA size 1250 base pairs in RNA 7E3, but not in RNA SP2/0. This is the correct dimensions for the RNA of the heavy and light chains, respectively. Because the cloned DNA fragments contain sequences that are expressed in hybridomas 7E3, the data obtained indicate that the clones containing the correct sequence of the variable regions of hybridoma 7E3. However, recent functional test shows that the above sequence after combining them with suitable sequences of the constant regions capable of directing the synthesis of antibodies with specificity and affinity similar to the specificity and affinity of murinova antibody 7E3.

Vectors and expression systems

V-genes of the assumed light and heavy chains, obtained by cloning from hybridoma 7E3, attach to genes and G4 constant regions of human rights in the previously described expression vectors (L. Sun et al., Proc. Natl. Acad. Sci. USA 84: 214-218 (1987)). Fragment 17-1A VKHindlll of pSV184 Hneo17-1AVKhCKreplace the HindIII fragment size 6.0 thousand base pairs, corresponding envisages the-hC4replace the EcoRI fragment size 3.5 thousand base pairs, corresponding to the intended gene variable regions of the heavy chain of 7E3. The structure of the resulting plasmid, designated as p7E3VKhCKand p7E3VHh C4, shown in Fig. 2.

For the expression of chimeric genes of the heavy and light chains of the two plasmids kurashina transferred into neproducyruth cell line SP2/0 murine myeloma. Plasmid light chain confers resistance against G418, and plasmid heavy chain confers resistance against mycophenolic acid and, thus, allow for dual selection upon receipt of clones carrying expressing genes from each plasmid. Colonies resistant to G418 and mycophenolic acid, propagated in a stable cell line and

store in the presence of both drugs. The liquid above the sediment tissue cultures of the indicated cell lines examined for the presence of antibodies with fluorescent immunoassay particle concentration using plastic beads coated with antibody anti-human IgG Fc, obtained from the goat, and the same antibody containing the label of fluorescein. Of the first ten proven lines for PR is CLASS="ptx2">

The analysis of activity linking platelets

After purification of antibodies C-7E3F6 on a column filled with A protein - separate, the antibody concentrate and compare with murinova 7E3 IgG in the analysis of activity linking. Fig. 3 shows that murinova 7E3 and-7E3F6 (estimated chimeric antibody) equally compete with containing radioactive label 7E3 for binding platelets; binding curves coincide, indicating that the binding properties murinova and chimeric 7E3 in accordance with this criterion are identical.

Inhibition of platelet aggregation with-7E3F6

Cleaned-7E3F6 compared with murinova 7E3 when conducting a functional analysis, which determine the ability of the test antibody to inhibit the aggregation of human platelets. The results of this analysis are presented in Fig. 4 and show that both antibodies at the same concentration equally suppress induced by collagen platelet aggregation. In addition with-7E3F6 equally inhibits ADP-induced platelet aggregation.

The results on the binding and inhibition of platelet aggregation show that: (1) from hybridoma clone 7E3 really paluchowska on murinova constant region does not affect the ability to communicate and functional characteristics of the variable regions 7E3.

Analysis using beads coated with fibrinogen

As it turned out, a chimeric antibody with-7E3F6 is positive when conducting quantitative functional analysis, which determine the ability of antibodies to inhibit the agglutination between platelets and coated with fibrinogen balls. B. Cooler et al., J. Clin. Invest. 73: 325-338 (1983).

Example 2

Production of chimeric IgG1 and IgG3

DNA fragments encoding the variable region of the heavy chain of murinova antibody 7E3, attached to the constant regions1and3people who are present in the expression vectors pSV2Hgtp17-1AVH-hC1and pSV2Hgtp17-1AVH-hC3(Sun et al., Proc. Natl. Acad. Sci. USA 84: 214-218 (1987)), replacing fragments of 17-1A variable regions of the heavy or light chain corresponding fragments of variable regions 7E3. The obtained chimeric genes heavy chain together with the chimeric genes of the light chain kurashina transferred into cells SP2/0 and obtain stable cell lines secreting antibodies1, K3, K

Example 3

Primary research on the use of chimeric 7E3 Fab for the treatment of humans

Obtaining fragments of chimeric 7E3 Fab

The fragment of the chimeric 7E3 (c7E3) poluchaut Fermo enzyme papain. The Fab fragment emit through multiple stages of purification, aimed at producing a product that is free from other fragments of fermentation and other contaminants (in particular, protein, nucleic acids, viruses). The final product is obtained in the form of sterile pyrogen free and no solution containing 2 mg of monoclonal chimeric 7E3 Fab per ml of 0.15 M solution of sodium chloride, 0.01 M solution of sodium phosphate with a pH of 7.2. In some preparations add Polysorbate 80 to a final concentration of 0.001% (wt./vol.). Immediately before use the product is filtered through a filter with a pore size of 0.22 microns, which weakly binds the protein. The product is stored at 2-8oC.

Pharmacokinetics: the leaching of c7E3 Fab from human plasma

Leaching of the fragment of the chimeric 7E3 (c7E3) Fab from plasma study, three patients suffering from chronic coronary artery disease. After a dose of 0.20 mg/kg c7E3 Fab, which is injected in the form of infusion for five minutes in different periods of time ranging from two minutes up to 72 hours, take blood samples. It was expected that some portions of the antibodies will be present in the plasma in the unbound state. To determine this, the unbound component antibodies in plasma free se Fab determine the solid-phase immunofermentative analysis (EA). In this analysis, we used the affinity of the selected antimarino 7E3 IgG, which is obtained from rabbit serum, for solid-phase capture and detection system based on a biotinylated derivative of the same drug antigen anti-7E3 obtained from rabbit. The results are shown in Table 1 (see end of description).

If in the form of unbound antibody in the plasma was determined if all the entered dose of 7E3, theoretical maximum concentration of antibodies would have been approximately 5,0 µg/ml (0.25 mg/kg, divided by 40 ml of plasma per kilogram). However, this theoretical maximum concentration could not be obtained, because most of the introduced antibody binds to platelets. In fact, during the earliest times of the measurements (2 minutes) average concentration of c7E3 Fab in plasma (n = 2) is 2,43 µg/ml; this value corresponds to the maximum observed plasma concentration (Cmax). The data obtained for later after the introduction of the antibodies of time intervals, show a rapid initial decrease of the content of 7E3 Fab in the plasma. By the time of the measurement after 1 hour and 24 hour remaining in the plasma entered anticell from time shown in Fig. 5 shows a fast initial leaching c7E3 Fab from plasma in all three patients.

Conduct a preliminary analysis pharmakinetics properties of the fragment c7E3 Fab. In order to satisfy the results obtained for the concentration in plasma, apply several models, including several mixed (random and fixed effects linear models, and standard dvukhkomponentnykh and decompartmentalized models. Information about the concentration of free antibody in the plasma inadequate to meet the standard pharmakinetics models. Because the subject of the impact of c7E3 Fab is a receptor located on platelets, it cannot be expected that the amount of free antibody in the plasma will not be any simple way related to its concentration at the place of his actions. The initial rapid removal of c7E3 Fab from plasma partially demonstrates the rapid binding of an antibody to the receptor GPIIb/IIIa. It was shown that one of the studied models for describing the values of the concentrations in plasma is most suitable linear model probabilistic effects. Using this model, determine preliminary values pharmakinetics parameters C1pVdand t1R>
The parameter - Value of

C1p(proportions/hour) - 15,6

Vd(L) - 6,8

t1/2(h) 0,1 (6 min)

(*) To match the received data is applied to a linear model with random effects

C1p= Value of the purification of the plasma is defined as the rate of decline in plasma concentration divided by the concentration, expressed as the rate per hour, i.e., if the velocity at a given time remains constant for one hour, then the calculated value will be proportional to the amount of medication, which is removed during this hour.

Vd= Volume of distribution is defined as the prescribed dose, which is divided into the measured plasma concentration multiplied by the volume of plasma. For calculations using plasma volume of 3 l, typical for a person weighing 70 kg.

t1/2= Half cleansing

Urine samples from people

Urine samples collected from three patients suffering from stable coronary artery disease, which intravenous 0.25 mg/kg c7E3 Fab (data cleansing plasma for the same three patients described earlier). Total urine output is taken through the following intervals after the injection: from 0 to 2 hours 2 to 6 hours 6 to 12 hours and from 12 hour to 24 hour. Kie free 7E3 Fab, using a slightly modified method of carrying out solid-phase immunofermentative analysis discussed earlier. In all cases, c7E3 Fab in urine not detected.

Preclinical toxicology

The study of preclinical toxicology spend 18 monkeys (Cyonomolgus and Rhesus), using chimeric 7E3 Fab. Appoint a bolus dose to 0.6 mg/kg followed by infusion in an amount up to 0.8 µg/kg/min for 96 hour (includes studies with heparin, aspirin, and recombinant tissue plasminogen activator). All monkeys for all doses and for all combinations of antibody 7E3 is safe and easily tolerated, with no induced bleeding complications or other harmful effects.

Escalating doses of chimeric 7E3 Fab in patients with stable angina pectoris

The study of escalating doses of conduct, selecting 52 patients suffering from stable angina (men aged from 43 to 75 years), which antiplatelet therapy was not administered for more than 10 days. Assign different dosing regimens. Patients receive either a single intravenous dose bolus in the amount of from 0.15 to 0.25 mg/kg of chimeric 7E3 Fab (20 patients), or they prescribe a bolus followed continuous nutrigenomics in response to 20 μm ADP (agonist) and the time of bleeding determined through 2 hours after the appointment of the dose with 7E3 Fab bolus (0.15 to 0.30 mg/kg). Blockade of receptor and platelet aggregation in response to agonists is determined in accordance with known methods (H. K. Gold et al., J. Clin. Invest. 86: 651-659 (1990)). The time of bleeding is determined by the Simplate method. With increasing doses there is a progressive strengthening of the blockade of the receptor, as indicated by the percentage of the number of blocked receptors (determined by the availability of the binding sites of the receptor). The strengthening of the blockade of the receptor is accompanied by suppression of platelet aggregation (defined as a percentage of the value before drug administration or from the base value) and increase in bleeding time.

The peak value for all three parameters observed for a dose of 0.25 mg/kg, This dose corresponds to a plasma concentration of 5 μg/ml concentration at which weak inhibition was observed in the platelet-rich plasma sample from a healthy subject, which is kept in the incubator for 15 minutes in a ditch of aggregometry in the presence of increasing concentrations of chimeric 7E3 Fab. (The degree of aggregation in the plasma of healthy human subject is defined as the percentage amount of light transmitted through the cuvette. Before assigning agonist plasma is EC, does not contain antibodies, agonists ADP, the transmittance increases progressively with the development of aggregation. However, if there is c7E3 Fab, there was a dose-dependent suppression of aggregation and complete inhibition was observed at a concentration of 5 μg/ml c7E3 Fab).

Determine the duration of action from the point of view of receptor blockade, inhibition of platelet aggregation and bleeding time. The peak value of the impact on the receptor blockade, platelet aggregation and bleeding time observed after 2 hours, and then slowly fades. The time of bleeding return to close to normal in 6 to 12 hours.

Since the peak value of the blockade of receptors and functional inhibition is achieved at a dose of 0.25 mg/kg, examine the duration of suppression of platelets during long-term infusion, which is carried out after the appointment of the dose, to determine whether you can increase the duration of suppression of platelets. The degree of blockade of the receptor, the degree of suppression of platelet aggregation and increase in bleeding time support during long-term infusion of five patients receiving 10 mg/min chimeric 7E3 Fab within 72 cha Similar results are observed after infusion for 12, 24 and 96 hours.

None of the patients noted an allergic reaction. No significant trends caused by treatment in Hematology or in the values obtained in the chemical laboratory. Not observed any serious cases of bleeding. Minor bleeding are rare and include temporary moderate bleeding from the nose and moderate bleeding from the gums of patients suffering from periodontal diseases. The test results show that chimeric 7E3 Fab can be assigned to patients using a dose regimens that lead to profound suppression of platelets up to several days.

Data on immunogenicity

When testing with moremovie 7E3 F(ab') and Fab (150 patients) immune response, which is determined using a sensitive enzyme immunoassay, occur in 16% (24/150) patients. All reactions have a low titer, usually in the range from 1:50 to 1:200. Subjected to the treatment group includes healthy volunteers, who injected a dose of 0.01 - 0.25 mg/kg murinova 7E3 F(ab')2, patients suffering from unstable angina, which enter of 0.05 - 0.20 mg/kg murinova 73 F(ab')2and the patient is renovage 7E3 F(ab') or 0.15 - 0.35 mg/kg murinova Fab, as well as patients suffering from stable angina, which with the help of intravenously injecting a single dose in the form of a bolus dose of 0.10 - 0.30 mg/kg murinova 7E3 Fab, and either a single dose bolus in the amount of 0.25 mg/kg 0.30 mg/kg followed by continuous infusion over a 12-36 hour (0.15 ug/kg/min or 10 µg/min) murinova Fab, or two injections murinova Fab with an interval of six hours (single bolus of 0.2 mg/kg 0.30 mg/kg, and then a bolus of 0.05 mg/kg).

Immunogenicity is significantly reduced for chimeric 7E3 Fab man - mouse. None of the 52 patients suffering from stable angina who were selected to conduct research with escalation of dose and which was introduced chimeric 7E3 Fab (see above), not detected immune response in the treatment process that is determined using a similar analysis, which is suitable for chimeric Fab.

Reversibility antiplatelet activity

Chimeric 7E3 Fab (1K) slowly separated from the platelets, and free of chimeric 7E3 Fab quickly washed out from the plasma in the circulation (see earlier). Thus, the antiplatelet effect of chimeric 7E3 is significantly reversible in the appointment of platelets from random is which appointed or murinova Fab, either chimeric Fab and who injected platelets from random donor at a time when the suppression of platelet aggregation in these patients is almost over. Recovery of platelet function determined by measuring the time of bleeding. The property of reversibility is useful in cases when, due to bleeding occurs the necessity of restoring the functions of platelets in the patient.

Example 4

Using chimeric antibody 7E3 for the prevention of thrombotic complications during election plastic surgery for coronary artery

Percutaneous crespella reconstruction of the coronary artery, for example, by the method of balloon or coronary atherectomy is an effective way of increasing the lumen prone to stenosis of the coronary arteries. For this operation there is a risk of acute occlusion of the coronary artery in the process of its implementation and after plastic surgery on the blood vessels. It is reported that the level of coronary occlusion varies from approximately 3 to 6% of the total number of cases of election plastic operations on vessels (K. M. Detre et al., Circulation 82: 739-750 (1991)) and it is a major cause of morbidity and mortality during hospitalization. caused by thrombosis, 10% to 20%.

Acute coronary occlusion during or immediately after plastic surgery on the coronary arteries, apparently, is caused by a combination of deep lesions of the arteries, causing a partial blockage "intimal flaps", which is in the process of clot formation, or simply the formation of a blood clot at the site of lesion of the vessel wall. In animal models, re-occlusion after successful thrombolyse preceded by periods of cyclic reduction and restore blood flow through the coronary artery, which is called "cyclic changes of blood flow". These cyclical changes in blood flow are a phenomenon almost entirely driven by platelets, and arise from the repeated accumulation and displacement of platelet aggregation in the center of the coronary stenosis and lesions of the endothelium. Examples of the cyclic changes of blood flow in humans after carrying out plastic surgery on the coronary arteries. Chimeric antibody 7E3 can be used to suppress the platelet function during plastic surgery on the blood vessels and thereby prevent the aggregation trombone thrombotic occlusions. These patients can be determined on the basis of anatomical (in particular, by the nature of the lesions in place of restenosis, which is controlled with the help angiography) or clinical risk factors (in particular, myocardial infarction, unstable angina, diabetes, women aged 65 years and older) that create a predisposition to acute coronary thrombosis and cause clinical syndromes acute myocardial infarction, unstable angina or sudden blockage of a blood vessel.

Chimeric antiplatelet antibody in elective percutaneous cresolate reconstruction of the coronary artery

The test is carried out in two stages. The main objective of the first stage is the definition of safe and optimal dose of chimeric 7E3 Fab for patients who have to spend elective percutaneous crispolto reconstruction of the coronary artery. The second stage is carried out with the aim to pre-evaluate the safety and efficacy of chimeric 7E3 (c7E3) when assigning a bolus injection followed different according to the duration of the infusion. The study in the second stage is subjected patients who underwent selective reconstruction of coronary arteries and which pogorzelica patients with unstable angina or persistent coronary artery disease with specific lesions of type B and C. Table 3 provides criteria for the identification of patients included in the high-risk group, and in Table 4 (see end of description) the typical lesions of the characteristics that define a method of angiography. Primary efficiency is defined as the suppression of the functions of platelets and prevent thrombotic complications. To conduct both stages of the test, you can choose men aged from 18 to 76 years of age and incapable of childbearing women aged 18 to 76 years.

Stage I

In stage 1 of the patients form groups that receive a single intravenous injection of a bolus of the fragment of the chimeric 7E3 (1K) Fab (which is produced and formulated in the form of a composition as described in Example 3). All treatment is subjected to 15 patients (3 women and 12 men). The average age of patients was 62 years (range from 46 to 76 years). Demographic characteristics are shown in Tables 5A and 5B (see end of description) for all patients who were prescribed a single dose, and patients from the group with individual dosages.

Five patients each (n = 5), receive a single dose of c7E3 Fab in the amount of 0.15 mg/kg, 0.20 mg/kg or 0.25 mg/kg approximately 30 minutes prior to the holding of the election is applying to ingredients during the operation give aspirin (standard dose) and spend their full anticoagulant treatment with heparin (standard dose).

While on stage I surgery percutaneous cresolate reconstruction of the coronary artery are considered selective for patients, six of the fifteen patients have residual unstable angina. The location of the extended parts in the coronary artery is summarized in Table 6 (below). Seven of the fifteen patients examined during phase 1, conducted percutaneous crespella reconstruction of coronary arteries from one defeat in a single vessel, six patients underwent percutaneous crespella reconstruction of the coronary artery with multiple lesions in the same vessel, and two patients underwent percutaneous crespella reconstruction of coronary arteries in multiple vessels (table 6, see the end of the description).

Performance criteria for optimum single dose of c7E3 were later defined as the minimum dose, the introduction of which can achieve 2 hours of average values of the following quantities: (1) lengthening the time of bleeding for at least 20 minutes; (2) blockade of the receptor GPIIb/IIIa, which is blocked for more than 80% of the receptor compared with the baseline; and (3) inhibition of platelet aggregation in response to 20 μm ADP to values not bolster spend prolonged infusion of c7E3 Fab in the amount of 10 μg/ml for 6, 12 or 24 hour. When conducting research in phase II trials expose a group of 32 patients (8 women and 24 men). The average age of patients who appoint c7E3 Fab, is 57 years (range from 38 to 76). The control group consists of nine patients (1 woman and 8 men). The average age of patients in the control group is 56 years (range from 37 to 74). The control group consists of patients included in the risk group, as already discussed earlier, which do not get with 7E3, however, surveillance as well as for patients who prescribe the medication. Demographic profile for all patients in phase II and for patients in groups with individual dosage are shown in Tables 5A and 5B.

Treatment with c7E3 Fab carried out for 30 minutes before balloon inflation when performing percutaneous cresolate reconstruction of the coronary artery. Aspirin and heparin is administered in accordance with clinical practice, it is recommended that after plastic surgery on the blood vessels were injected heparin in quantity 800 units per hour. 6-hour and 12-hour group consists of eleven patients, and 24-hour group consists of ten patients.

is uccio one coronary artery with one loss seven patients receive percutaneous crispolto reconstruction of one coronary artery with multiple lesions, and three patients spend percutaneous crispolto the reconstruction of several coronary arteries with multiple lesions (table 6). Type percutaneous cresolate reconstruction of the coronary artery for one patient is not installed. The location of the expanding parts in the coronary arteries for patients at stage 11 are summarized in Table 6. Of the nine control patients eight patients receive percutaneous crispolto reconstruction of one coronary artery with one loss and one patient percutaneous crispolto the reconstruction of several coronary arteries with multiple lesions. 32 patients, who were prescribed with 7E3 Fab, and 9 control patients have clinical or angiographic characteristics that can be classified as patients with a high risk of developing myocardial ischemia after carrying out percutaneous cresolate reconstruction of the coronary artery. Two patients, who were prescribed c7E3 Fab, and one patient from the control group have an unknown risk factors. The remaining 30 patients, who were prescribed c7E3 Fa characteristic, according to which they are exposed to the risk of ischemic complications, and most of them have more than one risk factor.

These risk factors for the control group and groups, which were introduced c7E3, are summarized in Table 7, and individual list of risk factors for each group are shown in Tables 8A-8D (see the end of the description).

Inhibition of platelet function

(Results of stage I)

To study the activity of chimeric 7E3 Fab in the inhibition of platelet function, consistently pursuing measures the availability of the binding sites of the receptor GPIIb/IIIa (recorded as the average number of blocked GPIIb/IIIa, expressed as a percentage), determine the average magnitude of the inhibition induced by the action of an agonist of platelet aggregation in response to the introduction of 20 μm ADP and the average bleeding time. Blockade of receptor and platelet aggregation in response to agonists carried out mainly by the described method (H. K. Gold et al. , J. Clin. Invest., 86: 651- 659 (1990)). When carrying out measurements on the blockade of receptor availability receptor determined at time 0 and the number of available receptors mistaken for a 0% blocked receptors (base value). Meant the or results obtained prior to dose). Bleeding time will determine the Simplate method.

In Fig. 6 shows the response after two hours on the introduction of a single bolus dose of chimeric 7E3 Fab for blockade of the receptor (Fig. 6A), platelet aggregation (Fig. 6B) and bleeding time (Fig. 6C). The continuous line in Fig. 6 shows the average values for the five patients from each group. When the dose is increased c7E3 Fab progressive increases blockade of receptors that are shown as the percentage of blocked receptors (Fig. 6A). The average value of the blocked receptors after two hours is 53,8% for the group with a dosage of 0.15 mg/kg, 80.2% of the group with a dose of 0.20 mg/kg and 86,6% for the group with a dosage of 0.25 mg/kg Increased receptor blockade is accompanied by inhibition of platelet aggregation, which is shown as a percentage relative to the value before the introduction of the dose (Fig. 6B). The average amount of suppression of platelet aggregation in two hours is 46.1%, 44.6% and 17.9% of the baseline values for groups, respectively, with a dosage of 0.15 mg/kg, 0.20 mg/kg and 0.25 mg/mg Similar to two hours after infusion observed a dose-dependent increase in bleeding time (the time dimension bleeding prerivaju 0.15 mg/kg, 0.20 mg/kg and 0.25 mg/mg, respectively. According to the data of measurements in the conditions of the experiment, the optimal dose for antiplatelet activity is 0.25 mg/kg

In Fig. 7 shows the duration of action after a single dose bolus in the amount of 0.25 mg/kg, i.e. the dose at which there is maximum exposure on platelets. Lines indicate average values from time 0 (baseline value) to 24 hours (delayed on the x-axis) for blockade of the receptor (upper graph, Fig. 7A), platelet aggregation (average chart, Fig. 7B) and bleeding time (bottom chart, Fig. 7C). The peak values for the blockade of the receptor, platelet aggregation and bleeding time observed after two hours, and then gradually reduced. Bleeding time returns to a value close to normal in 12 hours. None of the patients was not observed reduction in platelet count.

Inhibition of platelet function

(Results of stage II)

At the second stage, data on the blockade of GPIIb/IIIa and platelet aggregation receive not for all patients, and experiments on injection within 24 hours carried out only for two patients. For this reason, only data analysis, surface and 12 hours, the average value for the blockade of the receptor is maintained at a level in excess of 80% from baseline values during the entire period of infusion. The mean value of platelet aggregation induced by introduction of 20 μm ADP, for 6-hour and 12-hour groups is 2 hours 13% and 15% of baseline values, respectively, for the 12-hour group remains below 25% during the entire period of infusion. The average bleeding time measured after two hours, for all three groups is more than 30 minutes. In Fig. 8 analysis of the results obtained for the patients after the initial dose 0.25 mg/kg over 12 hours continuously injected by infusion of chimeric 7E3 Fab in the amount of 10 µg/min 12-hour period of infusion are shown as lightly shaded areas, and the lines correspond to average values. The degree of receptor blockade, inhibition of platelet aggregation and prolongation in bleeding time is maintained throughout the infusion period, and the reduction starts as soon as the infusion is stopped (see Table 9).

Clinical implications of stage I and stage II

None of the 47 patients who were prescribed c7E3 Fab, there has been cases of thrombosis at the time, and the La all 47 patients surgery percutaneous cresolate reconstruction of the coronary artery was successful, according to angiography lesions decreased to less than 50% of the diameter of the blood vessel. Two unsuccessful attempts extensions patient 01-012 the amount of narrowing of the left anterior coronary artery was reduced from 90% to 70%, but further expansion was technically impossible. The second patient (patient 01-019), which will be discussed below, the expansion operation was initially successful, but required installation of intracoronary stent, with the aim of stratification along the main longitudinal direction of the thrombosis was not observed). One of the 9 control patients (01-022) there was a sudden blockage of a blood clot within 15 minutes after the start of the operation, which required an emergency bypass bypass coronary artery, which it has transferred successfully. The other 8 control patients underwent successful surgery expansion, the value of the residual stenosis was 50% or less.

The patient 01-019 (group 12-hour infusion) held a balloon expansion 95% of the lesions in the left circumflex artery with residual narrowing of 50%. After the surgery the patient, apparently, was observed vasovagal syndrome, which led to bradycardia, hypotonia and time the RNA stent, the purpose of stratification along the main longitudinal direction. The stent is moved in the left main coronary artery, and the patient was referred for surgery emergency bypass grafting coronary artery. According to expert data obtained by angiography and during the operation, indicate the absence of intracoronary thrombosis. This patient also suffered a perioperative myocardial infarction. The patient recovered and was discharged 8 days after the operation.

Three patients, who were prescribed c7E3 Fab, there was a slight separate bouts of pain in the breast after surgery for percutaneous cresolate reconstruction of the coronary artery. The patient 01-009 (from a group with a single dose of 0.25 mg/kg) felt chest pain after 9 hours after the appointment of the dose, the patient 05-003 (group 12-hour infusion) suffered an attack of angina after 21 hours after the appointment of c7E3, and the patient 06-003 (group 12-hour infusion) suffered a stroke 2 days after the appointment of c7E3. According to medical experts, these cases of chest pain is not associated with symptoms of ischemia, which could indicate a re-corking.

The patient 02-004 (from a group with a common the conduct percutaneous cresolate reconstruction of the coronary artery. The next day, changes in the electrocardiogram, with a high content of cardiac enzymes (detected before), indicated that the patient has suffered a perioperative myocardial infarction, not corresponding to the Q-wave (peak content creatinekinase = 462, MB fraction = 64).

During the studies recorded only one death, which occurred in 52 days after the appointment of c7E3 Fab. The patient 06-002 (from a group with a 6-hour infusion), the history of which includes collagen disorders of the lung, congestive heart failure, and unstable angina, underwent successful surgery percutaneous cresolate reconstruction of the proximal left anterior descending coronary artery. During the operation, the patient developed persistent ventricular fibrillation, twice that required the application of electrical defibrillation, however, after this operation is finished without any consequences. After the patient left the Department catheterization, he has developed cyanosis, which initially led to the need of the use of diuretics and oxygen therapy. Next, however, this patient developed persistent respiratory failure, which was late the Ohm, respiratory distress syndrome, anemia (requiring multiple blood transfusions) and ischemia of the heart. This patient died on day 52 after the appointment of c7E3 Fab due to the failure of many organs.

Security: Surveillance for stage I and stage II

In Fig. 9 for patients from each dosing group shows the absolute changes in hematocrite number compared to the baseline value after 24 hours after injection. For comparison, the line corresponding to the zero change. Measurement data on hematocrite numbers for one patient from the control group (01-022) and one patient (01-019), which was appointed by c7E3 Fab, not shown, as both patients within the first 24 hours we had to spend a blood transfusion after an emergency operation on Obvodny coronary artery (see below). The second bottom line with a value of minus 12 indicates a change hematocrite number, which must be identified as corresponding to the minimum bleeding when using the criteria of thrombosis in myocardial infarction (Rao et al. , J. Am. Coil. Cardiol. 11: 1-11 (1988)). Changes in hematocrite the same number of patients in the control group and all groups to which the end of infusion. Changes in the number of platelets in not exposed to processing the control group and the groups who were prescribed c7E3 Fab, have the same distribution and do not reveal a clear dependence on the dose.

The discussion of the results of stage I and stage II

At the stage I studies established that c7E3 shows the same dependence of the parameters on the dose when performing percutaneous cresolate reconstruction of coronary arteries of patients who are prescribed aspirin and heparin, as observed for patients with stable angina when testing with escalating doses (Example 3). Chimeric 7E3 causes a dose-dependent blockade of receptor GPIIb/IIIa platelet, and this blockade of receptors correlates with the inhibition of platelet function. In addition, the results obtained in phase II, show that long, up to 24 hours, suppression functions Fab platelets can be achieved by continuous infusion. All patients recovery of platelet function begins after 6-12 hours after cessation of infusion, regardless of the duration of infusion.

Clinical consequences in patients exposed to c7E3, as shown by the angiographic and clinical data, znpy, exposed to c7E3, there were no cases of thrombosis during and after surgery. Next, for all patients, except two, operations, according to angiographic data, carried out successfully. All the patients selected for the research in stage II, and 6 of the 15 patients selected for research at stage I are patients who, in accordance with their clinical and angiographic characteristics are at increased risk. Individual clinical factors (such as unstable angina, diabetes, women older than 65 years) or specific angiographic characteristics of the lesions (such as signs of type B or C) result in the patient group with a high risk of complications, and the influence of many factors is cumulative.

17 patients at stage II, which was appointed by the antibody, suffered from unstable angina and had or had no additional clinical or angiographic signs of risk factors. Further, unstable angina was observed in 6 patients in stage I. In the number of published results indicate that unstable angina is the main factor complicated Recoleta reconstruction of the coronary artery) in 10-15% of patients (P. J. De Feyter: Editorial. Am. Heart J. 118: 860-868 (1989) and H. J. Rupprecht et al., Eur. Heart J. 11: 964-973 (1990)). Angiographic parameters also indicate a high probability of complications after carrying out percutaneous cresolate reconstruction of coronary arteries (S. G. Ellis, 1990, "Elective coronary angioplasty: technique and complications", In: "Textbook of Interventional Cardiology" (E. J. Topol, Ed.), W. B. Saunders Co., Philadelphia; P. J. De Feyter et al., Circulation 83; 927-936 (1991); S. G. Ellis and E. J. Topol, Am. J. Cardiol. 66: 932-937 (1/90); and ACC/AHA Task Force Report: Guidlines for percutaneous transluminal coronary angioplasty, J. Am. Coil. Cardiol. 12: 529- 545 (1988)). At twenty-nine patients subjected to treatment with c7E3 in phase II, there are some signs of complications, selected according to angiography. Of these, 12 patients had one characteristic lesion type B, 14 patients had two or more symptoms of type B, and three patients showing signs of defeat of type C. Then, many who took part in trials of patients with multiple lesions spread to one or more vessels that also potentially increases the risk of complications after surgery (M. Samson et al., Am. Hearfe J. 120: 1-12 (1990)). Based on both the quantity and severity of a certain angiographic risk factor, one might expect the appearance of ischemic complications in 10-20% of patients (S. G. Elliion 83: 927-936 (1991); S. G. llis and E. J. Topol, Am. J. Cardiol. 66: 932-937 (1990)).

The control group consists of patients included in the risk group. However, in the General case, the number and severity of risk factors in patients of the control group below. Five of the nine patients had one risk factor or one lesion type B (4 patients) or unstable angina (1 patient), while 26 out of 32 patients in stage II was introduced c7E3 had either lesion type, or two or more symptoms high risk. This difference in risk between the two groups is significant (according to the exact test of Fisher, p = 0,018). It is interesting to note that the control patients with sudden occlusion (patient 01-022, unstable angina, with two defeats B) was one of the three patients who were defined as patients with more than one risk factor (one patient from the control group had an unknown risk factor). Thus, while one out of three patients from the control group with the highest risk lesions observed thrombotic phenomena, none of the 26 patients who were injected c7E3 Fab, who were in that group with the highest risk of thrombotic phenomena cannot be safely implemented in patients which had been treated with intravenous heparin and oral administration of aspirin. Cases of bleeding in the control group and the treated group c7E3 comparable among themselves, and for both groups is not observed differences hematocrite number from a base value. Other unwanted effects have been rare and differed in minor or moderate severity. In the tests reported one death, and it happened almost two months after the appointment of c7E3 Fab in a patient who suffered from collagenosis lung and heart failure and who have developed after percutaneous cresolate reconstruction of the coronary artery respiratory failure complicated by sepsis, respiratory distress syndrome and in the end the failure of multiple organs.

Finally, none of the twenty patients with known results, was not observed immune responses directed against chimeric antibodies.

In conclusion, it should be noted that the chimeric 7E3 potentially safely inhibits the platelet function in patients who undergo percutaneous cresolate reconstruction of the coronary artery, who were prescribed aspirin and GNC is of the risk of bleeding and the response of the immune system. Among patients with a high risk of thrombotic complications in the group, which was appointed by c7E3, there are no cases of thrombosis, which indicates that c7E3 can reduce the risk of thrombotic complications in selected patients.

Example 5

Treatment of sudden blockage during plastic surgery on the vessel

Sudden blockage of a coronary artery during a plastic surgery on a blood vessel is a major factor in morbidity and mortality when carrying out the specified operation. They occur in approximately 3-6% of cases of election plastic surgery (K. M. Detre et al., Circulation 82: 739-750 (1991)), however, noted that they occur in 20-40% of patients undergoing plastic surgery for unstable angina or after acute myocardial infarction (S. G. llis al., Circulation 77: 372-379 (1988); P. J. De Feyter et al., Circulation 83: 927-936 (1991)). The mechanism of sudden obstruction is acute thrombosis of the artery place where plastic surgery creates or expands the area of the damaged layer of endothelium. There is generally a violation of a picture of blood flow caused by changes in the geometry of the vessel, the cause of which is often a gap of platelets, and tamparuli. Since the initiation of clotting necessary adhesion and aggregation of platelets, to treat sudden blockage of a coronary artery that arises when performing plastic surgery on the coronary arteries, using a fragment of the antibody 7E3 Fab.

History

The patient was a doctor-a man aged 45 years, who had previously distinguished himself in good health. A week before that, he was carried a plastic surgery on vessels, he began to experience discomfort in the chest and neck. When these symptoms began to recur and to go in a few days, he turned for advice to a colleague. Electrocardiogram revealed wave inversion T FWD atrium. The patient was placed in the cardiology Department of the local hospital, and he was appointed intravenous nitroglycerin and heparin and oral aspirin. A series of definitions in the next 24 hours the concentration of cardiac isoenzymes showed no increase in their level compared to normal. A series of recordings of electrocardiograms in the next two days showed a steady flattening of the teeth T of the front atrium, however, did not reveal any evolutionary changes characteristic of myocardial infarction. The second is and in General showed normal functioning with very little hypokinetic area in anterolateral the left wall of the ventricle and the other hypokinetic area in inferoposterior zone. The proportion of blood ejection from the left ventricle is 72%. Coronary arteriography shows the dominance of the left coronary system with a small and completely occluded right coronary artery. Significant narrowing of the lumen in the middle portion of the left anterior descending coronary artery. Small diffuse painful diagonal process appeared distal to the middle part of the narrowing of the lumen of the left anterior descending coronary artery

the arteries.

The patient returned to the cardiology Department, and he continued to introduce intravenous nitroglycerin and heparin within 48 hours. During this time he had no pain, levels of cardiac isoenzymes did not increase, but a daily electrocardiogram revealed only sustainable flattening of T-waves for the front atrium. The patient was transferred to the hospital on behalf of Hermann (Houston, Texas) for plastic surgery on the blood vessels.

Before performing plastic surgery on vessels, the patient continued to receive intravenous nitroglycerin and heparin, oral aspirin, and he began to orally enter the agent for blocking the calcium channel. Partial thromboplastin time sociolingo coagulation was 173 seconds. The patient was intravenously injected 5000 units of heparin. Left the mouth of the coronary artery enter the guide catheter French JL 3.5 8. Spent visualization of the left anterior descending coronary artery in the right caudal anterior oblique and anterior cranial inclined projections. First, in the left anterior descending artery was placed guide wire catheter Doppler (from "Cardiome-trics, Inc.", Mountain view, California) with a thickness 0,018 inch (0.45 mm). This wire guiding catheter is typically used to monitor blood flow in patients at significant risk for sudden blockage. Registered data on the speed of blood flow in the proximal and distal end of the left anterior descending coronary artery. On the wire guiding Doppler introduced coronary balloon catheter (from "Intrepid, Baxter, Inc.", Irvine, California) size 2.5 mm with guide wire into the coronary artery was preserved. The cylinder was placed so that it covers the defeat of the left anterior descending coronary artery. Held successive pressurizations in the form of short pulses with balloon to achieve a pressure of 6 atmospheres. Steut data measuring the speed of blood flow, peak flow rate increased from 12 cm/sec to 33 cm/sec.

Observation for several minutes after the expansion showed that the signal of the flow rate began to decrease. Introduction of contrast medium showed a re-narrowing of the place of plastic surgery due to the elastic thrust of the lung, a ruptured platelets and formation of thrombus. Again set the container in a place of defeat and spend another boost from the container. The artery is again expanded, and the value of the flow rate again was 34 cm/sec.

Control in the next few minutes for the signal of the flow rate again showed a decrease. Within five minutes the signal was very low, and average flow velocity was 3 cm/sec. The patient began to experience chest pains. When conducting an electrocardiogram monitor increased ST-segment for the front atrium. Angiography showed that the artery is completely occluded. Time activated clotting, which was determined for several minutes prior to angiography, was 344 seconds.

Introduced the fragment of the chimeric monoclonal antibody 7E3 Fab specific for the recipe is. the for approximately 1-2 minutes after the appointment of c7E3 Fab speed of blood flow through the coronary artery began to grow. Injection of contrast medium showed the return of the coronary artery in the open state, while its value in the criterion value current blood thrombosis in myocardial infarction (TIMI) was equal to one (TIMI 1). In the next 15 minutes the blood flow through the coronary artery has continued to increase and stabilized at the maximum value of blood flow, equal to 23 cm/sec. Several additional injection of contrast material showed improved blood flow. Chest pain in a patient weakened, and observed on the monitor screen of the ST segment decreased compared with the baseline.

Fifteen minutes after the appointment of c7E3 Fab conduct angiography by well-known methods. The signal velocity of blood flow at this point was 20 cm/sec. Continuous monitoring over the next five minutes showed no further improvement in blood flow through the coronary artery. During this time, the video angiograms testified that in place of plastic surgery on the vessel there is still a small amount of a blood clot. For this reason it was decided to enter the of about ten minutes. During this time there has been further improvement in blood flow that is determined using a wire guiding catheter Doppler. After intracoronary infusion of urokinase, at the thirty-third minute after the introduction of c7E3 Fab, had another angiogram of the coronary artery. The artery was opened, and the amount of blood flow matches the value of TIMI 3. Some modest, but definite, residual narrowing of the vessel lumen at the site of the lesion remained. It was further discovered that a blood clot has decreased in size, but not completely dissolved. It was decided to hold another balloon expansion, in order to reduce residual stenosis.

Again, using the wire guide, introducing a balloon catheter to the site of the lesion. Then hold inflation within the spirit of minutes to the value of 6 atmospheres. Then a balloon catheter is removed, and the guide is left in place. The signal value of the blood flow increased to a peak of 29 cm/sec and remained stable for several minutes. Angiogram showed adequate reduction of residual stenosis. Then I moved the guide wire proximally relative to the narrowing of the vessel lumen and the p is the overall catheter was removed, and the operation is thus completed.

The patient is then sent to the cardiology Department. Him in a few days orally was administered aspirin, nitrates, means blocking the calcium channel and intravenous heparin, so that the amount of partial thromboplastin time was in the range of 70-90 seconds. A series of electrocardiograms showed a decrease in the inversion of T-wave for the front atrium, and all subsequent electrocardiogram were normal. Concentrations of creatine kinase (ck) in a series of measurements consistently was less than 100 units per liter. The number of platelets before percutaneous cresolate reconstruction of the coronary artery was 248000, and subsequent values of the number of platelets in 2 hours, 6 hours, 12 hours, 24 hours and 48 hours after the appointment of c7E3 Fab was 304000, 279000, 246000, 185000 and 222000, respectively. Platelet aggregation induced by injection of 10 μm ADP was before the surgery 73% according to optical densitometry, and subsequent values in 2 hours, 6 hours, 12 hours, 24 hours and 48 hours was 0%, 13%, 26%, 45% and 51%, respectively. A week after plastic surgery on the vessel of the patient to implement contam TIMI 3. After a few days the patient was discharged home.

A discussion of the history of the disease

For a given patient, the combination of 0.25 mg/kg c7E3 Fab intravenous, 250000 units of intracoronary urokinase and re-expansion has enabled a successful treatment of acute ischemic coronary artery after a sudden blockage in plastic surgery on the coronary arteries. These results suggest that antiplatelet therapy, which inhibits the binding of the receptor glycoprotein IIb/IIIa platelet and the stitching of platelets, may provide an effective means for achieving sustainable resumption of blood flow during acute obstruction of the coronary arteries in similar clinical situations.

Example 6

A randomized, double-impersonal evaluation of the effectiveness of the use of anti-GPIIb/IIIa fragment of the chimeric antibody for the prevention of ischemic complications in plastic operations on vessels of patients included in the high-risk group.

Review

The number of operations for percutaneous revascularization of the myocardium increased significantly after the opening of the plastic surgery of the coronary arteries in 1977 (A. R. Gruentzig et al., N. Engl. J. Med., 316: 11, 26; 10-16(1992)), a serious disadvantage of it are acute complications. The treated vessel in 4-9% of cases suddenly closes during surgery or when the patient after surgery, and re-plugging or sudden blockage associated with significant complications and results in about a ten-fold increase in mortality (A. M. Lincoff et al., J. Am. Coll. Cardiol., 19: 926-938 (1992); A. N. Tenaglia et al., Am. J. Cardiol., in press (1993); K. M. Detre et al., Circulation, 82: 739-750 (1990); S. G. Ellis et al., Circulation, 77: 372-379 (1988)). Although the mechanism of sudden blockage of the vessel is often unclear, the main contribution comes from the formation of thrombus and dissection. The signs by which patients can be attributed to the group of high risk include the presence of clinical syndromes associated with the formation of a blood clot in the coronary artery (unstable angina, acute or recent myocardial infarction), diabetes, women and coronary morphological features (fractures, blood clots, branching), indicating the increased complexity of individual lesions (A. M. Lincroff et al., J. Am. Coll. Cardiol. , 19: 926-938 (1992); S. G. Ellis et al., J. Am. Coll. Cardiol., 17 (Suppl. B): 89B-95B (1991); B. Moushmoush et al., Cath. Cardiovasc. Diagn., 27: 97-103 (1992); and R. K. Myler et al., Circulation, 82 (Suppl. 11): 11-88-11-95 (1990).

Although Arda patients undergoing plastic surgery on vessels (L. Schwartz et al., N. Engl. J. Med., 318: 1714-1719 (1988): E. S. Barnathon et al., Circulation, 76: 125-134 (1987), its effect on platelet function is relatively weak and cases of ischemia continue to occur in 10-20% of patients included in the high-risk group who are prescribed aspirin (A. M. Tenaglia et al., J. Am. Coll. Cardiol. (193, in press)). On the contrary, the purpose of the Fab fragment of the chimeric antibody 7E3 people leads to significant blocking of the receptor GPIIb/IIIa and suppression of platelet aggregation (see Example 4 Inhibition of platelet function).

When conducting a preliminary study of patients undergoing plastic surgery on vessels, it was shown that with 7E3 Fab reduces the risk of sudden blockage of the vessel during and after percutaneous surgical intervention (see Example 4 and S. G. Ellis et al., Cor. Art. Dis. , 4: 167-75 (1993)). The present randomized study undertaken to further evaluate the effectiveness of fragments, chimeric antibodies that selectively bind to the receptor of glycoprotein IIb/IIIa inhibitors, for the prevention of ischemic complications (EPIC Study: the Study c7E3Fab in preventing ischemia-induced complications). In particular, the assessment of clinically is operative complications, undergoing plastic surgery on vessels, carried out in impersonal randomized study using a placebo as a control. The study involved 2099 patients in 56 centres, which prescribed the plastic surgery on the coronary arteries, or direct atherectomy in situations with increased risk; severe unstable angina with residual pain and registered changes in the electrocardiogram, developing myocardial infarction or clinical or coronary morphologic characteristics of the lesion, associated with a significant risk of postoperative complications. Patients prescribed either (a) placebo bolus plus infusion, (b) bolus c7E3 Fab and infusion of placebo or (C) a bolus and infusion of c7E3 Fab. The main results that lead to stop further testing, are a part of the results, including the manifestation of one of the following: death, nonfatal myocardial infarction, unplanned surgical revascularization or repeated plastic surgery on vessels, unplanned coronary stent placement or the intra-aortic balloon pump placement when resistant ischemia.

Methods

On the basis of previous trials conducted stratification of risk factors in conventional percutaneous surgery, patients are taken only with contraindications due to the high risk of bleeding. It is believed that the patient is at high risk if he is in one of the three clinical groups: (1) acute myocardial infarction within 12 hours after onset of symptoms that indicate the need for direct or rescue percutaneous surgical intervention; (2) early angina after myocardial infarction or unstable angina with at least two cases of residual angina associated with residual changes on the electrocardiogram in the preceding 24 hours despite therapy; or (3) with clinical and/or angiographic criteria in accordance with the criteria of the American heart Association/American College of cardiology (according to AHA/ACC) (T. J. Ryan et al. , J. Am. Coll. Cardiol., 12: 529-45 (1988)) and their modifications (S. G. Ellis et al., J. Am. Coll. Cardiol., 17 (Suppi. B): 89B-95B (1991)). These clinical or angiographic criteria of high risk include either two characteristic lesion type or one characteristic lesion type C or a sign of defeat B in women over the age of 65 years or patients with diabetes.

Below are described in more detail specific inclusion criteria patients at high risk:

(I) the territory of the receiving FDA-approved device in one of the following situations:

(A) Unstable angina and/or myocardial infarction, in which the electrocardiogram is not observed Q-wave, which are defined as:

1) residual angina: two or more cases of residual angina with ischemic ST-segment abnormalities, T-wave; or

2) recurrent angina: recurrent angina ischemic ST-segment abnormalities, T-wave during hospitalization, regardless of the standard pharmacological interventions; or

3) early postinfarct angina; angina within 7 days after the established myocardial infarction with residual angina, accompanied ischemic ST-segment changes, T-wave; or easily provoked angina,

where temporary ischemic ST-segment or abnormal T-wave is defined as:

a) 1 mm depression (80 milliseconds after the J-point) or increase (20 milliseconds after the J-point) ST-segment and/or

b) changes in T-wave (usually inversion) or at least two times less than the norm content of creatine kinase (ck) for all patients at the time of registration.

(B) Acute myocardial infarction Q-wave:

1) direct surgical intervention during myocardial infarction without predoslje failed thrombolytic therapy during acute myocardial infarction,

where registered myocardial infarction determined by the presence of at least two of the following three criteria:

(1) prolonged angina (over 30 minutes);

(2) the excess of the levels of creatine kinase more than two times the upper limit level (confirmed increased levels of band isoenzyme of creatine kinase in the myocardium);

(3) confirmed by electrocardiogram heart attack, which is defined as;

a) an increase in ST segment of at least 0.1 millivolt (measured after 0.2 seconds after the J-point) in at least one of three places:

(i) at least two of the bottom three-lead electrocardiogram (II, III, aVF); or

(ii) at least two of the six chest leads of the electrocardiogram (V1-V6); or

(iii) depression of the ST segment of the chest leads V1-V4, typical rear current wounds (usually mirrors); or

(iv) in the case of left ventricular blockade primary ST-changes in the lower or front-lead electrocardiogram;

b) a new significant Q-wave duration is not less than 0.04 seconds, or Q-wave with a depth of not less than one quarter of the amplitude of the corresponding R-wave, or both.

(C) Clinical/ the artery; signs of infection based on the criteria for ACC/AHA;

2) stenosis with one or more lesions of type C in the expanding artery;

3) age over 65 years combined with female and having at least one lesion type B;

4) diabetes mellitus and at least one lesion type B in the expanding artery; or

5) plastic operation on the vessels to eliminate induced myocardial lesions within 7 days after myocardial infarction, registered the increase of the content of the isoenzyme of creatine kinase in the band infarction.

II. Men aged from 18 to 80 years and women aged 18 to 80 years, not able to bear children (i.e., women who undergo surgical sterilization, or women after menopause, which is determined by the absence of menstruation for at least one year).

III. Written informed agreement that is made before carrying out the procedures in accordance with the selected methodology and purpose of the study tools.

Patients that meet the selection criteria, were then excluded those that fall under at least one of the following constraints:

(1) diagnosis of hemorrhagic diathesis in the history of Bo the test;

(3) recent (within 6 weeks of the registration period for testing) clinically significant bleeding gastrointestinal or genitourinary tract;

(4) stroke within 2 years prior to the registration or any other stroke, which caused a significant residual neurological failure;

(5) more than 50% blockage of the left main coronary artery;

(6) the estimated or recorded in the history of vasculitis;

(7) participation in other clinical studies, which conducted a study of the test drug or device within 7 days prior to infusion of the study means of the present invention;

(8) an oral appointment of anticoagulants within 7 days prior to randomization before learning tools, if only the thrombin time before randomization does not exceed 1.2 times control time;

(9) the appointment of intravenous dextran to plastic surgery or scheduled for plastic surgery;

(10) recorded in the medical history, previous destination marinovich monoclonal antibodies or available information about allergies towards murinova proteins; and

(that all participating institutions, and informed consent was obtained from all patients. A call for volunteers for a research was conducted in the period from December 1991 to November 1992 goal; was selected 2099 patients in 56 institutions in the USA.

Methodology research

All patients were administered aspirin and heparin. Aspirin was administered orally with a dose of 325 mg for at least two hours before surgery and was prescribed 325 mg per day after surgery. Heparin (porcine) were intravenously administered bolus with an initial dose of from 10,000 to 12,000 units, and then the dose is gradually increased to 3000 units at intervals of 15 minutes, but the dosage is not exceeded 20000 units; the aim was to maintain the activated clotting time in therapeutic range, which in General is in the process of operation from 300 to 350 seconds (K. G. Dougherty et al., Abstracts of th 63rd Scientific Sessions, 111-189 (1991); B. Rath et al., Br. Heart J., 63: 18-21 (1990); J. D. Ogilby et al., Cath. Cardiovasc. Diag. , 18: 206-9 (1989)). Heparin continued to enter through infusion in quantities of 1000 units per hour for at least 12 hours. According to clinical indications may intravenously and vnutriaortalina be entered nitrates. Chimeric 7E3 Fab (1K) comes in the form with the rija, 0.01 M phosphate and 0.001% Polysorbate 80, pH 7,2. Only prescribed medicines after discharge is aspirin in the amount of 325 mg per day.

Patients blind selection is divided into three equal groups according to the principle of dual identification results of the experiment. One group of patients should receive a bolus of c7E3 Fab with a dose of 0.25 mg/kg followed by continuous infusion of c7E3 Fab with a dose of 10 mcg/min the Second group receives a bolus dose of 0.25 mg/kg c7E3 Fab and continuous 12-hour infusion of placebo solution. The third group receives the bolus placebo and continuous 12-hour infusion of placebo solution. The bolus is administered at least 10 minutes before the start of the operation and is administered for 5 minutes and the infusion continued for 12 hours or until, unless there is a clinical contraindication.

Blood samples for counting the number of platelets taking over 30 minutes, 2, 12 and 24 hours after the beginning of drug administration and then daily until discharge of the patient from the hospital, to thoroughly test possible thrombocytopenia. To assess life-threatening bleeding and reducing the number of platelets use a pre-designed algorithm D. C. Sane et al. , Ann. Intern. Med., 111: tiv, a blood transfusion is administered in accordance with local practices in each institution. Plastic surgery on vessels performed by standard methods. Before and after the enlargement of the lumen of the vessels conduct angiographic studies using 150-300 mcg of nitroglycerin in the form of intracoronary injection. After operation of the protective sheath of vascular catheters retain for at least six hours after injection of the test tools. In addition, protective catheters remain on the ground for at least another four hours after stopping the infusion of heparin until then, until it reaches acceptable to stop bleeding activated partial thromboplastin time.

The termination of the experiment

Independent clinical advice for dealing with cases stop the experiment examines all the episodes, which can lead to the termination of the experiment or serious complication. This advice remains anonymous to the doctors who conduct the treatment, during the whole period of the experiment and considering the report, the electrocardiogram, and, if necessary and appropriate medical card. For making sakhnovski further test is a complex clinical case of termination of the tests, which includes appearance within 30 days after the blind selection of one of the following events:

(1) death due to any cause;

(2) non-fatal myocardial infarction; or

(3) emergency surgery:

(a) a Second plastic surgery. Repeated percutaneous surgical intervention to prevent recurrent acute ischemia (balloon plastic surgery or coronary atherectomy). Planned (i.e., operations that are carried out in several stages) are not considered as criteria for stopping the experiment.

(b) Obvodnogo coronary artery. Urgent (non-selective) surgical intervention for the treatment of acute recurrent ischemia.

(C) placement of a coronary endovascular stent. Intracoronary stent placed to quickly bring the extensible vessel in the open state.

(d) placement of the pump for intra-aortic balloon counterpulsation. Balloon pump placed in the case of recurrent ischemia in patients who are not considered as potential candidates for re plastic surgery on vessels or for surgical intervention.

the grow flow by TIMI criteria is 2 or 3; stenosis determined by the surgeon is not more than 50%; and electrocardiogram does not detect signs of ischemia.

The end of the experiment due to myocardial infarction is determined as follows:

1. In patients subjected to a blind selection within 24 hours after myocardial infarction, for the diagnosis of non-fatal heart attack requires one of two isozyme criteria: (a) the content of creatine kinase or strips of creatine kinase in the myocardium at least 3 times the upper normal limit, showing an increase by at least 33% compared with the previous value in the valley" (defined as 25% increase compared with the previous peak value, which, however, at least twice the upper normal limit); or (b) at least 100% increase in the content of creatine kinase or strips of creatine kinase in the myocardium, which is at least 3 times the upper normal level, with a further 50% reduction relative to the previous peak value and the value in the valley" less than two times the upper normal level. Registered a new case of prolonged angina (>20 minutes), Sopra a second heart attack. In the absence of the registered angina beginning of a second heart attack set as the time of measurement of enzyme "in the valley", which immediately precedes the new increase. Definition 1 applies only to those patients whose beginning recurrent angina and/or the value of the content of the enzyme "in the valley" is observed within 24 hours after myocardial infarction, which was recorded before testing. In all cases, except when it is not defined, use the content of a strip of creatine kinase in cardiac muscle; otherwise, use the value of the total content of creatine kinase.

2. In patients who did research for more than 24 hours after an acute myocardial infarction or without recent myocardial infarction, myocardial infarction, occurred during hospitalization, install one of the following criteria: (a) new Q-EUPEC a duration of at least 0.04 seconds or more depth of one quarter of the amplitude of the corresponding R-eubsa in two or more contiguous leads of the electrocardiogram; or (b) the content of a strip of creatine kinase in the myocardium at least 3 times the upper Normala early re-infarction in patients which at the onset of registered acute myocardial infarction, equal to, or time of occurrence of new cases of persistent angina (>20 minutes) or time determining levels of enzyme "in the valley" prior to new growth. To this definition can be applied, any time exceed 24 hours after the initial heart attack.

3. After discharge from the hospital for the diagnosis of myocardial infarction requires one of the following two criteria: (a) a new significant Q-wave duration is not less than 0.04 seconds, or Q-wave depth of not less than one quarter of the amplitude of the corresponding R-wave, or both in two or more contiguous leads of the electrocardiogram; or (b) the levels of creatine kinase or strips of creatine kinase in the myocardium at least 2 times the upper normal limit.

Another component of the basic case of termination of studies is the need for emergency re-operative intervention, which is defined as an unplanned return the patient to the operating room for surgical intervention; this does not include elective surgery, passing and W is nil emergency surgery on the coronary arteries, for the treatment of recurrent angina or repair of failed plastic surgery. The introduction of intracoronary stent is seen as the main reason for stopping research in the case when the stent is placed to treat threatened or actually incurred a sudden blockage of the vessel, which carried out plastic surgery. The placement of the balloon pump is considered as a case of causing the termination of the research, if the pump is placed due to recurrent angina in a patient who is not expected to be repeated surgery for revascularization.

Cases of bleeding are classified as significant, weak or insignificant, using the criteria of the study group on thrombosis in myocardial infarction (A. K. Rao et al., J. Am. Coll. Cardiol., 11: 1-11 (1988)). Significant bleeding defined as intracranial bleeding or bleeding caused by the decrease in hemoglobin of more than 5 g/DL (or, when hemoglobin is not available, to reduce hematocrite number of at least 15%). Light bleeding is either spontaneous or occur in the form of macroscopic hematuria or bloody vomiting, caused by a sharp decrease in hemoglobin b the ore 10%), and if the site of bleeding is not installed, a decrease in hemoglobin of more than 4 g/DL (or, when hemoglobin is not available, determine the reduction hematocrite number of at least 12%). In patients who are transfusion of blood, to determine the total reduction in hemoglobin and to establish whether there was a significant or light bleeding, the amount paid by transfusion units add to the observed reduction hematocrite number divided by three (C. S. Landefeld et al., Am. J. Med., 82: 703-13 (1987)).

Information processing and statistics

Blind patient selection is conducted by phone from the coordinating data center at Duke University. Selected by a blind method patients stratified depending on the location of the institution and depending on whether any of the patient's acute myocardial infarction. On the basis of previously obtained data plan quota would be 2100 patients in order to fix a 33% decrease in the number of fixed stops in the experiment (assessment it is 15% in the group receiving placebo) with the index of 0.8 and p = 0.05.

Data is collected possiblydue. The organizers of the research do not know about the code of randomization, and the results obtained until then, until a selection all patients and until all cases stop the experiment will not be discussed in the special Committee on the termination of studies.

Baseline characteristics are presented graphically in the form of averages and in line with values of 25% and 75% for continuous variables or percentages in the below tables for discrete variables. When the analysis of the main stops of the experiment, whereas the time elapsed before the first occurrence of any one of the complex components in the event of interruption of studies within the first 30 days after conducting the selection process. If no such cases in a 30-day interval is not fixed, then observation of the patient continue after 30 days. For graphical representation of results using survival curves Kaplan-Meier (E. L. Kaplan et al. , J. Am. Stat. Assn., 53; 457-81 (1958)). All comparisons of the results of the treatment according to the principle of purpose/treatment. In the event of major cases, stop the experiment is carried out test check/ranking for the trend assessment (dose-dependent) and rasmol bolus and perform the injection (J. D. Kalbfieichsch and R. L. Prentice, The Statistical Analysis of Failure Time Data, John Wiley and Sons, New York (1980)). Further the plan of the experiment involves pairwise comparison on the principle of registration/ranking for each of the groups treated with c7E3 Fab, if it is important to determine the trend. An interim safety analysis of the experiment carried out at the time, when you get data about one third and about two-thirds of patients. Nominal set level of alpha values to assess the significance of the experimental results for the dependence of the dose at each interim analysis in advance is chosen so that the level of all errors of type 1 did not exceed 0.05. When conducting the final analysis the level of significance when comparing chosen equal being 0.036. A similar strategy (test to determine trends with subsequent relevant pairwise comparisons) used when conducting the final analysis, with the aim of establishing the relationship between treatment and each component of the composite case stop the experiment, although these comparisons are only for purposes of explanation. The final analysis is also performed using the specified strategy, in order to compare treatments with the number who indicate ratios and confidence intervals for key subgroups (age, sex, weight, clinical subgroup) and represent them graphically.

Results

Baseline clinical characteristics for groups of patients show that patients are at increased risk from the point of view of the probability of occurrence of acute complications in plastic surgery due to the high proportion of patients with diabetes, recent myocardial infarction, elderly and a large number of women in combination with symptoms that meet the selection criteria (see Table 11). Most patients find one or two diseased vessel, the left ventricle is functioning properly. Details of surgical intervention are shown in Table 12. No significant differences in the planned treatment is not found.

Composite core cases of termination of the experiment and its components are shown in Table 13. Compared with placebo, significantly ranked effect c7E3 Fab (p = 0.009), while reducing the probability of a compound event of termination of the experiment is 11% (p = 0,43) for the group that received only the bolus, and 35% for the group that received a bolus and p and experiment due to ischemia, in Table 13. Thus, it was found that blockade of glycoprotein IIb/IIIa reduce the likelihood of myocardial infarction, emergency holding bypass grafting coronary artery and emergency re-percutaneous revascularization, while in the short-term blockade, which is achieved by assigning a single bolus, there is only a slight trend in the same direction. Three deaths in the group of patients receiving bolus and infusion, are observed in patients who died after conducting a blind selection, but prior to the introduction of the drug in spite of these deaths included in the analysis in accordance with the principle goal/treatment.

As c7E3 Fab prevents the occurrence of nonfatal myocardial infarction, interest severity prevented myocardial infarction. As can be seen from Table 14, is prevented as heart attacks with Q-wave and heart attacks associated with high levels of enzymes, when this occurs the dose.

Extensive MI without Q-wave is defined as myocardial infarction, confirmed by the presence of the peak value of the band creatine kinase P CLASS="ptx2">

Neabry MI without Q-wave is defined as myocardial infarction, confirmed by the presence of a peak bandwidth of creatine kinase in the myocardium or the total content of creatine kinase, which is 3-5 times the upper normal limit.

Define the time of nonfatal ischemic complications are different for each of the three groups to conduct emergency repeated plastic surgery, i.e., events that can be precisely established (see Fig. 10). The majority of cases in the group that receives placebo, occurs in the first hours after carrying out the specified operation, while in the group receiving bolus, there is some grace period (approximately 6-12 hours), just after which begin to show signs of complications, corresponding to the time of maximal blockade of the receptor. Observed a marked delay in the onset of ischemic complications, and decrease the absolute frequency of their occurrence in the group that receives the bolus plus infusion.

Distribution caused by bleeding complications during hospitalization are shown in Table 15. As in the study of efficiency for the basic cases of the termination of the research, there hung infusion, there is an increased number as the main cases of bleeding, and blood transfusion, while patients who were prescribed only bolus, noted only moderate growth. Most cases of bleeding observed during bypass grafting coronary artery or in the groin at the site of puncture of the vessel, although the rate of surgical repair of vessels are distributed evenly (1% in the group with the placebo group bolus plus infusion and 2% in the group with a single bolus). Similarly, six of the patients have intracranial hemorrhage, with two such cases are marked in the group with placebo, one group with bolus and 3 cases in the group of patients who are prescribed bolus plus infusion, while one of the patients, the drug is not received, as celebrated incident occurred after carrying out a blind selection, but before the plastic surgery on the blood vessels).

The frequency of re-occurrence of clinical events is low, and the main differences in the results obtained correspond to the assigned treatment. Installed after 30 days, the cases of heart failure(2,3%, 2,4%, 2,3%), prolonged ischemia(3,0%, 3,6%, 4,1%), ventricular fibrillation (3,0%,and the bolus plus placebo.

When assessing the effectiveness of treatment in subgroups (see Fig. 11) depending on, whether registered in patients of acute myocardial infarction, unstable angina or anatomic risk factor, a beneficial effect with 7E3 Fab observed for all three registered subgroups. Similarly, the effectiveness of the treatment is of uniform subgroups, selected by age or gender. As a function of weight, effective positive effects of bolus plus infusion with 7E3 Fab observed for all patients, although therapeutic effect to be more pronounced for patients with more weight.

The risk of severe bleeding is increased in patients with less weight compared with patients with more weight for the treated group only bolus, and for the group receiving bolus plus infusion. In the most easy tertile patients severe bleeding occurred in 21% vs. 15% vs. 7%, respectively, for patients who were injected bolus+infusion, bolus and placebo, while in the most severe tertile the corresponding figures for bleeding 8%, 7% and 7%. Transfusion of preserved red blood cells in the lightest tertile and placebo, while in the most severe tertile the corresponding figures are 11%, 7% and 4%.

Discussion of results

The obtained results confirm the important role played by platelets, apparently, managed platelet mediators and performed by platelet function in the event of a severe ischemic complications in patients who undergo percutaneous cresolate reconstruction of the coronary artery. The test method is EPIC specially designed for the reception of patients at significant risk for sudden obstruction (re-blockage) of a blood vessel and associated complications. Analysis of previously accumulated results shows that some patients may be classified as high risk on the basis of clinical signs of a blood clot in a vessel, such as acute myocardial infarction (R. S. Stack et al. , J. Am. Coll. Cardiol., 11: 1141-1149 (1988)), heavy unstable angina (R. K. Myler et al., Circulation, 82 (Suppl. II): II-88 II-95 (1990)) or registered angiographic appearance of a blood clot (D. D. Sugrue et al., Br. Heart J., 56: 62-66 (1986); B. D. Hettleman et al., J. Am. Coll. Cardiol. , 15: 154A (1990)). Other patients are at increased risk due to mechanical factors such as small size, is); N. A. Ruocco et at., J. Am. Coll. Cardiol., 69: 69-76 (1992); S. G. Ellis et al., J. Am. Coll. Cardiol. , 63: 30-4 (1989)). The studies reported significant number of patients of both types, which allows to shed some light on the possible involvement of inhibition of platelets for each group of patients. It is expected that the inclusion of these patients included in the high-risk group, will lead to an ischemic complications in 15% of patients in the group, which prescribe a placebo, despite the use of aspirin and high doses of heparin for each patient; as a result, it was obtained the value of this indicator is close to the expected.

Appointment with 7E3 Fab leads to a reduction of 35% in the number of cases of severe complications, the main observed effect is the reduction of cases of myocardial infarction, urgent plastic surgery on the blood vessels and an emergency bypass grafting coronary artery. Doing c7E3 bolus results in delayed onset of these events, which corresponds to the duration of exposure on platelet aggregation. However, ischemic complications begin to appear after 4-6 hours. This int% returned to baseline value after bolus injection murinova 7E3 Fab.

In addition to postponing the combined purpose of bolus and infusion, which leads to profound and prolonged suppression of platelet aggregation (see Example 4), leads to the prevention of the onset of acute ischemic complications. Complex case, stop the experiment will allow to assess the impact of this therapeutic approach for ischemic events in the period of the operation.

One of the most important discoveries of the present study is consistent decrease the likelihood of complications for the different cases of termination of the test. The reduction of the probability of occurrence of a myocardial infarction is significant and is consistent with a similar decrease

clinical need for emergency operations. Classification of non-fatal myocardial infarction is an important factor in assessing the results of the percutaneous reconstruction of the coronary artery. The most common is the determination of the level strip of the enzyme creatine kinase myocardial above the upper normal limit, and ranges from 4 to 21% for all published in the literature results for a series of patients (L. W. Klein et al., J. Am. Coll. Cardiol., 17: 621-6 (1991); A. C. Hunt et al., Eur. Heart J., 12: 690-3 (1991); P. Paul enzyme cannot be uniquely associated with a particular clinical phenomena, not marked and long-term adverse outcomes. Thus preventing the increase of the content of the selected enzyme, regardless of clinical events may not have predictive value. Order to obtain objective results in this delicate issue when evaluating events as myocardial infarction, regularly measure levels of enzyme and removed the electrocardiogram, employ anonymous expert advice and use the criterion of at least three times the threshold level content-specific cardiac enzymes. What c7E3 Fab reduces the full spectrum of myocardial infarction, including those associated with a moderate increase in the content of the enzymes, with a high increase in the content of enzymes, development of Q-wave, gives us confidence about the importance of what complications were prevented, especially because at the same time reduces the need for emergency operations revascularization of the coronary arteries.

Although not expected to detect and no detectable effect on mortality, it should be noted 3 deaths in patients belonging to the group, designate where the s in the analysis of cases, stop the experiment in accordance with the principle goal/treatment. All other deceased patients actually received prescribed therapy. With regard to mortality when performing plastic surgery on vessels, in order to see 25% of the positive or negative effects of treatment method requires carrying out observations of more than 20,000 patients.

The beneficial effects of the blockade of the receptor glycoprotein IIb/IIIa on the probability of the occurrence of stop the experiment when performing plastic surgery patients included in the high-risk group, quite convincingly, and is consistent with the positive results of recent observations using the same antibodies in patients undergoing plastic surgery on the blood vessels in the resistant state is unstable ischemia (M. L. Simoons et al., J. Am. Coll. Cardiol., 21: 269A (1993)).

Since this study was first undertaken large-scale study of the blockade of the receptor IIb/IIIa, there was the likelihood of developing thrombocytopenia. But with 7E3 Fab observed only a small important from a clinical point of view, the fall in the platelet count. In particular, the analysis shows that thrombocytopenia (platelet count < 100000/µl) was observed for larger placebo (3.4 percent). Thus, there is a likelihood of thrombocytopenia (platelet count < 100000/µl) for the group treated with bolus and infusion, compared with the group receiving placebo (for a given pair p = 0,062). Severe thrombocytopenia (platelet count < 50000/µl) developed in 11 (1,6%) patients in the group with treatment with bolus and infusion, and 5 (0,7%) patients in the group treated with placebo. Only 4 patients (< 1%) in each of these groups marked by severe thrombocytopenia and deadly or fatal complications. All cases of reduction in the platelet count were temporary and remained generally within the first few days.

The under-treated patients there was a significant increase in the number of complications associated with bleeding and blood transfusion. This increase is mainly as a result of bleeding in places of the femoral puncture and does not lead to significant difference in the minimum value hematocrite number or the number of life-threatening complications in all three groups. Trends persist no matter include in the analysis of patients subjected to surgical intervention. During Anonim the lower threshold for the purpose of transfusion, they have to stop the bleeding. Our previous studies have shown that improved technique for eliminating bleeding and blood transfusion in patients undergoing thrombolytic therapy, may lead to effective reduction is necessary to prescribe blood products (T. C. Wall et al., J. Am. Coll. Cardiol., 21: 597-603 (1993)).

The relationship between the beneficial effects of treatment and the risk of hemorrhage as a function of the patient's weight was more difficult than expected. Although the frequency of occurrence of major cases, stop the experiment and the risk of heavy bleeding is not much change in comparison with the weight of patients in the placebo group, there is a strong tendency to decrease weight increase of the number of major cases of termination of the experiment and increase the proportion of severe bleeding in both groups, which was appointed by c7E3 Fab.

Conclusion on clinical suitability of this approach to blocking of the receptor glycoprotein IIb/IIIa in patients before implementation, during and after surgery percutaneous revascularization depends on the relative importance of prevention Samoylenko risk which have been registered for the purposes of this test, this balance in favor of the appointment of antibodies. Prognostic aspects of acute myocardial infarction or the need for urgent repeat revascularization are very serious and the risk of blood transfusion, fortunately, continues to decline (J. G. Donahue et al., N. Engl. J. Med., 327: 369-73 (1992); R. Y. Dodd, N. Engl. J. Med., 327: 419-21 (1992); K. E. Nelson et al., Ann. Intern. Med., 117: 554-9 (1992)). Attempts to develop practical algorithms to reduce unnecessary bleeding and blood transfusion, and more effective dosage when conducting antithrombotic therapy, including the impact of anti-thrombin and antiplatelet effects, should lead to further strengthening of positive clinical factors observed in this study. In practice, the parenteral administration of funds, potentially antiplatelet effect in patients undergoing surgery with the use of technical means, should focus on the dosage used antithrombotic therapy (in particular, the dose of heparin were not consistent with the weight of patients depending on the patient's weight. Using a more accurate assessment methods and techniques to oasam study may help us to gain more information about the origin associated with bleeding complications.

A high degree of consistency obtained in the treatment effects in all groups of patients is a strong argument in favor of the formation of thrombus plays a more important role for many patients, although in some cases sudden blockages may in some cases be caused mainly thrombotic phenomena, and in other cases mechanical phenomena. Postponing the onset of complications, which contributes to therapy with bolus and prevention of these complications in the appointment of bolus and infusion, suggests that in many cases the surface of the torn artery lost most of its thrombogenicity after 18-24 hours after surgery. Therapeutic approaches should take into account the need to maintain long-term antithrombotic effects in patients at significant risk of sudden blockage of blood vessels.

Summing up, it should be noted that this study demonstrates a favorable impact of long-term blockade of the receptor glycoprotein IIb/IIIa in patientrelated re embolism or sudden blockage of the vessel. Although this beneficial effect is achieved by increasing the risk of bleeding, validity of the positive clinical results favored this approach in the treatment of patients for which it is known that they are at increased risk for severe ischemic complications, which is determined on the basis of clinical and angiographic criteria prior to surgery. The present study represents the first assessment of significant therapeutic approach to the suppression of the integrative functions of proteins of the cell membrane, paving the way biotechnology for development in the future for other purposes selectin and integrin, as well as approaches to the same glycoprotein IIb/IIIa, which does not use antibodies or use peptides.

Example 7

Reducing the incidence of clinical restenosis during operative intervention with early assignment of the fragment of the chimeric antibody anti-GPIIb/IIIa

The process of re-narrowing of the lumen after holding balloon plastic surgery on the blood vessels and percutaneous surgical intervention operations on coronary vessels - the phenomenon is very R the I within 6 months of re-operation revascularization, what in the United States spent more than $ 2 billion annually (J. J. Popma et al. , Circulation, 84: 1426-1436 (1991); E. J. Topol et al., Circulation, 87: 1489-1497 (1993); J. - P. R. Herrman et al., Drugs, 46: 249-262 (1993)). The main mechanism that leads to action restenosis, is trauma to the vessel under the action of a cylinder or other alternative device in place of surgical intervention, followed by formation of the platelet-thrombus and the change in phenotype of the cell medium smooth muscle from their state of rest or contractile status when they acquire the ability to move, to grow and secrete (J. S. Forrester et al., J. Am. Coll. Cardiol., 17: 758-769 (1991); J. H. Ip, J. Am. Coll. Cardiol. , 17: 77B-88B (1991); W. Casscells, Circulation, 86: 723-729 (1993)). Although experimental models for the modulation of the growth characteristics neointima that occurs after injury to the vessel, were successfully tested various pharmacological tools and a few angiographic studies confirm their beneficial action is still not carried out large-scale clinical trials on patients, which would have shown the effectiveness of these tools and to date no known pharmacological methods of treatment, which would lead to smart the Circulation, 86: 100-110 (1992)).

Plastic surgery on the coronary vessels are usually accompanied by the introduction as an aid aspirin oral and intravenous heparin. However, this antithrombotic approach is weak and weakly inhibits the aggregation of platelets. Various agonists, including thrombin, collagen, and adenosine diphosphate, is able to stimulate platelets despite therapy with aspirin. Methods of molecular biology in the study of platelets found that glycoprotein IIb/IIIa, which is the integrative protein of the cell membrane, is responsible as a receptor for the process of platelet aggregation (E. F. Plow et al., Prog. Hemostas. Thromb., 296: 320-331 (198); B. S.-Paste, J. Clin. Invest., 76: 101-108 (1985)). The fragment of the chimeric antibody 7E3 Fab selectively binds to the integrin IIb/IIIa platelet. After preliminary studies have confirmed a primary data on the safety and effectiveness of the fragment of the chimeric monoclonal Fab antibody, in many centres were held twice impersonal research involving 2099 patients (see Example 6). In addition to the initial efficiency, the main result is the reduction of the probability of developing acute phase of severe ischaemic about is to reduce the likelihood of clinical restenosis, what is defined ischemic complications or the need for repeat revascularization procedures within 6 months of medical surveillance.

Methods

Details of selection of candidates and methods are provided in Example 6. In brief, register patients, which should be performed plastic surgery on the coronary vessels or direct atherectomy and have developed a myocardial infarction or who have recently had a heart attack, suffer from unstable angina or at risk of angiographic lesions in accordance with the criteria of the American heart Association/American College of cardiology (ACC/AHA Task Force Report, J. Am. Coll. Cardiol., 12; 529-545 (1988)). As the criteria by which patients were rejected from participation in the experiment, were hemorrhagic diathesis, age over 80 years, stroke within 2 years or extensive surgical intervention within 6 weeks. Methods were approved by the academic boards of all participating centers, and informed agreement were obtained for all patients.

Patients orally were prescribed aspirin (325 mg per day), the first dose is administered at least two hours prior to conducting operational at least 300 seconds. In addition to the appointment of aspirin and heparin in patients blind selection is determined in one of three groups with alternative regimens: (1) bolus and placebo infusion placebo for 12 hours; (2) active c7E3 (from Centocor, Malvern, Pennsylvania) in the form of a bolus dose of 0.25 mg/kg and infusion of placebo for 12 hours; or (3) active c7E3 bolus with the same dose and immediate infusion of c7E3 with a dose of 10 mcg/min for 12 hours. The bolus is administered at least 10 minutes before performing surgery on the coronary vessels.

The main criterion for stopping the experiment is an integral and include the appearance within 30 days of the following events: death regardless of the cause, myocardial infarction, Obvodnogo coronary artery in acute ischemia, repeated percutaneous surgical intervention in acute ischemia, the need for the placement of endoluminal stent or the introduction vnutriaortalina balloon pump for the treatment of ischemia. All of these cases deal of the independent Advisory Council on the investigation of cases of termination of the experiment, which remains anonymous for conducting the treating physicians during the entire time of the study and requires an agreed re the, the principle of double anonymity is preserved. In addition to the appropriate ischemic complications, such as death or myocardial infarction, patients are monitored to determine the need for repeat revascularization, which consists in carrying out percutaneous reconstruction of the coronary artery or bypass grafting of the coronary artery, or both types of operations. In contrast to acute cases of termination tests, placement of the stent or intra-aortic balloon pump is not considered as an outcome, because the focus in the case of sudden ischemia facing surgery revascularization, not its substitute. Criteria for the diagnosis of myocardial infarction after discharge from the hospital is either a new appearance on the electrocardiogram significant Q-wave duration is not less than 0.04 seconds, or have depth component not less than one quarter of the amplitude of the corresponding R-wave in two or more contiguous leads of the electrocardiogram; or the increase in the concentration of creatine kinase or strips of creatine kinase in the myocardium, which is more than twice the upper normal limit. Data on repeated revascularization gather with informationlatina completed by 97.2%.

To separate the data of the six-month observations from the occurrence of the acute phase, the analysis includes all events that have passed from the base level up status after 6 months, events after followed within 30 days of stopping the experiment in patients with initially successful surgical intervention (determined by the value of the final narrowing of the vessel, which is consistent with clinical expert report, less than 50%, in the absence of ischemic complications), and events that occurred within 48 hours in patients with initially successful surgical intervention. If you stop the experiment for 30 days and then taken away, because they are observed in many cases of surgical intervention on the coronary vessels. 48-hour limit is set for the reason that, as is well known, practically all cases of sudden blockage that may occur after the surgical intervention on the coronary vessels that fall within this time interval (K. M. Detre et al., J. Am. Coll. Cardiol. , 13: 230A (1989); A. M. Lincoff et al., J. Am. Coll. Cardiol., 19: 926-938 (1992); P. J. de Feyter et al., Circulation, 83: 927-936 (1991)).

Blind sampling conducted by telephone in the Coordinating center Duke Univers is ARCT attack. Data in the form of individual reports about the conduct of the 6-month observations collected by the facilitators, and their quality before processing the data is evaluated and documented by anonymous experts. The customer is not aware of the test results until, until you have received information about all the patients, the events considered by the Committee on the cessation of research and data collection is finished.

Statistical analysis

Comparisons of all treatments are carried out according to the principle of purpose/treatment. The probability of occurrence of events assessed by the method of Kaplan-Meier (E. L. Kaplan and P. Meier, J. Am. Stat. Assn., 53: 457-481 (1958)), and survival curves are used for the graphical representation of test results. Test for dependent doses to the probability of occurrence of events from placebo to bolus and bolus plus infusion (with values 0, 1 and 2, respectively), make use of aggregate statistics score/ranking. Paired comparisons between placebo and one option for the purpose c7E3 also performed using statistical evaluation/ranking. Model proportional hazards (Cox) were selected so that they satisfy the possible Association between baseline characteristics and redeye treatment, and separately for the treatment group, in order to identify differences between individual treatment areas. Next, regression models, proportional hazards (Coxmust meet all composite instances of the completion of the research after 48 hours of observation, to study factors that may be related to subsequent events or influences treatment. Factors that are included in this analysis are the treatment, revascularization of one or more lesions, duration of surgery, myocardial infarction or unstable angina near baseline values or other criteria risk factors, gender, age > 65 years or < 65 years of age, weight and diabetes.

Results

Registration began December 1, 1991 and completed on 18 November 1992, with the participation of 2099 patients. Features of the study all patients above (see Example 6, table 10). Baseline characteristics of patients initially successful plastic surgery or atherectomy, and therefore can be registered for future clinical trials of restenosis in Table 16. Significant differences between patients who udaetsya.

In patients receiving bolus se or bolus with infusion, there has been a significant increase in complications associated with bleeding, mainly in the first 48 hours, and about two times more blood transfusions (placebo 7%, a single bolus of 13%, bolus and infusion - 15%, p < 0,01). 12-hour infusion is not completed for 48 patients (7,0%) who received placebo, for 85 patients (12.5%), and assigned a single bolus, and 107 patients (15.8 per cent) of the group receiving bolus and infusion.

When using c7E3 no significant increase in thrombocytopenia and not marked by the appearance of hypersensitivity or allergic reactions. Positive response antihimera.ru human antibodies occur in 5.2% of patients assigned to the bolus and 6.5% of patients who are prescribed bolus and infusion. The majority of patients with positive responses antihimera.ru human antibodies have a low titer. All 32 patients in whom there is a positive response antichimeric antibodies person in the group, which was appointed by the bolus has a titre of not more than 1: 1600. Thirty-four of the forty patients who received bolus and infusion, have noted the positive response of the RS and the injection and in one patient from group receiving bolus, values titers positive response antihimera.ru human antibodies are in the range from 1:6400 and 1:51200.

After 30 days there is a 35% reduction of major ischemic complications (death, myocardial infarction, urgent revascularization) for patients receiving c7E3 bolus and infusion (8,3%) compared with those patients who were exposed to placebo (12,8%, p= 0.009) (see Example 6, table 13). Data after 6 months are shown in Table 17, which shows the cases of death, myocardial infarction and the need for bypass grafting coronary artery or repeated plastic surgery on the coronary vessel to undergo revascularization operation of the vessel (a) for all registered patients (b) patients for whom surgery is done successfully, and (c) for those patients whose first operation was successful and there were no complications during the first 30 days. After 6 months, there's been a 23% reduction in ischemic complications and revascularization (27% vs. 35%, p = 0.0001; see Table 17 - All registered patients, a Composite outcome: Death, myocardial infarction, Obvodnogo bypass the initial effect is mainly due to less need for shunting or re-operations, because the number of revascularization subjected to the operation of vessels decreased by 26% for the appointment of c7E3 bolus and infusion (16,4%) versus treatment with placebo (22,3%; p = 0.007; see Table 17). The group who received only the bolus, shows an intermediate result, which is in accordance with the applied in this study, the criteria is slightly better than the result for placebo.

Separately analyze data obtained after 6 months for all patients that have not reported cases of termination of studies within the first 30 days. These results are shown in Table 18. These findings indicate a 21% reduction in the need for repeat surgery for revascularization by assigning c7E3 bolus and infusion during the course of the acute phase.

Data for all cases of complications (death, nonfatal myocardial infarction, or need for coronary revascularization) for all patients who participated in the experiment, shown also in Fig. 12. Data for patients who underwent a successful surgery and had complications within 30 days, also shown in Fig. 13.

Among patients with acute complication pouech subjects groups (82,0% placebo 79,7% bolus, 81.4 per cent of the bolus and infusion). Analyzing cases of complications after 48 hours in patients with initially successful operation, identify the vessel, which was subjected to repeated revascularization within the first 30 days. As is shown in Fig. 14, there is a slight difference between substrate ischemic complications between groups with c7E3 bolus and the group with bolus and infusion after the termination of observations within 30 days.

Instead of a composite outcome (death, myocardial infarction, and revascularization) and any revascularization, including vessels that have not previously been exposed to, it is useful to focus on revascularization only vessels target, previously subjected to the operation. For all patients within 6 months there was a significant decrease, amounting to 26%, revascularization vascular targets in the group treated with bolus and infusion, compared to other tested groups (see Fig. 15). It should be noted that the appointment of a single bolus ceteris paribus have negligible effects on the vessel revascularization target.

The analysis compares subgroups of patients whose underlying conditions established acute disease to who are experiencing persistent angina and defined angiographic morphologic complications (table 19). This comparison reveals a significant impact on the reduction of compound outcomes for both subgroups, however, decrease the need for repeat coronary intervention significantly only for patients with stable angina (table 19). This observation is consistent regardless of whether the results are compared with the baseline values, or after 48 hours for patients with successfully conducted operations.

Discussion of results

These results are based on large-scale randomized studies conducted in different centres, show declines in the number of clinical events and less restenosis for patients who have been subjected to operations in the coronary arteries, which by assigning bolus and infusion were conducted blockade of integrin IIb/IIIa platelet. The degree of a favorable outcome after 6 months is reflected in a General decline, amounting to 23%, ischemic complications, including death, nonfatal myocardial infarction, need for revascularization, and decreased by 26% re-vessel revascularization target. These results extend the boundaries of beneficial effects of c7E3 bolus and livaningo the need for re-operations on coronary vessels.

Fragment of a monoclonal Fab, which is used in this study has the potential ability to bind surface integrin IIb/IIIa platelet with minimal dissociation constant. Previous studies of patients undergoing plastic surgery with the use of c7E3, showed that after injection of the antibody-binding sites IIb/IIIa remain occupied for at least another within 36-48 hours, and the evidence for the suppression of platelet aggregation - at least within 72 hours (see Example 4; see also S. G. Ellis et al., Cor. Art. Dis., 4: 1675-175 (1993); J. E. Tcheng et al., Circulation, 88: (1993). Although both of these effects weaken and return to its original state, long-term effect of c7E3 on platelet aggregation, it is interesting because the bolus plus infusion of placebo does not exert clinically significant effects on acute complications or results after 6 months in accordance with the adopted in the present study, criteria and conditions of its carrying out. This observation suggests that the reduction of the probability of occurrence of acute ischemic complications or clinical restenosis by assigning antibodies anti-GPIIb/IIIa can the drugs) and more prolonged suppression of integrin GPIIb/IIIa may be accompanied by a further improvement in results.

It should also be noted that, according to reports, she are also capable of binding to the vitronectin receptor (R. O. Hynes., Cell 69: 11-25 (1992)), probably because this receptor contains a component GPIIb/IIIa. The specified integrin (vitronectin) may play a role in the modulation of stenosis or restenosis, so that the binding of anti-GPIIb/IIIa with the vitronectin receptor may contribute to the observed effect. Other inhibitors of receptor GPIIb/IIIa have a degree of specificity towards the target and homologous integrins (J. Sutton et al., Clinical Research AFCR, 41: 118A (1993)). To determine the clinical consequences of the interaction of these molecules should conduct comparative studies.

In this study, a systematic six-month repeated angiographic study, with the aim of quantifying the degree of narrowing of the tested groups was not conducted. However, repeated angiography was carried out in many studies stenosis (J. S. Forrester et al., J. Am. Coll. Cardiol., 17: 758-769 (1991); J. H. Ip et al., J. Am. Coll. Cardiol. , 17: 77B-8B (1991); W. Casscells, Circulation, 86: 723-729 (1993); E. J. Topol et al., N. Engl. J. Med., 329: 228-233 (1993); A. G. Adelman et al. , N. Engl. J. Med., 329: 228-233 (1993); P. W. Serruys et al., Circulation, 84: 1568-1580 (1991)), it has an important drawback, since the diagnosis of the vessel to the target,e must occur. In contrast, this study proposes the modeling of clinical practice, conducted on a large number of patients. The overall aim of the study restenosis is the demonstration of a significant reduction in the need for repeat surgery for revascularization because the benefits from angiography as such, which is celebrated in a number of several studies restenosis, do not always correspond directly to the clinical results. Further, as death and myocardial infarction are not commonplace after carrying out percutaneous reconstruction of the coronary arteries, the main result that can be modulated by applying an effective pharmacological intervention, is repeated revascularization of the vessel, which we observed in this study. Because the patients in the studies were subjected to similar effect, except that prescribed bolus and infusion, this was done by strict preservation of anonymity until completion of the experiment, we can conclude that reducing the likelihood of reduction of the vessel lumen to explain the observed clinically meaningful result.

This study is an Pervoye the subsequent operations of revascularization, which can be interpreted as less clinical stenosis. It achieves this through blockade of IIb/IIIa using bolus and infusion, and indicates the true passivation struck the walls of the vessel. Even in the case when the infusion se conduct during the entire 12 hours that the agent has continued antiplatelet effect in a few days, if this is not observed cases of acute ischemia. It is important to note that an independent beneficial effect on the vessel revascularization target after 6 months indicates residual effects acute pharmacological intervention using se and is clinical evidence of passivation of the walls of the blood vessel.

Detection of lower probability of occurrence of re-stenosis using blockade IIb/IIIa platelet emphasizes the role played by a bunch of platelet-thrombus upon the occurrence of restenosis, which is considered as a potentially major factor in the development of postoperative or endothelial lesions neointima (R. S. Schwartz et al., J. Am. Coil. Cardiol., 20: 1284-1293 (1992); E. J. Topol, Mayo Clin. Proc., 68; 88-90 (1993); J. T. Willerson et al. , Proc. Natl. Acad. Sci. USA, 88: 10624-10628 (1991)). Although the growth of cells medium smooth muscle may-8B (1991); W. Casscells, Circulation, 86: 723-729 (1993)), our results indicate that the potential antiplatelet and antithrombotic approaches to this important clinical phenomenon can be particularly fruitful. It should be noted that, as shown in the present study, the use of aspirin in the process of conducting operations on coronary artery (L. Schwartz et al. , N. Engl. J. Med., 318: 1714-1719 (1988) as the only antiplatelet funds is not sufficient to suppress the response of platelets to the injured vessel.

Example 8

Additional opening after conducting a randomized, double-anonymous studies using placebo

The size of the plastic catheter and associated with bleeding complications

Associated with bleeding complications judged by the criterion EPIC (see Examples 6 and 7), in order to determine the correlates of whether the size of the case during the PTCA/DCA with caused by bleeding complications. The size of the sheath and guide catheter to determine clinically. Explore further cases of heavy bleeding, bleeding in the groin, blood transfusion, recovery vessels, the lowest hematocrite number.

We can predict that the size of cehl the Isla method of treatment and use of heparin in the process of catheterization (p = 0,0004). The trend towards a greater number of recovery operations of vessels in the case of large size is not significant (p = 0,0004). Heavy bleeding (10.5%), blood transfusion (11,8%) and the lowest values hematocrite number (34) will not change with a change in the size of the case. Patients who received se Fab, the number of bleeding in the groin area exceeds the same indicator for patients who c7E3 not been assigned (55% vs. 30%, p < 0,0001) (see tab. A).

The results show that the large size of the case cause increased bleeding in the groin area, but do not lead to complications during PTCA/DCA. Adding c7E3 Fab is associated with increased bleeding in the groin area, but it can be minimized by using covers and catheters smaller.

The coincidence of cases of ischemia and bleeding after inhibition of receptor GPIIb/IIIa when conducting operations on coronary artery

Test EPIC (evaluation of the effect of c7E3 for the prevention of ischemic complications) (see Examples 6 and 7) shows that therapy with c7E3 Fab, a potential antagonist receptor GPIIb/IIIa platelet prevent ischemic complications in carrying out the claim, however, the number of cases of bleeding that requires blood transfusion, increasing from 7% for the group receiving placebo, to 14% in the group treated with c7E3 Fab. To further study this phenomenon conduct a comparison between the indices of bleeding (lowest value hematocrite number, the rate of bleeding, changes hematocrite number, the number of units of red blood cells transfusion of banked blood) and main stops in the experiment (death, myocardial infarction, Obvodnogo coronary artery or percutaneous crespella reconstruction of the coronary artery, with the aim of eliminating acute ischemia, or the placement of a coronary stent in case of unsuccessful operation). There is a strong relationship between bleeding and main stops in the experiment (p = 0.0001 for all indexes bleeding). This relationship is observed for all types of therapy: placebo bolus c7E3 Fab and the bolus plus infusion of c7E3 Fab. Thus, patients with significant bleeding is more likely prone to ischemic complications. This strong relationship may be related to the fact that when stopped the experiment (WinCE, bleeding caused by hypotension, can make an important contribution to ischemic complications after the surgery. In support of this, patients who develop hypotension (except hypotension, which develops after the occurrence of the event that caused a stop in the experiment) after successful percutaneous cresolate reconstruction of the coronary artery is more likely to experience complications, as well as more at risk of bleeding and the occurrence of the event, which entails a stop in the experiment (see tab. B).

Therefore, bleeding is likely leads to ischemic complications in some patients, and measures for the further reduction of bleeding (for example, modification of the dose of heparin) can further strengthen anti-ischemic efficacy of inhibition of GPIIb/IIIa operations on coronary vessels).

Economic advantages and disadvantages of inhibition of aggressive platelets in plastic operations in high risk

For 2100 patients when conducting a randomized test EPIC (see Examples 6 and 7) inhibition of aggressive platelet large doses is during percutaneous cresolate reconstruction of the coronary artery at high risk, however, this doubles the likelihood of heavy bleeding. To assess the economic consequences of the use of this combination of clinical effects conduct economic analysis. Within six months after registration, collect data on the costs of hospitalization and cost of raw materials for each participant in the experiment. The average base cost of hospitalization in the absence of complications are 9300 USD. The impact of major complications to the basic cost of hospitalization assessed using linear regression models with many variables:

The average cost of hospitalization = $9065 + $5923 emergency, RTS + $AlN CABG + $ony infarction + $AlN bleeding

This model shows that reducing the share of emergency percutaneous cresolate reconstruction of coronary arteries from 4.5% to 0.8%, the share of emergency bypass grafting coronary artery from 3.6% to 2.4%, the fraction of a second heart attack from 8.6% to 5.2%, therapy using the fragment of the chimeric 7E3 can save an average of $682 per patient compared with therapy, which is used as placebo. However, doubling the likelihood of si which leads to a total savings of $440 per person. The observed average difference between the use of high doses of c7E3 (X = $10970 + 7284) and placebo (X = $11376 + 12555) is $406, which is in good agreement with model calculations.

Thus, significantly reducing ischemia-induced complications when performing percutaneous cresolate reconstruction of coronary arteries in patients included in the high-risk group, the inhibition of aggressive platelets by using a fragment of the chimeric anti-GPIIb/IIIa brings not only medical results, but also leads to an overall cost reduction. Measures to reduce heavy bleeding in the appointment of c7E3 in addition to providing additional therapeutic benefits, can contribute to increased savings of up to $700 per person.

Time activated clotting increases when conducting operations on coronary artery in the presence of antagonist GPIIb/IIIa platelet

Time activated clotting use when performing percutaneous cresolate reconstruction of the coronary artery, with the aim of monitoring the degree of inhibition of thrombin and degree of anticoagulation and to minimize adverse thrombotic phenomena. Adding potential inhibitors of platelets, such centivany blood during percutaneous cresolate reconstruction of the coronary artery was not addressed previously. To date, the effect of c7E3 for the time activated clotting was not known. Explore the possible influence of the antagonist GPIIb/IIIa platelet amount of time activated clotting during surgery. In this study, 2099 patients who had to spend percutaneous crispolto reconstruction of the coronary arteries, are selected by a blind selection and they prescribe placebo (n = 696) or antagonist GPIIb/IIIa, c7E3Fab (n = 1403). Despite the fact that both groups receive the same amount of heparin, and some patients receive very high doses of heparin (14,000 units), compared with the group receiving placebo, patients groups, which designate c7E3 Fab, have adjusted weight is significantly higher values (p < 0,001) for time activated clotting (see tab. C).

Summing up, it should be noted that the time activated clotting of blood under the action of the antagonist c7E3 Fab against GPIIb/IIIa platelet count increases by 35-40 seconds. This is an important result for the choice of dose conjunctive heparin therapy and surgery on the coronary arteries using therapies directed against GPIIb/IIIa.

The influence of sex polerowanie plastic operations on vessels using monoclonal antibodies against the receptor GPIIb/IIIa platelet

It was studied the difference between sex of patients in research for the prevention of ischemic complications during plastic operations on vessels using c7E3 Fab monoclonal antibodies against the receptor GPIIb/IIIa platelet (see Examples 6 and 7). Patients receive percutaneous crispolto reconstruction of the coronary artery and appointed immediately before surgical intervention one of the following treatments using the blind method of dividing into groups: c7E3 bolus followed by infusion of c7E3 for 12 hours; only c7E3 bolus; only a placebo. Despite the fact that women were older, had less weight and they were more cardiovascular risk factors than men, there is no difference between men in terms of frequency of occurrence of a fatal outcome (2.2 percent vs. 1.3 percent), myocardial infarction (7.5 percent versus 6.3 percent), emergency percutaneous cresolate reconstruction of the coronary artery (2.6 percent versus 3.1%), emergency bypass grafting coronary artery (1.7 percent versus 3.2%), placement of the stent, intra-aortic balloon pump or from the point of view of the negative impact of these ischemic complications during the first 30 days (10.5 percent against sudiste system), and potential statistical relationships between the sexes did not change this result. The purpose of the bolus and infusion c7E3 Fab leads to the same decrease of the probability of occurrence of ischemic complications in both sexes.

Do women have more severe bleeding (12.6 percent vs. 9, 8%), and they had more frequent transfusion (PRBC) blood (19.5% vs. 9.0 percent), and they noted a higher rate of bleeding (hematocrite number/3 + units PRBC, against 2,4 1,9). When using a regression model to calculate the rate of bleeding, despite accounting for the known parameters (location of treatment, age, weight, baseline value hematocrite numbers, hypertension) sexual characteristics remained statistically an independent predictor (p = 0,0041).

In conclusion, it should be noted that when using c7E3 bleeding in women is observed more often than men, but no marked increase in cases of complications when performing plastic surgery on blood vessels for groups with a high risk factor.

Equivalents

For specialists in the art obvious or using standard techniques received many equivalents to the mother, these equivalents are covered by the following claims.

1. Method of inhibiting stenosis and/or restenosis, which is to assign to a patient suffering from a disease of the coronary arteries, an effective amount of a chimeric immunoglobulin or chimeric fragment of an antibody, having specificity for glycoprotein IIb/IIIa, and the specified immunoglobulin or fragment contains the region of binding antigen, not owned by the person and specific for the glycoprotein IIb/IIIa, and constant region of human immunoglobulin.

2. The method according to p. 1, characterized in that the immunoglobulin or the fragment of the immunoglobulin inhibits platelet aggregation.

3. The method according to p. 1, characterized in that the region of binding of an antigen derived from a monoclonal antibody 7E3.

4. The method according to p. 1, characterized in that the fragment of an antibody is a Fab fragment, Fab' or F(ab')2.

5. Method of inhibiting stenosis and/or restenosis, which is to assign to a patient suffering from a disease of the coronary arteries, an effective amount of a compound that selectively binds to the glycoprotein IIb/IIIa.

6. The method according to p. 5, the best of what efficinetly to the glycoprotein IIb/IIIa.

7. The method according to p. 6, characterized in that the immunoglobulin or immunoglobulin fragment inhibits platelet aggregation.

8. Method of inhibiting stenosis and/or restenosis after surgery on the coronary arteries in humans, which is to assign the specified person an effective amount of a chimeric immunoglobulin or chimeric fragment of an antibody, having specificity for glycoprotein IIb/IIIa, and the specified immunoglobulin or fragment contains the region of binding antigen, not owned by the person and specific for the glycoprotein IIb/IIIa, and constant region of human immunoglobulin.

9. The method according to p. 8, characterized in that the operation is a plastic operation on the vessel.

10. The method according to p. 8, characterized in that the immunoglobulin or immunoglobulin fragment inhibits platelet aggregation.

11. The method according to p. 8, characterized in that the region of binding of an antigen derived from a monoclonal antibody 7E3.

12. The method according to p. 8, wherein the fragment of the immunoglobulin selected from the group consisting of Fab, Fab' or F(ab')2.

13. Method of inhibiting stenosis and/or realook effective number of connections, which selectively binds to GPIIb/IIIa.

14. The method according to p. 13, characterized in that the compound is an immunoglobulin or a fragment of an antibody, having specificity for glycoprotein IIb/IIIa.

15. The method according to p. 14, characterized in that the immunoglobulin or the fragment of the immunoglobulin inhibits platelet aggregation.

16. The way to reduce the likelihood or prevent mild ischemic complications of angioplasty in humans, which is to assign the specified person an effective amount of a chimeric immunoglobulin or the fragment of the chimeric immunoglobulin having specificity for glycoprotein IIb/IIIa, and the specified immunoglobulin or fragment contains the region of binding antigen, not owned by the person that are specific to the glycoprotein IIb/IIIa, and constant region of human immunoglobulin.

17. The way to reduce the likelihood or prevent mild ischemic complications when performing plastic surgery on the blood vessels in humans, which is to assign the specified person an effective amount of a compound that selectively binds to the glycoprotein IIb/IIIa.

19. The method according to p. 18, characterized in that the immunoglobulin or the fragment of the immunoglobulin inhibits platelet aggregation.

20. The method according to p. 8, characterized in that the procedure is the placement of the stent.

21. The method according to p. 13, characterized in that the procedure is the placement of the stent.

22. The method according to p. 13, characterized in that said compound selectively binds to GPIIb/IIIa and the vitronectin receptor.

23. The method according to p. 22, characterized in that the procedure is angioplasty.

24. The method according to p. 22, characterized in that the procedure is the placement of the stent.

25. The method according to p. 22, characterized in that the compound is a chimeric immunoglobulin or the fragment of the chimeric immunoglobulin having specificity for glycoprotein IIb/IIIa-receptor for vitronectin and comprising a binding region of an antigen that is not owned by the person, and at least part of a constant region of human immunoglobulin.

26. The method according to p. 25, characterized in that the region of binding of an antigen derived from a monoclonal antibody 7E3.

2 the I fragments Fab, Fab' or F(ab')2.

28. Method of inhibiting stenosis and/or restenosis, which is to assign the patient with disease of the coronary arteries, an effective amount of Fab fragment of the chimeric immunoglobulin mouse-man, containing the variable regions of murine light and heavy chains mnogokanalnoj antibody 7E3.

29. The method according to p. 28, characterized in that the patient should receive heparin.

30. The method according to p. 29, wherein the heparin is administered in quantities depending on the weight of the patient.

31. Method of inhibiting stenosis and/or restenosis after surgery on the coronary arteries in humans, including the appointment of a person an effective amount of Fab fragment of the chimeric immunoglobulin mouse-man, containing the variable regions of murine light and heavy chains of the monoclonal antibody 7E3.

32. The method according to p. 31, wherein the patient is additionally given heparin.

33. The method according to p. 32, wherein the heparin is administered in quantities depending on the patient's weight.

 

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