WO2011013668A1 - Pharmaceutical composition for treatment of ischemic events - Google Patents

Abstract

Disclosed is a therapeutic pharmaceutical composition comprising a thrombolytic agent and an inhibitor capable of inhibiting a signaling that is mediated by a receptor of a vascular endothelial growth factor (VEGF). The therapeutic pharmaceutical composition can be used for the treatment of severe ischemic events including cerebral infarction, cardiac infarction and pulmonary embolism.

Description

Pharmaceutical composition for the treatment of ischemic events

The present invention relates to a pharmaceutical composition for the treatment of ischemic events, specifically comprising a thrombolytic agent and an inhibitor that inhibits signal transduction mediated by the receptor for vascular endothelial growth factor (VEGF), The present invention relates to a pharmaceutical composition for the treatment of severe ischemic events including cerebral infarction, myocardial infarction, and pulmonary embolism.

Serious ischemic events, including cerebral infarction, myocardial infarction, and pulmonary embolism, result from local blood flow blockage or ischemia. For example, in cerebral infarction, the central part of ischemia in the acute phase is irreversible and cell death occurs even when blood flow is resumed, but there is a reversible incomplete ischemic region around it, especially called penumbra . The central part of the ischemia expands unless treated, and the penumbra gradually disappears. As a result, the cerebral infarction portion is pathologically enlarged, clinically dysfunctional, and in the worst case, death occurs. The purpose of treatment in the acute phase of cerebral infarction is to restore blood flow in the penumbra. The recovery depends on the degree of ischemia and its duration. That is, how quickly the blood flow to the penumbra is resumed determines the early recovery of cerebral infarction.

Tissue-type plasminogen activator (hereinafter sometimes referred to as “t-PA”) is effective as a thrombolytic therapy that reopens the blood supply to the penumbra by dissolving the thrombus causing ischemia. So it is approved as a treatment for the acute phase of ischemic events.
However, administration of t-PA to patients after the acute phase of an ischemic event is not effective. For example, cerebral infarction results in cerebral hemorrhage complications and prognostic exacerbations. The administration of t-PA to patients after a period of time, ie, 3 hours or more after the onset of cerebral infarction is contraindicated. In addition, in myocardial infarction, administration of t-PA to patients who have passed 6 hours or more after the onset of myocardial infarction is contraindicated.
In this way, the risk of cerebral hemorrhage is particularly high in thrombolytic therapy used for treatment of ischemic stroke, and any thrombolytic therapy that reduces the severity of ischemic symptoms may increase the risk of cerebral hemorrhage. It has a problem in that it has (see Non-Patent Document 2).

Therefore, the rapid development of a therapeutic pharmaceutical composition that can be administered to patients after the acute phase of severe ischemic events including cerebral infarction, myocardial infarction, and pulmonary embolism without causing complications such as cerebral hemorrhage This is the current situation.

This invention makes it a subject to solve the said various problems in the past and to achieve the following objectives. That is, the present invention relates to a therapeutic pharmaceutical composition that can be administered to patients after acute passing of severe ischemic events including cerebral infarction, myocardial infarction, and pulmonary embolism without causing complications such as cerebral hemorrhage. The purpose is to provide.

In order to solve the above-mentioned problems, the present inventors have conducted intensive studies. When blood flow is resumed by administration of a thrombolytic drug, the expression of vascular endothelial growth factor (VEGF) increases, thereby activating the VEGF receptor signaling system and constructing the vascular wall It has been found that this is due to accelerated protein degradation.
Therefore, cerebral hemorrhage can be achieved by using the thrombolytic agent and an inhibitor that inhibits signal transduction mediated by the VEGF receptor, for example, an antibody against at least one of VEGF and VEGF receptor and other binding factors. It was found that the thrombolytic drug can be administered to patients who have caused severe ischemic events including cerebral infarction, myocardial infarction, and pulmonary embolism without causing complications of the present invention. .

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> A therapeutic pharmaceutical composition or a treatment method using the composition as one embodiment is provided for the treatment of severe ischemic events including cerebral infarction, myocardial infarction, and pulmonary embolism. The The composition comprises a thrombolytic agent and an inhibitor that inhibits signal transduction mediated by at least one receptor for vascular endothelial growth factor (VEGF). The inhibitor inhibits signal transduction mediated by the VEGF receptor, for example, by inhibiting the binding of VEGF to the receptor for vascular endothelial growth factor (VEGF).
<2> The composition or method according to <1>, wherein the inhibitor decreases binding of VEGF to the VEGF receptor.
<3> The composition or method according to <2>, wherein the inhibitor is a specific binding partner for at least one of VEGF and a VEGF receptor.
<4> The composition or method according to <3>, wherein the specific binding partner is at least one of the following (1) to (4). (1) antibody, (2) aptamer, (3) VEGF peptide or small molecule mimic of VEGF peptide that binds to VEGF receptor but does not activate VEGF receptor, (4) is used for stimulation of VEGF receptor VEGF receptor peptides or small molecule mimics of VEGF receptor peptides that reduce the effective level of VEGF.
<5> At least one of an antibody that binds to a VEGF receptor and antagonizes the binding of VEGF to the VEGF receptor, and an antibody that binds to the VEGF and causes the removal of the VEGF from the blood The composition or method according to <4>, wherein
<6> In one embodiment, the composition or method according to <1>, wherein the inhibitor inhibits the release of VEGF from platelets.
<7> The composition or method according to <6>, wherein the inhibitor reduces binding of ADP to adenosine diphosphate (ADP) receptor.
<8> The composition or method according to <7>, wherein the inhibitor is a specific binding partner for at least one of ADP and ADP receptor.
<9> The composition or method according to <8>, wherein the specific binding partner is at least one of the following (1) to (4). (1) an antibody, (2) an aptamer, (3) an ADP peptide or a small molecule mimic of an ADP peptide that binds to an ADP receptor but does not activate the ADP receptor, (4) an ADP receptor peptide that binds ADP, or It is a small molecule mimic of the ADP receptor peptide.
<10> In one embodiment, the inhibitor is at least an inhibitor that interacts with a component of the VEGF receptor signaling pathway, and an inhibitor that interacts with an enzyme that modifies a component of the VEGF receptor signaling pathway The composition or method according to <1>, which is any one of the above.
<11> The composition or method according to <10>, wherein the inhibitor is a tyrosine kinase inhibitor and an agonist of tyrosine phosphatase.
<12> In one embodiment, the inhibitor is the composition or method according to <1>, wherein the inhibitor decreases production of at least one of VEGF and a VEGF receptor.
<13> The composition or method according to any one of <10> and <12>, wherein the inhibitor is at least one of an antisense nucleic acid, a small interfering RNA (siRNA), and a ribozyme.
<14> In one embodiment, the composition or method according to <1>, wherein the inhibitor is combined with a thrombolytic drug.
<15> The composition or method according to <14>, wherein the inhibitor binds to the thrombolytic drug as a fusion protein.
<16> The composition or method according to <1>, wherein the thrombolytic drug is at least one of urokinase, streptokinase, tissue-type plasminogen activator (t-PA), and analogs thereof.
<17> The composition or method according to any one of <1> to <5>, wherein the VEGF receptor is VEGF receptor type 2 (VEGFR-2).
<18> The composition or method according to any one of <1> to <17>, wherein the composition or method is administered to a patient after an acute phase of an ischemic event including cerebral infarction, myocardial infarction, and pulmonary embolism.
<19> The composition or method according to <18>, wherein the acute phase of the ischemic event is 3 to 6 hours from the onset of the ischemic event.
<20> The composition or method according to <18>, wherein the acute phase of cerebral infarction is within 3 hours from the onset of the cerebral infarction.
<21> A polyclonal antibody or a monoclonal antibody, wherein the specific binding partner specifically binds to at least one of VEGF and the VEGF receptor, and inhibits the signal transduction of the VEGF, and an antigen of the antibody The composition or method according to any one of <2> to <5>, selected from the group consisting of a binding fragment, a recombinant antibody or chimeric antibody containing the antigen-binding fragment, and derivatives thereof. .
<22> In one embodiment, the specific binding partner binds to at least one of VEGF-A and VEGF-A receptor, and inhibits a signal transduction pathway mediated by VEGF-A receptor <3 > To <5>.
<23> In one embodiment, the specific binding partner is the composition or method according to the above <22>, wherein the specific binding partner is an anti-VEGF-A neutralizing antibody or a derivative thereof.
<24> A polyclonal antibody or a monoclonal antibody, wherein the specific binding partner specifically binds to at least one of ADP and the ADP receptor and inhibits the signal transduction of the ADP, and an antigen of the antibody The composition or method according to any one of <7> to <9>, wherein the composition or method is selected from the group consisting of a binding fragment, a recombinant antibody or chimeric antibody containing the antigen-binding fragment, and derivatives thereof. .
<25> From the above <10>, wherein the inhibitor is (E) -3- (3,5-Diisopropyl-4-hydroxyphenyl) -2-((3-phenyl-n-propyl) amino-carbonyl) acrylonitrile) <11> The composition or method according to any one of the above.
<26> A kit comprising a thrombolytic agent and an inhibitor that inhibits signal transduction mediated by a receptor for vascular endothelial growth factor (VEGF).

According to the present invention, it is possible to solve the above-described problems and achieve the above-mentioned object, and to prevent serious complications including cerebral infarction, myocardial infarction, and pulmonary embolism without causing complications such as cerebral hemorrhage. It is possible to provide a therapeutic pharmaceutical composition that can be administered to a patient after an acute phase of a bloody event.

FIG. 1A is a schematic diagram showing a procedure for producing a conventional rat cerebral infarction model. FIG. 1B is a schematic diagram showing a procedure for producing a rat cerebral infarction model in Example 1. FIG. 2A is a photograph of a coronal section of an animal 24 hours after the onset of cerebral infarction due to thrombus injection. FIG. 2B is a photograph of a coronal section of an animal to which t-PA was administered 1 hour after the onset of cerebral infarction due to thrombus injection. FIG. 2C is a photograph of a coronal section of an animal administered with t-PA 4 hours after the onset of cerebral infarction due to thrombus injection. FIG. 3 shows the results of Western blotting showing that the expression of VEGF is suppressed after the combined administration of t-PA and anti-VEGF antibody. FIG. 4A is a bar graph showing the volume of cerebral infarction of a TTC-stained coronal section 24 hours after the onset of rats administered with t-PA and an anti-VEGF antibody in combination 4 hours after the onset of cerebral infarction due to thrombus injection. Vertical axis: volume of cerebral infarction ^(mm 3). FIG. 4B is a bar graph showing the volume of edema of a TTC-stained coronal section 24 hours after the onset of rats administered with t-PA and an anti-VEGF antibody in combination 4 hours after the onset of cerebral infarction due to thrombus injection. Vertical axis: volume of edema (mm ³ ). FIG. 4C is a bar graph showing the amount of cerebral hemorrhage of a TTC-stained coronal section 24 hours after the onset of rats administered with t-PA and an anti-VEGF antibody in combination 4 hours after the onset of cerebral infarction due to thrombus injection. Vertical axis: cerebral hemorrhage (mg / dL). FIG. 4D is a band graph showing the motor function scale 24 hours after the onset of rats administered with t-PA and an anti-VEGF antibody in combination 4 hours after the onset of cerebral infarction due to thrombus injection. Vertical axis: Motor function scale. FIG. 5A is a bar graph showing the volume of cerebral infarction in a TTC-stained coronal section 24 hours after the onset of rats administered with t-PA and SU1498 in combination 4 hours after the onset of cerebral infarction due to thrombus injection. Vertical axis: cerebral infarction volume (mm ³ ). FIG. 5B is a bar graph showing the volume of edema of TTC-stained coronal sections 24 hours after the onset of rats administered with t-PA and SU1498 in combination 4 hours after the onset of cerebral infarction due to thrombus injection. Vertical axis: volume of edema (mm ³ ). FIG. 5C is a bar graph showing the amount of cerebral hemorrhage of a TTC-stained coronal section 24 hours after the onset of rats administered with t-PA and SU1498 in combination 4 hours after the onset of cerebral infarction due to thrombus injection. Vertical axis: cerebral hemorrhage (mg / dL). FIG. 5D is a band graph showing a motor function scale 24 hours after the onset of rats administered with t-PA and SU1498 in combination 4 hours after the onset of cerebral infarction due to thrombus injection. Vertical axis: Motor function scale.

(Therapeutic pharmaceutical composition)
The therapeutic pharmaceutical composition of the present invention comprises at least a thrombolytic agent and an inhibitor that inhibits signal transduction mediated by a receptor for vascular endothelial growth factor (VEGF), and if necessary, other Contains the ingredients.

<Thrombolytic drug>
Examples of the thrombolytic agents include serious ischemic events including cerebral infarction, myocardial infarction, and pulmonary embolism (“severe ischemic events”, “ischemic disease”, “ischemia”, “ischemic attack”, etc. Can be appropriately selected according to the purpose, for example, tissue type plasminogen activator (t-PA) or Derivatives thereof, urokinase (see Murray V. et al., J Inter Med. 2010, Feb; 267 (2): 191-208), streptokinase, single-chain urokinase-type plasminogen activator (u-PA), desmoteplase And other proteases that act on fibrin. Other factors known to be able to cleave fibrin are also included in the present invention. These may be used alone or in combination of two or more.
Among these, the thrombolytic drug preferably contains urokinase, tissue-type plasminogen activator (t-PA), and derivatives or analogs thereof from the viewpoint of increasing the success rate of thrombolysis.
There is no restriction | limiting in particular as a manufacturing method of the said thrombolytic agent, According to the kind etc. of the said thrombolytic agent, it can select suitably, For example, a gene recombination method, a synthesis method, etc. are mentioned. Moreover, you may use a commercial item.
The t-PA derivative is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the t-PA includes sugar chains, oligonucleotides, polynucleotides, polyethylene glycol, and other pharmaceutically acceptable products. And the like which are combined with additives and treatment agents. In addition, one or several amino acids may be substituted in the amino acid sequence of t-PA.
Specific examples of the t-PA derivative include a t-PA derivative in which a part of amino acids are substituted in the amino acid sequence of the t-PA such as monteplase, pamitepase, and reteplase; and the t-PA such as tenecteplase and lanoteplase. And a t-PA derivative in which a part of the amino acid is substituted and the sugar chain is further modified.

The content of the serum dissolving drug in the therapeutic pharmaceutical composition is not particularly limited and can be appropriately selected depending on the type of the serum dissolving drug.

The “acute phase” in the present invention is an early stage of development of a serious ischemic event including cerebral infarction, myocardial infarction, and pulmonary embolism. For example, in cerebral infarction, cranial nerve function associated with a decrease in cerebral blood flow Although the disorder is recognized, it refers to a time when recovery is possible only by rapid resumption of blood flow by the thrombolytic drug. Here, the acute phase generally refers to 3 to 6 hours from the onset of infarction, but in cerebral infarction, it is preferably within 3 hours from the onset.

“Patient” in the present invention includes humans but is not limited to humans.

<Inhibitor that inhibits signal transduction mediated by vascular endothelial growth factor (VEGF) receptor>
The inhibitor that inhibits signal transduction mediated by the VEGF receptor (hereinafter sometimes simply referred to as “inhibitor”) is not particularly limited and may be appropriately selected depending on the intended purpose. Inhibitors that reduce the binding of VEGF to VEGF receptors, inhibitors that inhibit the release of VEGF from platelets, inhibitors that interact with components of the VEGF receptor signaling pathway, components of the VEGF receptor signaling pathway An inhibitor that interacts with an enzyme that modifies VEGF, an inhibitor that reduces the production of at least one of VEGF and VEGF receptors, and the like.

<< Inhibitors that reduce VEGF binding to VEGF receptors >>
The inhibitor that decreases the binding of VEGF to the VEGF receptor is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a specific binding partner (for example, a specific binding partner for VEGF and / or VEGF receptor ( And the like, which are sometimes referred to as “binding inhibitors” that inhibit the binding between VEGF and the VEGF receptor.

-Specific binding partner-
The specific binding partner (binding inhibitor) is not particularly limited as long as it can inhibit the binding between the VEGF and the VEGF receptor, and can be appropriately selected according to the purpose. It is preferably one that specifically binds to at least one of the VEGF receptors. Thereby, the signal transduction mediated by the VEGF receptor can be inhibited.
Examples of the specific binding partner include a receptor or a ligand that specifically binds to at least one of the VEGF and the VEGF receptor.
The receptor or ligand is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include proteins such as antibodies, carbohydrates, nucleic acids, lipids, and other biopolymers.

“Antibody” in the present invention refers to an antibody that is generally defined, and includes a Fab fragment, a single chain Fv structure, a bispecific antibody in which two different Fab fragments are bound to one Fc, and a similar structure. (See Bauerle PA. Et al., Cancer Res. 2009, Jun 15; 69 (12): 4941-4). In order for an antibody to be useful, it is necessary to minimize the antigenicity of the antibody in humans, and thus the antibody is human in sequence (transgenic animals expressing the human antibody repertoire (Jakobovits A. et al., Nat Biotechnol. 2007, Oct; 25 (10): 1134-43) or a human antibody gene recombination library (Benhar I. et al., Expert Opin Biol Ther. 2007, May; 7 ( 5): 763-79)), or humanized (see Almagro JC. Et al., Front Biosci. 2008, Jan 1: 13: 1619-33) or isolated from humans Too (.. Collarini EJ et al, J Immunol 2009, Nov 15; 183 (10):. 6338-45 reference) may be. Similarly, camels (Deschach N. et al., J Immunol. 2010, May 15; 184 (10): 5696-704) and sharks (Weslowski J. et al., Med Microbiol Immunol. 2009, Aug; 3): low molecular weight antibodies (nanobodies) can also be used, such as the antibodies discovered in 157-74) (natural variants).

Mimics of antibodies can also be used and include, for example, protein families based on fibronectin, transferrin, glutathione transferase, lens etc. (Wurch T. et al., Curr Pharm Biotechnol. 2008). Dec; 9 (6): 502-9). Other mimetics include non-peptide binding factors such as aptamers consisting of nucleic acids (see Guo KT. Et al., Int J Mol Sci. 2008, Apr; 9 (4): 668-78). Similarly, small molecule peptides (see Holtzman JH. Et al., Biochemistry. 2007, Nov 27; 46 (47): 13541-53), peptide mimetics (eg, beta amino acids (Petersson EJ. Et al., J Am) Chem Soc. 2008, Jan 23; 130 (3): 821-3), or D-amino acid (Van Regennortel MH. Et al., Curr Opin Biotechnol. 1998, Aug; 9 (4): 377-82). Or structural stability Sutame chemical crosslinking agent (Kutchukian PS et al, J Am Chem Soc 2009, Apr 8; 131 (13):... 4622-7 reference), etc.) and the like.
That is, “antibody mimic” refers to all binding agents having the same function as the antibody. In the present invention, such an antibody mimic can be used instead of an antibody.
For convenience, in the disclosed invention, the inventors focused on antibodies in the embodiments as excellent examples of such binding factors, but the present invention is not limited thereto.

--Specific binding partner that specifically binds to VEGF--
The VEGF is a group of glycoproteins involved in angiogenesis and angiogenesis. When the VEGF binds to a VEGF receptor present on the surface of vascular endothelial cells as a ligand, the VEGF signaling system is activated. In severe ischemic events including cerebral infarction, myocardial infarction, and pulmonary embolism, the activation of the VEGF signal transduction system promotes the degradation of proteins that make up the vascular wall. For example, in cerebral infarction It was confirmed in the present invention that a complication of cerebral hemorrhage occurs.
Examples of the VEGF family include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF) -1, and PIGF-2. Each member of the VEGF family has several additional subtypes. For example, human VEGF-A has 121 amino acids (VEGF-A ₁₂₁ ), 165 amino acids (VEGF-A ₁₆₅ ), 189 (VEGF-A ₁₈₉ ), 206 (VEGF-A ₂₀₆ ), 145 (VEGF-A ₁₄₅ ), 183 (VEGF-A ₁₈₃ ) and the like are known. Further, human VEGF-B is known to have 167 amino acids (VEGF-B ₁₆₇ ), 186 amino acids (VEGF-B ₁₈₆ ), and the like.
The specific binding partner that specifically binds to VEGF may bind to any of the VEGF families.

The specific binding partner that specifically binds to VEGF is not particularly limited and may be appropriately selected depending on the purpose. For example, a polyclonal antibody or a monoclonal antibody that recognizes VEGF, an antigen-binding fragment of the antibody , A chimeric antibody or a recombinant antibody (hereinafter sometimes referred to as “anti-VEGF antibody” or the like) containing the antigen-binding fragment, or a derivative thereof, a part of a recombinant VEGF receptor (Chu QS. Expert) Opin Biol Ther. 2009, Feb; 9 (2): 263-71), a VEGF variant that competitively binds to VEGF receptor for VEGF but does not activate the VEGF receptor (Siemeister G. et al. , Proc Natl Acad Sci US A.19 8, Apr 14; 95 (8): see 4625-9), and the like. Among these, the specific binding partner that specifically binds to VEGF is preferably a monoclonal antibody, and the anti-VEGF-A neutralizing antibody is a VEGF receptor of VEGF-A that is involved in vascular disruption during angiogenesis. It is more preferable in that the binding to can be efficiently inhibited.

The method for producing a specific binding partner that specifically binds to VEGF is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a gene recombination method and a synthesis method. Moreover, you may use a commercial item.
In addition, the specific binding partner that specifically binds to VEGF may be at least one of the anti-VEGF antibody and the like and derivatives thereof, such as polyethylene glycol, other pharmaceutically acceptable additives, Other components such as a treatment agent may be combined or added. The content of other components in the specific binding partner that specifically binds to VEGF is not particularly limited and may be appropriately selected depending on the purpose.

--- Polyclonal antibody ---
The polyclonal antibody is injected into any animal host of mammals (eg, mouse, rat, rabbit, sheep or goat) or birds (eg, chicken) using the VEGF or a fragment thereof as an immunogen. When a VEGF fragment is used as an immunogen, an excellent immune response may be induced when linked to a carrier protein such as bovine serum albumin or keyhole limpet hemocyanine.
The immunogen is preferably injected into the animal host according to a predetermined schedule incorporating one or more booster immunizations.
The immunogen may be injected into the animal host in a mixture with complete or incomplete Freund's adjuvant or other immunopotentiators.
The polyclonal antibody is purified from the antiserum by, for example, affinity chromatography using VEGF bound to an appropriate solid support or a fragment thereof, and binding between VEGF and the VEGF receptor is inhibited, In some cases, it has been confirmed that inhibition of binding can inhibit VEGF signaling.
Examples of the polyclonal antibody include rabbit anti-rat VEGF antibody IgG (RB-222, 19 kDa to 22 kDa) prepared using human recombinant VEGF ₁₆₅ as an immunogen. The RB-222 can recognize VEGF165 and VEGF121.

--- Monoclonal antibody ---
The monoclonal antibodies may be prepared using the technique of Kohler and Milstein (Eur. J. Immunol. 6: 511-519 (1976)) and improved techniques thereof. These methods involve the preparation of immortal cell lines that can produce antibodies with the desired specificity.
The immortal cell line may be prepared from spleen cells derived from an animal host immunized by the same method as the method for producing the polyclonal antibody. The spleen cells are immortalized by various methods to prepare an immortalized cell line capable of producing an antibody.
The spleen cells are immortalized by, for example, fusion with myeloma cells derived from the same or different species of the immunized animal. Various fusion techniques known to those skilled in the art may be used.

For example, the spleen cells and myeloma cells are mixed with a nonionic surfactant for several minutes and then plated at a low concentration in a selective medium that supports the growth of hybrid cells but does not support the growth of myeloma cells. A preferred selection technique uses HAT (hypoxanthine, aminopterin, thymidine) selection. Hybrid colonies are usually observed after a sufficient time of about 1 to 2 weeks. A single colony is selected, and the single colony is cultured in a medium such as HAT (hypoxanthine, aminopterin, thymidine-added medium), and the culture supernatant is tested for binding activity to the VEGF and these fragments. Further, the inhibition of the binding between the VEGF and the VEGF receptor and the activity of inhibiting the VEGF signal transduction due to the inhibition of the binding are also tested. Hybridomas with high reactivity and specificity are preferred. By repeating the cloning by the limiting dilution method, a hybridoma clone that stably produces a large amount of highly reactive and specific antibody is selected. Monoclonal antibodies may be isolated from the supernatants of colonies of cell lines derived from selected growing hybridoma clones.
In addition, various techniques may be used to improve yield, such as injecting the hybridoma cell line into the peritoneal cavity of a suitable vertebrate host such as a mouse.
The monoclonal antibody may be recovered from the hybridoma cell ascites or blood. Contaminants such as impure proteins from cell debris may be removed from the antibody by conventional techniques such as chromatography, gel filtration, precipitation and extraction.

The monoclonal antibody is, for example, bevacizumab obtained by genetically recombining the mouse monoclonal antibody against VEGF, or a Fab fragment of bevacizumab, and genetic modification is performed so that the binding to VEGF is further strengthened. And anti-VEGF-A neutralizing antibody of a monoclonal antibody preparation such as Ranibizumab. The monoclonal antibody preparation has already been clinically applied to malignant tumors and has been confirmed to be safe for humans.

--- Antigen-binding fragment ---
The antigen-binding fragment of the antibody refers to the part of the antibody that participates in antigen binding. The antigen binding site is formed by amino acid residues in the variable (V) region at the N-terminus of the heavy (H) chain and light (L) chain.
The antigen-binding fragment of the antibody includes, in addition to the Fab fragment or F (ab ′) 2 fragment obtained by degrading an intact polyclonal antibody or monoclonal antibody with the proteolytic enzyme papain or pepsin, respectively, Includes Fv fragments containing non-covalent VH and VL region heterodimers containing antigen binding sites that retain much of the binding capacity.

--- Recombinant antibody ---
The recombinant antibody may be prepared by expression cloning of an antibody gene including transformation into a suitable bacterial host, transfection into a suitable mammalian cell host, and the like.
The recombinant antibody can be prepared in large quantities using, for example, gene expression systems derived from prokaryotes and eukaryotes.

--- Chimeric antibody ---
The chimeric antibody is a fusion protein supported by a constant domain of a homologous or heterologous antibody so that the antigen-binding site of the recombinant antibody can specifically bind to VEGF.
The chimeric antibody includes a short chain variable region antibody (scFv) comprising an antibody heavy chain variable region (VH) operably linked to an antibody light chain variable region (VL), camelidae (Camelidae, camel, dromedary, A camel heavy chain antibody (HCAb) or its heavy chain variable region domain (VHH), which is a class of IgG without light chain produced by animals (including llamas).

--- Derivatives ---
The derivative of the anti-VEGF antibody or the like having binding inhibitory activity between the VEGF and the VEGF receptor is not particularly limited and may be appropriately selected depending on the purpose. For example, the anti-VEGF antibody or Examples include chains, oligonucleotides, polynucleotides, polyethylene glycols, and other pharmaceutically acceptable additives and treatment agents bound thereto.

Specific examples of the derivative such as the anti-VEGF antibody include RNA aptamer pegaptanib that binds to the exon 7 portion of the VEGF gene and inhibits the production of VEGF.

In addition, sugar chains, oligonucleotides, polynucleotides, polyethylene glycol, and other pharmaceutically acceptable additives and treatment agents may be added to the anti-VEGF antibody and the like.
These sugar chains, oligonucleotides, polynucleotides, polyethylene glycol, additives and treatment agents are not particularly limited and can be appropriately selected depending on the purpose.

--Specific binding partner that specifically binds to VEGF receptor--
The VEGF receptor (VEGFR) is a kind of receptor tyrosine kinase, and is involved in expression of actions such as promotion of proliferation and migration of vascular endothelial cells by the ligand VEGF.
The VEGF receptor includes VEGFR-1 (sometimes referred to as Flt-1), VEGFR-2 (sometimes referred to as KDR and Flk-1), and VEGFR-3 (sometimes referred to as Flt-4). ), Soluble VEGFR-1, soluble VEGFR-2, soluble VEGFR-3, and the like are known. The VEGF family binds to a specific receptor, VEGF-A binds to VEGFR-1 and VEGFR-2, VEGF-B, PlGF-1, and PlGF-2 bind to VEGFR1, VEGF-C and VEGF-D Binds to VEGFR-2 and VEGFR-3, and VEGF-E binds to VEGFR2.
The specific binding partner that specifically binds to the VEGF receptor may bind to any of the VEGF receptors.

The specific binding partner that specifically binds to the VEGF receptor is not particularly limited and may be appropriately selected depending on the purpose. For example, the VEGF analog, the VEGF antagonist, and the VEGF receptor A polyclonal or monoclonal antibody that recognizes, an aptamer, an antibody that antagonizes the binding of VEGF to the VEGF receptor, an antibody that binds to the VEGF and causes the removal of the VEGF from the blood, an antigen-binding fragment of these antibodies, A chimeric antibody or a recombinant antibody comprising the antigen-binding fragment (hereinafter sometimes referred to as “anti-VEGFR antibody etc.”), derivatives thereof, VEGF receptor that binds to VEGF receptor but does not activate VEGF receptor, Small molecule mimics of peptides or VEGF peptides Reducing the effective level of VEGF to be used for stimulation of VEGF receptor, VEGF receptor peptide or VEGF receptor small molecule mimetic peptides (small molecule mimic), may be selected from the group consisting of. Among these, the specific binding partner that specifically binds to the VEGF receptor is preferably a monoclonal antibody, more preferably an anti-VEGFR-1 neutralizing antibody or an anti-VEGFR-2 antibody.
Here, the “small molecule mimic of VEGF peptide” means a molecule that is smaller than a peptide consisting of the complete form of VEGF and that has the same function as the VEGF peptide.
The “small mimic of VEGF receptor peptide” means a molecule that is smaller than a peptide consisting of the complete form of VEGF receptor and that has the same function as the VEGF receptor peptide.

There is no restriction | limiting in particular as a manufacturing method of the specific binding partner couple | bonded specifically with the said VEGF receptor, According to the objective, it can select suitably, For example, a gene recombination method, a synthesis method, etc. are mentioned. Moreover, you may use a commercial item.
The specific binding partner that specifically binds to the VEGF receptor may be at least one of the anti-VEGFR antibody and the like, or a derivative thereof, such as polyethylene glycol and other pharmaceutically acceptable additions. Other components such as agents and treatment agents may be combined or added. The content of other components in the specific binding partner that specifically binds to the VEGF receptor is not particularly limited and can be appropriately selected depending on the purpose.

--- Polyclonal antibody, monoclonal antibody, antigen-binding fragment ---
The polyclonal antibody, monoclonal antibody, and antigen-binding fragment can be produced in the same manner as the polyclonal antibody, monoclonal antibody, and antigen-binding fragment that recognize VEGF using the VEGF receptor or these fragments as an immunogen. it can.

--- Recombinant antibody ---
The recombinant antibody can be produced in the same manner as the recombinant antibody recognizing VEGF.

--- Chimeric antibody ---
The chimeric antibody recognizes the VEGF except that it is a fusion protein supported by the constant domain of the same or different antibody so that the antigen binding site of the recombinant antibody can specifically bind to the VEGF receptor. Examples thereof include antibodies similar to the chimeric antibody.

--- Derivatives ---
The derivative of the anti-VEGFR antibody or the like having binding inhibitory activity between the VEGF and the VEGF receptor is not particularly limited and may be appropriately selected depending on the purpose. For example, the anti-VEGF antibody or the like Examples include chains, oligonucleotides, polynucleotides, polyethylene glycols, and other pharmaceutically acceptable additives and treatment agents bound thereto.

In addition, sugar chains, oligonucleotides, polynucleotides, polyethylene glycol, and other pharmaceutically acceptable additives and treatment agents may be added to the anti-VEGF antibody and the like.
Examples of the additive, treating agent, sugar chain, oligonucleotide, polynucleotide, polyethylene glycol, and the like are the same as those of the anti-VEGF antibody.
These sugar chains, oligonucleotides, polynucleotides, polyethylene glycol, additives and treatment agents are not particularly limited and can be appropriately selected depending on the purpose.

<< Inhibitor that Inhibits VEGF Release from Platelets >>
The inhibitor that inhibits the release of VEGF from the platelet is not particularly limited and may be appropriately selected depending on the purpose. For example, an adenosine diphosphate (ADP) receptor (Bambace NM) present on platelets. Et al., Platelets. 2010, 21 (2): 85-93), and the like.
The inhibitor for reducing the binding of ADP to the ADP receptor is not particularly limited and may be appropriately selected depending on the purpose. For example, a specific binding partner (for at least one of ADP and ADP receptor ( And the like, which are sometimes referred to as “binding inhibitors” that inhibit the binding between ADP and the ADP receptor).

The specific binding partner is not particularly limited as long as it specifically binds to at least one of the ADP and the ADP receptor and can inhibit the binding between the ADP and the ADP receptor. For example, ADP receptor competitive inhibitor, polyclonal or monoclonal antibody that recognizes ADP or ADP receptor, aptamer, binds to ADP receptor but does not activate ADP receptor ADP peptide or ADP peptide small molecule mimic, ADP binding, ADP-binding ADP receptor peptide or ADP receptor peptide small molecule mimic, and the like.
Here, the “small molecule mimic of ADP peptide” means a molecule that is smaller than a peptide consisting of the complete form of ADP and that has the same function as the ADP peptide.
In addition, the “small molecule mimic of the ADP receptor peptide” means a molecule that is smaller than a peptide consisting of the complete form of the ADP receptor and that has the same function as the ADP receptor peptide.

<< Inhibitor that interacts with components of VEGF receptor signaling pathway, Inhibitor that interacts with enzymes that modify components of VEGF receptor signaling pathway >>
At least one of an inhibitor that interacts with a component of the VEGF receptor signaling pathway and an inhibitor that interacts with an enzyme that modifies a component of the VEGF receptor signaling pathway (hereinafter referred to as “VEGF receptor signaling inhibition”). May be referred to as “agents.”) As bleeding caused by administering t-PA to patients after acute phase of severe ischemic events including cerebral infarction, myocardial infarction, and pulmonary embolism Any VEGF receptor-mediated signal transduction inhibitor may be used provided that it can be inhibited.
The component of the VEGF receptor signal transduction pathway is not particularly limited and can be appropriately selected according to the purpose. For example, phospholipase (PLCγ), protein kinase C (PKC), Raf, map kinase kinase (MEK) ), Extracellular signal-regulated kinase (ERK), PI3 kinase (PI3K), pyruvate dehydrogenase kinase (PDK1), Akt and the like.
The enzyme that modifies the components of the VEGF receptor signaling pathway is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include PLCγ inhibitory enzyme, PKC inhibitory enzyme, Raf inhibitory enzyme, MEK inhibitory enzyme, ERK inhibitory enzyme, PI3K inhibitory enzyme, PDK1 inhibitory enzyme, Akt inhibitory enzyme and the like can be mentioned.
In addition, the VEGF receptor signaling inhibitor is capable of functioning other biomolecules, such as receptor kinases, provided that the side effects are in an acceptable range for treatment of patients who have caused ischemic events. Examples include those that inhibit other enzyme activities.

Specific examples of the VEGF receptor signaling inhibitor include SU1498 ((E) -3- (3,5-Diisopropyl-4-hydroxyphenyl) -2-((3-phenyl-n-propyl) amino- carbonyl) acrylonitrile), SU5614 (5-Chloro-3-((3,5-dimethylpyrrol-2-yl) methylene) -2-indolinone), SU11248 (N- (2- (diethylamino) ethyl) -5-(( Z)-(5-fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidine) methyl) -2,4-dimethyl-1H-pyrrole-3-carbamide), A D2171 (4-((4-Fluoro-2-methyl-1H-indol-5-yl) oxy) -6-methyl-7- (3- (pyrrolidin-1-yl) propoxy) quinazoline), PTK787 / ZK222584 ( N- (4-Chlorophenyl) -4- (pyridin-4-ylmethyl) phthalazin-1-amine succinate), sorafenib (4- (4-((4-chloro-3-methyl) phenyl) carbamoylamine) N-methyl-pyridine-2-carboxamide), GW78660B (5- (4-((2,3-dimethyl-2H-inda ol-6-yl) methylamino) -2-pyrimidinyl) amino) -2-methyl-monohydrochloride), CBO-P11 (cyclic (DF-PQIMRIPHPHQGQHIGE); Cyclo-VEGI-Pro-P93-D-P93 , A cyclic peptide having the 79th to 93rd amino acid sequence of VEGF-A), Je-11 ((RTELNVGIDFNWEYPAS _{) 2} K-NH ₂ , an immunoglobulin-like equivalent to the 247th to 261st amino acid sequence of VEGFR-2 Domain-derived peptide dimer), V1 (ATWLPPR, peptide of 8 amino acids binding to VEGFR-2), VEGFR-2 kinase inhibitor I ((Z) -3-((2,4-Dimethyl) 3- (ethoxycarbonyl) pyrol-5-yl) methylidyl) indolin-2-one), VEGFR-2 kinase inhibitor II ((Z) -5-Bromo-3- (4,5,6,7-tetrahydro-1H-) indol-2-ylmethylene) -1,3-dihydroindol-2-one), VEGFR-2 kinase inhibitor III or SU5416 (3-((2,4-Dimethylpyrrole-5-yl) methyllidene) -indolin-2-one) VEGFR-2 kinase inhibitor IV (3- (3-Thienyl) -6- (4-methoxyphenyl) pyrazolo (1,5-a) pyrimidine), VEGFR-2 3 tyrosine kinase inhibitor (3- (Indole-3-yl ) -4- (3,4,5-trimethoxyphenyl) -1H-pyrrole-2,5-dione), GW654652 (N 2 - (5- (ethylsulphonyl) - 2-methoxyphenyl) -N4-methyl-N4- (3-methyl-1H-indazol-6-yl) pyrimidine-2,4-diaminine). An agonist of tyrosine phosphatase can also reduce signal transduction via VEGF receptor tyrosine kinase (see Xu D. et al., Front Biosci. 2008, May 1; 13: 4925-32). These may be used alone or in combination of two or more.

Among these, the VEGF receptor signaling inhibitors include SU1498, SU5416, SU11248, AZD2171, PTK787 / ZK222584, sorafenib, GW78660B, and other VEGFR-2 kinase inhibitor preparations that are involved in angiogenesis. This is preferable in that it does not affect VEGFR-1-positive cells known to do.
Specific examples of the VEGFR-2 kinase inhibitor preparation include cediranib (AZD2171), sunitinib (SU11248), varatinib (PTK787 / ZK222584), sorafenib (pazobib), pazobib Is mentioned.

<< Inhibitor that reduces production of at least one of VEGF and VEGF receptor >>
There is no restriction | limiting in particular as an inhibitor which reduces the production of at least any one of the said VEGF and a VEGF receptor, According to the objective, it can select suitably, For example, antisense nucleic acid, small molecule interference RNA (siRNA) ( Morris KV.Oligoncleotides.2009, Dec; 19 (4): 299-306), ribozyme (Franzen S. et al., Curr Mol Mol Ther. 2010, Apr; 12 (2): 223-32) and the like. Can be mentioned. These may be used alone or in combination of two or more.
These inhibitors can reduce the production of at least one of the VEGF and VEGF receptors by inhibiting the transcription or translation of VEGF.

In addition, as described above, the therapeutic pharmaceutical composition may use the thrombolytic agent and an inhibitor that inhibits signal transduction mediated by the VEGF receptor, It may be a single factor having both a thrombolytic effect and an inhibitory effect on signal transduction mediated by the VEGF receptor.
Such factors include, for example, the Fab as the thrombolytic drug (Siller-Matula JM et al., Br J Pharmacol, 2010 Feb 1; 159 (3), 502-17, Epub 2009 Dec 24), Bispecific antibodies (fusion proteins) with Fab as an inhibitor can be made. According to this bispecific antibody, Fab as the thrombolytic agent acts on von Willebrand factor (vWF), and Fab as the inhibitor acts on VEGF or VEGF receptor. The two functions can be performed simultaneously by one factor. Further, the thrombolytic drug and the inhibitor may be a directly bound fusion protein (Baeuler PA et al., Curr Opin Mol There, 2009, Feb; 11 (1): 22-30).
The von Willebrand factor is a high-molecular glycoprotein that is produced in vascular endothelial cells and bone marrow megakaryocytes and is present in plasma, intravascular subcutaneous tissue and platelets.

The content of the inhibitor in the therapeutic pharmaceutical composition is not particularly limited and may be appropriately selected depending on the type of the inhibitor.

<Other ingredients>
There is no restriction | limiting in particular as said other component, According to an administration method, a dosage form, etc., it can select suitably from the carriers accept | permitted pharmacologically.
For example, when the therapeutic pharmaceutical composition is used as an oral solid preparation, excipients such as lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid; water, ethanol, propanol , Simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone, etc .; dry starch, sodium alginate, agar powder, hydrogen carbonate Disintegrating agents such as sodium, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose; lubricants such as purified talc, stearate, borax, polyethylene glycol; Coloring agents such as iron oxide; sucrose, orange peel, citric acid, and the like flavoring / flavoring of tartaric acid.
For example, when the therapeutic pharmaceutical composition is used as an oral solution, a flavoring / flavoring agent such as sucrose, orange peel, citric acid or tartaric acid; a buffering agent such as sodium citrate; a stable agent such as tragacanth, gum arabic and gelatin And the like.
For example, when the therapeutic pharmaceutical composition is used as an injection, pH adjusting agents and buffers such as sodium citrate, sodium acetate, and sodium phosphate; sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, etc. Stabilizers; isotonic agents such as sodium chloride and glucose; local anesthetics such as procaine hydrochloride and lidocaine hydrochloride; surfactants such as dimethyl sulfoxide (DMSO) and polyethylene glycol.
The therapeutic pharmaceutical composition may contain a sugar chain, an oligonucleotide, a polynucleotide, and the like. These sugar chains, oligonucleotides, polynucleotides, polyethylene glycol, additives and treatment agents are not particularly limited and can be appropriately selected depending on the purpose.
There is no restriction | limiting in particular as content of the said other component in the said therapeutic pharmaceutical composition, According to the objective, it can select suitably.

<Administration>
The administration timing of the therapeutic pharmaceutical composition is not particularly limited and may be appropriately selected according to the purpose. However, after the onset of a serious ischemic event including cerebral infarction, myocardial infarction, and pulmonary embolism 3 hours or more are preferable, and 3 to 6 hours are more preferable. The therapeutic pharmaceutical composition is advantageous in that it can be administered to patients after an acute phase of an ischemic event, and further, it can improve complications such as cerebral hemorrhage and prognostic exacerbation due to administration of the thrombolytic drug. is there.
The administration method of the therapeutic pharmaceutical composition is not particularly limited and can be appropriately selected according to the type and content of the thrombolytic drug or the inhibitor in the therapeutic pharmaceutical composition, for example, Oral administration method, injection method, inhalation method and the like.
The dose of the therapeutic pharmaceutical composition is not particularly limited, and various factors such as the age, weight, constitution, symptom of the administration subject and the presence or absence of administration of a drug containing other ingredients as active ingredients are considered. Can be selected as appropriate.
The animal species to be administered is not particularly limited and can be appropriately selected according to the purpose. For example, human, monkey, pig, cow, sheep, goat, dog, cat, mouse, rat, bird, etc. Among these, it is preferably used for humans.

The thrombolytic agent and the inhibitor in the therapeutic pharmaceutical composition may be administered in combination at the same time or may be administered separately. Further, the same composition may be used, the inhibitor may be administered prior to administration of the thrombolytic agent, or the thrombolytic agent may be administered within 30 minutes after administration of the inhibitor. Good.
In addition, when the thrombolytic agent is t-PA, plasmin activated by the t-PA is involved in the processing of the VEGF, so that the inhibitor is administered in the brain prior to the administration of the t-PA. When the ischemic event is delivered to the site of the ischemic event, the VEGF or the VEGF receptor binds to the inhibitor, and the site of the ischemic event such as the cerebral circulatory system is bound to the VEGF or the VEGF receptor. Therefore, the signal transduction of VEGF is more strongly inhibited. Therefore, the t-PA may be administered after the inhibitor is administered, and the t-PA may be administered within 30 minutes after the inhibitor is administered.

The dosage and administration method of the thrombolytic drug are not particularly limited and may be appropriately selected depending on the intended purpose. However, the dosage and administration method according to the instructions of each pharmaceutical manufacturer are preferable.
For example, when the thrombolytic drug is alteplase, which is one of the t-PA preparations, the dosage and administration method are not particularly limited and can be appropriately selected according to the purpose. The upper limit is a method of intravenously administering 60 mg to 90 mg per individual. Specifically, a method of intravenously injecting 10% of the total dose by bolus administration for 1 to 2 minutes and the remaining 90% by infusion administration for 1 hour can be mentioned.

There is no restriction | limiting in particular as a dosage and administration method of the said inhibitor, Although it can select suitably according to the objective, The dosage and administration method according to each pharmaceutical manufacturer's instruction | indication are preferable.
For example, when the inhibitor is the anti-VEGF-A neutralizing antibody or a derivative thereof, a method of intravenously administering 5 mg / kg to 10 mg / kg is preferable.
When the anti-VEGF-A neutralizing antibody is bevacizumab, it is preferable to dissolve 5 mg / kg to 10 mg / kg in 100 mL of physiological saline and administer intravenously over 90 minutes.
For example, when the inhibitor is cediranib, the dosage and administration method include a method of orally administering 10 mg to 45 mg per individual per day.
For example, when the inhibitor is sunitinib, the dosage and administration method include a method of orally administering 25 mg to 75 mg per individual once a day.
For example, when the inhibitor is sorafenib, the dosage and administration method thereof are not particularly limited and can be appropriately selected according to the purpose. For example, 400 mg to 800 mg per individual Examples include a method of oral administration once a day.
For example, when the inhibitor is the varatinib, the dosage and administration method thereof are not particularly limited and can be appropriately selected depending on the purpose. For example, 500 mg to 1,500 mg per individual is used. And a method of oral administration once a day.
For example, when the inhibitor is pazopanib, the dosage and administration method thereof are not particularly limited and can be appropriately selected according to the purpose. For example, 400 mg to 1,200 mg per individual is used. And a method of oral administration once a day.

When the thrombolytic drug and the inhibitor in the therapeutic pharmaceutical composition are administered simultaneously, the dosage and administration method of the therapeutic pharmaceutical composition are not particularly limited, depending on the purpose. It can select suitably, According to the kind of said thrombolytic agent and the said inhibitor in the said composition, content, etc., it can select suitably.

<Application>
The therapeutic pharmaceutical composition can also be administered to patients after an acute phase of severe ischemic events including cerebral infarction, myocardial infarction, and pulmonary embolism, and can improve complications such as cerebral hemorrhage and prognostic deterioration Therefore, it can be suitably used for treating severe ischemic events including cerebral infarction, myocardial infarction, and pulmonary embolism.
In the treatment of the severe ischemic event, an inhibitor that inhibits signal transduction mediated by the VEGF receptor at the same time as the step of administering a thrombolytic agent and the step of administering the thrombolytic agent or in advance. It is preferable to use a treatment method including the step of administering.
A kit containing the thrombolytic drug and the inhibitor is also included in the present invention. There is no restriction | limiting in particular as the density | concentration of the said thrombolytic agent and the said inhibitor in the said kit, Although it can select suitably according to the objective, It is preferable that it is a desired amount used for each administration. The inhibitor in the kit may be an inhibitor that inhibits signal transduction caused by the binding of VEGF to the VEGF receptor, or an antibody that inhibits the binding between VEGF and the VEGF receptor, Other binding factors may be used.

Hereinafter, the present invention will be specifically described with reference to examples of the present invention, but the present invention is not limited to these examples. The following examples were carried out after approval by the Niigata University Animal Experiment Ethics Committee.

(Example 1: Preparation of rat cerebral infarction model)
<Experimental animals>
In order to prepare a rat cerebral infarction model, Sprague-Dawley rats (male, 8 weeks old, obtained from Charles River, Japan) were used.

<Production of rat cerebral infarction model>
With reference to FIG. 1A and FIG. 1B, the preparation method of the rat cerebral infarction model of this invention is demonstrated.
In the conventional middle cerebral artery occlusion model, a nylon thread is applied to the bifurcation of the external carotid artery (ECA) 1 and the common carotid artery (CCA) 3, or from the external carotid artery (ECA) 1 to the origin of the middle cerebral artery (MCA) 2. The middle cerebral artery was blocked by invading (FIG. 1A).
However, a rat cerebral embolism model shown in FIG. 1B was prepared in this example in order to reproduce the concurrent cerebral hemorrhage caused by administering a thrombolytic drug beyond the possible treatment time of thrombolytic therapy. The thrombus was obtained by coagulating rat autologous blood and thrombin as a gel in a polyethylene tube catheter (PE-50, manufactured by Becton Dickinson) with a diameter of 0.35 mm. After being allowed to stand overnight, it was cut into a length of 1 mm. It was. The thrombus was injected from the rat external carotid artery (ECA) 1 into the rat middle cerebral artery (MCA) 2 under 1% to 1.5% by weight halothane anesthesia using the catheter. Thereafter, before blood clot injection and 30 minutes or 24 hours after blood clot injection, brain surface blood flow values (CBF) were measured using a laser Doppler blood flow meter (AFL21, Advance Co., Ltd., Tokyo). An animal having a cerebral blood flow value of less than 50% compared to that before infusion of thrombus was used as a rat cerebral infarction model animal in the following experiment.

<Plug dissolution therapy>
In thrombolytic therapy for rat cerebral infarction model, t-PA (alteplase, manufactured by Mitsubishi Tanabe Pharma Corporation), a thrombolytic drug, was intravenously injected into the femoral vein for 1 hour or 4 hours after thrombus injection (30 minutes). 10 mg / kg, 10% bolus administration and 90% infusion administration).

<TTC staining>
Rats euthanized with halothane overdose 24 hours after thrombus infusion were perfused with PBS to produce unfixed coronal coronal sections. The coronal sections were stained with TTC in PBS (pH 7.4) containing 2% by mass of triphenyltetrazolium salt (TTC) for 15 minutes at 37 ° C., and scanned using a scanner (CanoScanner, Canon). .
The volume of cerebral infarction and edema was measured by Swanson, R .; A. (J. Cereb. Blood Flow Metab., 10: 290-293 (1990)).

<Result>
2A to 2C are photographs of coronal sections showing the cerebral infarction reducing effect and cerebral hemorrhage-inducing effect of t-PA administration. A black part shows a healthy tissue, and a white part shows a cerebral infarction part.
Extensive cerebral infarction was observed in the operative cerebrum after 24 hours without administration of t-PA after thrombus injection (FIG. 2A).
When t-PA was administered 1 hour after thrombus injection, a reduction in the cerebral infarction portion was observed as compared to animals not treated with t-PA (FIG. 2B).
However, when t-PA was administered 4 hours after thrombus injection, enlargement of the cerebral infarction part and bleeding in the part were observed as compared to animals administered t-PA 1 hour later (FIG. 2C). ).
From the above results, it was shown that the rat cerebral infarction model can reproduce cerebral hemorrhage complications and exacerbation of cerebral infarction accompanying t-PA administration after acute cerebral infarction in humans.

(Example 2: Inhibition of VEGF expression using anti-VEGF antibody)
In order to suppress or reduce cerebral hemorrhage complications and exacerbation of cerebral infarction associated with administration of t-PA after acute cerebral infarction in humans, 100 μg of rabbit anti-rat VEGF antibody IgG (RB-222, Lab Vision- Neomarkers (hereinafter sometimes referred to as “anti-VEGF antibody”) was administered as a bolus with t-PA. In a control experiment, 100 μg of rabbit anti-human IgG (R5G10-048, manufactured by OEM Concepts, hereinafter sometimes referred to as “control antibody”) was administered as a bolus with t-PA.

<Western blot method>
Western blotting is performed using a whole cell extract as a sample by Shimahata, T. et al. (J. Cereb. Blood Flow Metab., 27: 1463-1475 (2007)).
For detection of VEGF, an anti-VEGF antibody (SC-152, manufactured by Santa Cruz Biotechnologies, dilution ratio 1: 200) is used as a primary antibody, and a peroxidase-conjugated anti-rabbit IgG antibody (dilution ratio 1:10) is used as a secondary antibody. , 000) was used.
In addition, in order to confirm that the amount of applied protein is uniform in any sample, the anti-β-actin antibody (SC-1616, SC-1616, (Santa Cruz Biotechnologies, dilution ratio 1: 2,000) and the secondary antibody were reacted to detect β-actin.

<Result>
FIG. 3 is a Western blot diagram showing that VEGF expression was suppressed after co-administration of t-PA and anti-VEGF antibody.
Lane 1 shows a sample of an animal that did not develop cerebral infarction due to thrombus injection, and lane 2 shows a sample of an animal that did not develop cerebral infarction due to thrombus injection and administered t-PA and a control antibody. 3 shows a sample of an animal to which only a control antibody was administered 1 hour after the onset of cerebral infarction due to thrombus injection, and lane 4 represents an animal to which t-PA and a control antibody were administered 1 hour after the onset of cerebral infarction due to thrombus injection. Samples are shown, lane 5 shows a sample of an animal administered with t-PA and an anti-VEGF antibody in combination 1 hour after the onset of cerebral infarction due to thrombus injection, and lane 6 shows a sample of t -Shows samples of animals administered PA and control antibody, lane 7 is an animal administered together with t-PA and anti-VEGF antibody 4 hours after cerebral infarction due to thrombus injection It illustrates a sample.

In lane 3 and lane 4, VEGF expression was observed. In lane 6, very much VEGF expression was observed. On the other hand, in lane 1, lane 2, lane 5, and lane 7, almost no VEGF expression was observed.
The difference in the amount of VEGF detected in these lanes did not correlate with the difference in the amount of β-actin. Comparison of lane 3, lane 4, and lane 5 showed that when t-PA was administered 4 hours after the onset of cerebral infarction due to thrombus injection, the expression level of VEGF was greatly increased. Moreover, it was found from the comparison between lane 4 and lane 5 and the comparison between lane 6 and lane 7 that the combined administration of t-PA and anti-VEGF antibody significantly suppressed the expression of VEGF.

It is known that ischemic vascular endothelial cell damage and subsequent cerebral blood barrier dysfunction are related to cerebral hemorrhage after t-PA administration. In addition, VEGF activates MMP-9, and activated MMP-9 is known to degrade proteins involved in the brain blood barrier, such as Zona oculusens-1 and basement membrane type IV collagen. Therefore, without being bound by theory, the mechanism of action of the combined administration of t-PA and anti-VEGF antibody is to suppress the increase in VEGF by t-PA administration after acute cerebral infarction. It may be explained by preventing cerebral hemorrhage by preventing cerebral blood barrier dysfunction such as -9 activation.

(Example 3: Evaluation of influence of combined administration of t-PA and anti-VEGF antibody)
The co-administration of t-PA and anti-VEGF antibody was performed as described in Example 2. The effect of the combined administration of t-PA and anti-VEGF antibody 4 hours after the onset of cerebral infarction due to thrombus injection is the effect of cerebral infarct volume, edema on the TTC-stained coronal section 24 hours after the onset of cerebral infarction due to thrombus injection. Volume, cerebral hemorrhage, and motor function scales were measured and evaluated.
The volume of cerebral infarction and edema of the TTC-stained coronal section is described in Swanson, R. et al. A. (J. Cereb. Blood Flow Metab., 10: 290-293 (1990)), statistical significance was verified by ANOVA (ANOVA), and post hoc comparison (post hoc comparison) was , Performed by Tukey method.
As for the amount of cerebral hemorrhage, the hemoglobin concentration (unit: g / dL) per 1 dL of the operated brain tissue was measured with a spectrophotometer.
The motor function scale is described by Andersen, M .; (Stroke, 30: 1464-1471 (1999)) (Stage 0: no movement disorder, Stage 1: flexion of the forelimb opposite to the surgical side, Stage 2: body to the paralyzed side Decrease in resistance to pushing, stage 3: spontaneous rotation to the paralyzed side, stage 4: death). Statistical significance in comparing motor function scales was verified by ANOVA (ANOVA), and post hoc comparison (post hoc comparison) was performed by Tukey method.

<Results of cerebral infarction and edema volume and cerebral hemorrhage>
4A to 4C are bar graphs showing cerebral infarction volume, edema volume, and cerebral hemorrhage volume, respectively, of a TTC-stained coronal section 24 hours after the onset of cerebral infarction due to thrombus injection. The white bar is the group that received only control antibody 4 hours after the onset of cerebral infarction due to thrombus injection, and the black bar is the group that received t-PA and control antibody 4 hours after the onset of cerebral infarction due to thrombus injection. The gray bar is a group to which t-PA and anti-VEGF antibody were administered 4 hours after the onset of cerebral infarction due to thrombus injection. The number of individuals in each group was 6.
From these results, the combined administration of t-PA and anti-VEGF antibody was not able to reduce the volume of cerebral infarction and edema compared to the administration of t-PA and anti-VEGF antibody, but the amount of cerebral hemorrhage was reduced. It was possible to reduce (P = 0.013).

<Motor function scale evaluation results>
FIG. 4D is a band graph showing the motor function scale 24 hours after the onset of cerebral infarction due to thrombus injection. The different colored parts of the band represent the number of individuals in each of the five stages. The left band shows a group (number of individuals 23) administered only with control antibody 4 hours after the onset of cerebral infarction due to thrombus injection, and the middle band shows t-PA and control 4 hours after the onset of cerebral infarction due to thrombus injection. The group to which the antibody was administered (20 individuals) was shown, and the right band represents the group to which t-PA and anti-VEGF antibody were administered 4 hours after the onset of cerebral infarction due to thrombus injection (12 individuals).
From the comparison of the left and middle bands, the group administered t-PA and the control antibody 4 hours after the onset of cerebral infarction had a worse prognosis than the group administered only the control antibody. From this result, it was confirmed that the rat cerebral infarction model reproduces cerebral hemorrhage complications and exacerbation of cerebral infarction accompanying t-PA administration after acute cerebral infarction in humans.
From the comparison of the middle band and the right band, the combined administration of t-PA and anti-VEGF antibody had a better prognosis than the combined administration of t-PA and control antibody (P = 0.0001). Furthermore, from the comparison of the left and right bands, the combined administration of t-PA and anti-VEGF antibody improved the prognosis over the control antibody alone (P = 0.045).
In addition, when a rat individual administered with t-PA and anti-VEGF antibody in combination was pathologically examined, the presence of the antigen-antibody complex was not observed in the liver, pancreas and kidney.

From the above experimental results, the combined administration of t-PA and anti-VEGF antibody can prolong the time until administration of t-PA in patients who have developed cerebral infarction, and is associated with cerebral hemorrhage complications. It was shown that the motor function and the survival rate can be improved while preventing the above.

(Example 4: Combined administration of t-PA and SU1498)
The possibility of substituting an anti-VEGF antibody with a VEGF receptor kinase inhibitor was examined below.
Specific inhibitors for the VEGF receptor include SU1498 ((E) -3- (3,5-diisopropyl-4-hydroxyphenyl) -2-((3-phenyl-n-propyl) aminocarbonyl) acrylonitrile, Calbiochem. Catalog No. 572888) manufactured by the company was used. SU1498 was dissolved in 1 mL of DMSO (dimethyl sulfoxide) per kg of the patient's body weight to give 20 mg / kg, and was administered as a single bolus with t-PA 4 hours after cerebral infarction. As a control experiment, only the solvent DMSO was administered at 1 mL per kg patient body weight.

<Evaluation of effects of combined administration of t-PA and SU1498>
The effect of the combined administration of t-PA and SU1498 4 hours after cerebral infarction is the same as in Example 3, and the deposition of cerebral infarction in the TTC-stained coronal section 24 hours after the onset of cerebral infarction by thrombus injection It was evaluated by measuring edema volume, cerebral hemorrhage volume, and motor function scale.

<Results of cerebral infarction and edema volume and cerebral hemorrhage>
5A to 5C show the volume of cerebral infarction and the volume of edema, respectively, of TTC-stained coronal sections 24 hours after the onset of rats administered with t-PA and SU1498 at 4 hours after the onset of cerebral infarction due to thrombus injection. And a bar graph showing the amount of cerebral hemorrhage. The black bar is a group administered with t-PA and DMSO 4 hours after the onset of cerebral infarction due to thrombus injection, and the gray bar is a group administered with t-PA and SU1498 4 hours after the onset of cerebral infarction due to thrombus injection It is. The number of individuals in each group was 6.
From these results, the combined administration of t-PA and SU1498 failed to reduce the volume of cerebral infarction and edema, but reduced the amount of cerebral hemorrhage compared with the administration of t-PA and DMSO. (P = 0.005).

<Motor function scale evaluation results>
FIG. 5D is a band graph showing a motor function scale 24 hours after the onset of rats administered with t-PA and SU1498 in combination 4 hours after the onset of cerebral infarction due to thrombus injection. Different colored bands represent each of the five levels. The left band shows the group administered t-PA and DMSO 4 hours after the onset of cerebral infarction due to thrombus injection, and the right band shows the group administered t-PA and SU1498 4 hours after the onset of cerebral infarction due to thrombus injection Indicates. The number of individuals was 10 in both cases.
From the comparison of the left and right bands, the group administered t-PA and SU1498 4 hours after the onset of cerebral infarction tended to improve the prognosis more than the group administered t-PA and DMSO.

Based on the above experimental results, the combined administration of t-PA and SU1498 is similar to the combined administration of t-PA and anti-VEGF antibody in the time until administration of t-PA in patients with cerebral infarction. It has been shown that the motor function and survival rate can be improved while preventing cerebral hemorrhage complications.

The therapeutic pharmaceutical composition can also be administered to patients after an acute phase of severe ischemic events including cerebral infarction, myocardial infarction, and pulmonary embolism, and can improve complications such as cerebral hemorrhage and prognostic deterioration Therefore, it can be suitably used for treating severe ischemic events including cerebral infarction, myocardial infarction, and pulmonary embolism.

1 External carotid artery (ECA)
2 Middle cerebral artery (MCA)
3 Common carotid artery (CCA)

Claims (20)

1. Treatment of severe ischemic events, including cerebral infarction, myocardial infarction, and pulmonary embolism, including thrombolytic agents and inhibitors that inhibit receptor mediated by vascular endothelial growth factor (VEGF) receptors A therapeutic pharmaceutical composition, characterized by being used in the above.
2. The composition according to claim 1, wherein the inhibitor reduces the binding of VEGF to the VEGF receptor.
3. The composition according to claim 2, wherein the inhibitor is a specific binding partner for at least one of VEGF and a VEGF receptor.
4. The composition according to claim 3, wherein the specific binding partner is at least one of the following (1) to (4).
   (1) antibodies,
   (2) aptamer,
   (3) a VEGF peptide that binds to the VEGF receptor but does not activate the VEGF receptor or a small molecule mimic of the VEGF peptide,
   (4) VEGF receptor peptides or small molecule mimics of VEGF receptor peptides that reduce the effective level of VEGF used to stimulate VEGF receptors.
5. The inhibitor is at least one of an antibody that binds to a VEGF receptor and antagonizes the binding of VEGF to the VEGF receptor, and an antibody that binds to the VEGF and causes the removal of the VEGF from blood. Item 5. The composition according to Item 4.
6. The composition according to claim 1, wherein the inhibitor inhibits release of VEGF from platelets.
7. 7. The composition of claim 6, wherein the inhibitor reduces ADP binding to the adenosine diphosphate (ADP) receptor.
8. The composition according to claim 7, wherein the inhibitor is a specific binding partner for at least one of ADP and ADP receptor.
9. The composition according to claim 8, wherein the specific binding partner is at least one of the following (1) to (4).
   (1) antibodies,
   (2) aptamer,
   (3) an ADP peptide or a small molecule mimic of an ADP peptide that binds to an ADP receptor but does not activate the ADP receptor;
   (4) ADP receptor peptide that binds ADP or a small molecule mimic of ADP receptor peptide.
10. The inhibitor according to claim 1, wherein the inhibitor is at least one of an inhibitor that interacts with a component of the VEGF receptor signaling pathway and an inhibitor that interacts with an enzyme that modifies a component of the VEGF receptor signaling pathway. Composition.
11. The composition according to claim 10, wherein the inhibitor is a tyrosine kinase inhibitor and an agonist of tyrosine phosphatase.
12. The composition according to claim 1, wherein the inhibitor decreases production of at least one of VEGF and a VEGF receptor.
13. The composition according to claim 10, wherein the inhibitor is at least one of an antisense nucleic acid, a small interfering RNA (siRNA), and a ribozyme.
14. The composition according to claim 1, wherein the inhibitor binds to the thrombolytic agent.
15. The composition according to claim 14, wherein the inhibitor binds to the thrombolytic drug as a fusion protein.
16. 2. The composition according to claim 1, wherein the thrombolytic drug is at least one of urokinase, streptokinase, tissue-type plasminogen activator (t-PA), and analogs thereof.
17. The composition according to claim 1, wherein the VEGF receptor is VEGF receptor type 2 (VEGFR-2).
18. The composition according to any one of claims 1 to 17, which is administered to a patient after an acute phase of an ischemic event including cerebral infarction, myocardial infarction, and pulmonary embolism.
19. The composition according to claim 18, wherein the acute phase of the ischemic event is 3 to 6 hours from the onset of the ischemic event.
20. The composition according to claim 18, wherein the acute phase of cerebral infarction is within 3 hours from the onset of the cerebral infarction.

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