JP2011046684A - Pharmaceutical composition for treatment of cerebral infarction, containing immunological pharmaceutical preparation - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for the treatment of cerebral infarction, administrable even to a patient after cerebral infarction acute stage passage. <P>SOLUTION: The therapeutic pharmaceutical composition for the treatment of cerebral infarction comprises a thrombolytic agent and a binding inhibitor for inhibiting the binding of a vascular endothelial growth factor (VEGF) to a receptor of VEGF. The therapeutic pharmaceutical composition for the treatment of cerebral infarction is, if necessary, administered to patients after cerebral infarction acute stage passage. The therapeutic pharmaceutical composition for the treatment of cerebral infarction, if necessary, contains tissue type plasminogen-activator (t-PA) or its derivative. The therapeutic pharmaceutical composition for the treatment of cerebral infarction, if necessary, contains an anti-VEGF-A neutralizing antibody or a derivative thereof. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  TECHNICAL FIELD The present invention relates to a pharmaceutical composition for treating cerebral infarction, specifically, for treating cerebral infarction comprising a thrombolytic agent and a binding inhibitor between vascular endothelial growth factor (VEGF) and the VEGF receptor. The present invention relates to a pharmaceutical composition.

  Cerebral infarction is caused by local blood flow blockage or ischemia in the brain. The central part of ischemia in the acute stage of cerebral infarction is irreversible even when blood flow is resumed, leading to cell death, but there is a reversible incomplete ischemic region around it, and it is particularly called a 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 acute cerebral infarction. However, administration of t-PA to patients after acute cerebral infarction is not effective, but rather leads to complications of cerebral hemorrhage and worsening prognosis. Administration of t-PA to patients after more than 3 hours is contraindicated.

  Therefore, the present situation is that rapid development of a pharmaceutical composition for treating cerebral infarction that can be administered to a patient after an acute stage of cerebral infarction is desired.

N. Engl. J. et al. Med. 333: 1581-1587 (1995)

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, an object of the present invention is to provide a pharmaceutical composition for treating cerebral infarction that can be administered to a patient who has passed the acute phase of cerebral infarction.

In order to solve the above-mentioned problems, the present inventors have conducted intensive studies. When resumed, vascular endothelial growth factor (VEGF) expression increases, which activates the VEGF receptor signaling system and promotes degradation of the proteins that make up the vascular wall. I found out.
Therefore, by using the thrombolytic agent and a binding inhibitor that inhibits the binding of the VEGF and the VEGF receptor in combination, after an acute phase of cerebral infarction, that is, after 3 hours or more from the onset of cerebral infarction It was found that the thrombolytic drug can be administered to these patients, and the present invention has been completed.

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 pharmaceutical composition for the treatment of cerebral infarction, comprising a thrombolytic drug and a binding inhibitor that inhibits binding between vascular endothelial growth factor (VEGF) and the receptor for VEGF.
<2> The composition according to <1>, wherein the composition is administered to a patient after an acute stage of cerebral infarction.
<3> The composition according to <2>, wherein the acute phase of cerebral infarction is within 3 hours from the onset of cerebral infarction.
<4> The composition according to any one of <1> to <3>, wherein the thrombolytic drug contains tissue-type plasminogen activator (t-PA) or a derivative thereof.
<5> The binding inhibitor includes a polyclonal antibody or a monoclonal antibody that specifically binds to at least one of VEGF and the VEGF receptor, and has an activity of inhibiting signal transduction of the VEGF, and an antigen of the antibody Any one of <1> to <4>, characterized in that it is selected from the group consisting of a binding fragment, a recombinant antibody or chimeric antibody containing the antigen-binding fragment, and derivatives thereof. It is a composition.
<6> The composition according to any one of <1> to <5>, wherein the binding inhibitor binds to VEGF-A.
<7> The composition according to <6>, wherein the VEGF-specific binding partner is an anti-VEGF-A neutralizing antibody or a derivative thereof.

  ADVANTAGE OF THE INVENTION According to this invention, the said various problems in the past can be solved, the said objective can be achieved, and the pharmaceutical composition for cerebral infarction treatment which can be administered also to the patient after cerebral infarction acute phase passing can be provided.

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 administered with t-PA 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 in a TTC-stained coronal section 24 hours after the onset of a rat 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 infarction volume (mm ³ ). FIG. 4B is a bar graph showing the volume of edema in 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 a motor function scale 24 hours after the onset of a rat 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.

(Pharmaceutical composition for treatment of cerebral infarction)
The pharmaceutical composition for the treatment of cerebral infarction of the present invention contains at least a thrombolytic agent and a binding inhibitor of vascular endothelial growth factor (VEGF) and the VEGF receptor, and if necessary, other components Containing.

<Thrombolytic drug>
The thrombolytic agent is not particularly limited as long as it can be applied to thrombolysis in the acute phase of cerebral infarction, and can be appropriately selected according to the purpose. For example, tissue type plasminogen activator (t-PA) Alternatively, derivatives thereof, urokinase, streptokinase, single chain urokinase type plasminogen activator (u-PA), desmoteplase and the like can be mentioned. These may be used alone or in combination of two or more.
Among these, it is preferable that the thrombolytic drug contains tissue-type plasminogen activator (t-PA) or a derivative thereof because the success rate of thrombolysis can be increased.
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 drugs that are acceptable. And the like which are combined with additives and treatment agents. Moreover, in the amino acid sequence of t-PA, one or several amino acids may be substituted.
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 pharmaceutical composition for treating cerebral infarction is not particularly limited, and can be appropriately selected according to the type of the serum dissolving drug.

  The “cerebral infarction acute phase” in the present invention is an early stage of the onset of cerebral infarction, and cerebral nerve dysfunction associated with a decrease in cerebral blood flow is observed, but can be recovered only by rapid resumption of blood flow with the thrombolytic agent. Say time. Here, the acute phase of cerebral infarction generally means within 3 hours from the onset of cerebral infarction.

  The “patient” in the present invention includes, but is not limited to, a human.

<Binding inhibitor>
The binding inhibitor is not particularly limited as long as it can inhibit the binding between vascular endothelial growth factor (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 of VEGF can be inhibited.
Examples of the binding inhibitor 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, carbohydrates, nucleic acids, lipids, and other biopolymers.

<< Binding inhibitor 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 cerebral infarction, it was confirmed in the present invention that the activation of the VEGF signal transduction system promotes the degradation of proteins constituting the blood vessel wall, resulting in complications of cerebral hemorrhage.
Examples of the VEGF family include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, placental growth factor (PIGF) -1, and PIGF-2. There are several subtypes in each member of the VEGF family. 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 binding inhibitor that specifically binds to VEGF may bind to any of the VEGF families.

  The binding inhibitor 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, The antibody may be selected from the group consisting of a chimeric antibody or a recombinant antibody (hereinafter sometimes referred to as “anti-VEGF antibody etc.”) containing the antigen-binding fragment, and derivatives thereof. Among these, the binding inhibitor that specifically binds to VEGF is preferably a monoclonal antibody, and the anti-VEGF-A neutralizing antibody is a VEGF-A receptor for VEGF-A that is involved in vascular disruption during angiogenesis. It is more preferable in that the binding can be efficiently inhibited.

There is no restriction | limiting in particular as a manufacturing method of the binding inhibitor couple | bonded specifically with the said VEGF, 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 binding inhibitor that specifically binds to VEGF may be the anti-VEGF antibody or the like, or at least one of these derivatives, polyethylene glycol, and other pharmaceutically acceptable additives and treatments. Other components such as an agent may be combined or added. There is no restriction | limiting in particular as content of the other component in the binding inhibitor couple | bonded specifically with the said VEGF, According to the objective, it can select suitably.

-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 such 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.

  Further, 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).

-Derivative-
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 drugs that are combined with pharmaceutically acceptable additives and treatment agents.

  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.

<< Binding inhibitor 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. Each of the VEGF families binds to a specific receptor, VEGF-A is in VEGFR-1 and VEGFR-2, VEGF-B, PlGF-1, and PlGF-2 are in VEGFR1, VEGF-C and VEGF-D. Binds to VEGFR-2 and VEGFR-3, and VEGF-E binds to VEGFR2.
The binding inhibitor that specifically binds to the VEGF receptor may bind to any of the VEGF receptors.

  The binding inhibitor that specifically binds to the VEGF receptor is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it recognizes the VEGF analog, VEGF competitive inhibitor, and VEGF receptor. A group consisting of a polyclonal antibody or a monoclonal antibody, an antigen-binding fragment of the antibody, a chimeric antibody or a recombinant antibody containing the antigen-binding fragment (hereinafter sometimes referred to as “anti-VEGFR antibody etc.”), and derivatives thereof May be selected. Among these, the binding inhibitor that specifically binds to the VEGF receptor is preferably a monoclonal antibody, and more preferably an anti-VEGFR-1 neutralizing antibody or an anti-VEGFR-2 antibody.

The method for producing a binding inhibitor that specifically binds to the VEGF receptor 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.
The binding inhibitor 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 additives. Or other components such as a treatment agent or the like may be combined or added. The content of other components in the binding inhibitor that specifically binds to the VEGF receptor is not particularly limited and may be appropriately selected depending on the intended purpose.

-Polyclonal antibodies, monoclonal antibodies, antigen-binding fragments-
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.

-Derivative-
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 drugs that are combined with pharmaceutically acceptable additives and treatment agents.

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.

  The content of the binding inhibitor in the pharmaceutical composition for treatment of cerebral infarction is not particularly limited and can be appropriately selected depending on the type of the binding 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 composition for treating cerebral infarction 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, Disintegrating agents such as sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose; lubricants such as purified talc, stearate, borax, polyethylene glycol; Emissions, colorants such as iron oxide; sucrose, orange peel, citric acid, and the like flavoring / flavoring of tartaric acid.
For example, when the composition for treating cerebral infarction 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; tragacanth, gum arabic, gelatin, etc. And the like.
For example, when the composition for treating cerebral infarction is used as an injection, a pH regulator and buffer such as sodium citrate, sodium acetate, sodium phosphate; sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid Stabilizers such as sodium chloride and glucose; local anesthetics such as procaine hydrochloride and lidocaine hydrochloride; and surfactants such as DMSO (dimethyl sulfoxide) and polyethylene glycol.
The composition for treating cerebral infarction may contain a sugar chain, an oligonucleotide, a polynucleotide and the like. These can use the same thing as the said binding inhibitor. These sugar chains, oligonucleotides, polynucleotides, polyethylene glycol, additives and treatment agents are not particularly limited and can be appropriately selected depending on the purpose.
The content of the other components in the pharmaceutical composition for treating cerebral infarction is not particularly limited and can be appropriately selected depending on the purpose.

<Administration>
The administration time of the pharmaceutical composition for treating cerebral infarction is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 3 hours or more after cerebral infarction onset, and more preferably 3 to 6 hours. . The pharmaceutical composition for treatment of cerebral infarction is advantageous in that it can be administered even to patients after an acute stage of cerebral infarction, and can further improve cerebral hemorrhage complications and prognosis exacerbation due to administration of the thrombolytic agent. .
The administration method of the pharmaceutical composition for treating cerebral infarction is not particularly limited, and is appropriately determined depending on the type and content of the thrombolytic drug or the binding inhibitor in the pharmaceutical composition for treating cerebral infarction. For example, oral administration method, injection method, inhalation method and the like can be mentioned.
The dosage of the pharmaceutical composition for the treatment of cerebral infarction is not particularly limited, and various factors such as the age, weight, constitution, symptom of the administration target individual, and the presence / absence of administration of a drug containing other ingredients as active ingredients 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 binding inhibitor in the pharmaceutical composition for treatment of cerebral infarction may be administered in combination at the same time or separately.
When the thrombolytic agent is t-PA, plasmin activated by the t-PA is involved in VEGF processing, and therefore, a binding inhibitor is delivered into the brain prior to administration of t-PA. This more strongly inhibits the binding between the VEGF and the VEGF receptor, and thereby leads to a stronger inhibition of the VEGF signaling. Therefore, t-PA may be administered after administering the binding inhibitor.

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 may be appropriately selected according to the purpose. 0.6 mg / kg to 0.9 mg / kg, and the upper limit includes a method of intravenous administration of 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 binding 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 binding 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.

  In the pharmaceutical composition for the treatment of cerebral infarction, when the thrombolytic agent and the binding inhibitor are administered simultaneously, the dosage and administration method of the composition are not particularly limited, depending on the purpose. And can be appropriately selected according to the type and content of the thrombolytic agent and the binding inhibitor in the composition.

<Application>
The pharmaceutical composition for the treatment of cerebral infarction can be administered to a patient after an acute stage of cerebral infarction and can improve complications of cerebral hemorrhage and exacerbation of prognosis, and thus can be suitably used for the treatment of cerebral infarction.
In the treatment of the cerebral infarction, it is preferable to use a treatment method comprising a step of administering a thrombolytic agent and a step of administering the binding inhibitor at the same time as or prior to the step of administering the thrombolytic agent. .

  EXAMPLES The present invention will be specifically described below 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 Co., Ltd.) 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 external carotid artery (ECA) 1 to the middle cerebral artery (MCA) 2 of the rat under 1% to 1.5% by mass of 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), which is 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 administering 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 administered 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 t-PA administration 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 may be 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 the primary antibody, and a peroxidase-conjugated anti-rabbit IgG antibody (dilution ratio 1:10) is used as the secondary antibody. , 000) was used.
Further, in order to confirm that the amount of applied protein is uniform in any sample, 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 the combined 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 was administered t-PA and a control antibody without developing cerebral infarction due to thrombus injection. 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 shows an animal sample 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 the t 4 hours after the onset of cerebral infarction due to thrombus injection. -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 the onset of 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. From the comparison of lane 3, lane 4, and lane 5, it was found 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 injury and subsequent cerebral blood barrier dysfunction are related to cerebral hemorrhage after t-PA administration. Moreover, VEGF activates MMP-9, and activated MMP-9 is known to degrade proteins involved in the brain blood barrier such as zona oculus dens-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 the administration of t-PA 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)
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 infarction volume, 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), 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 case of administering 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) to which only the control antibody was administered 4 hours after the onset of cerebral infarction due to thrombus injection, and the middle band is t-PA and the control 4 hours after the onset of cerebral infarction due to thrombus injection. The group (20 individuals) to which the antibody was administered is shown, and the right band represents the group (12 individuals) to which t-PA and anti-VEGF antibody were administered 4 hours after the onset of cerebral infarction due to thrombus injection.
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 associated with 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 administration of 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 the administration of t-PA in patients with 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.

  Since the pharmaceutical composition for treatment of cerebral infarction of the present invention can be administered even to a patient after an acute stage of cerebral infarction, it can be suitably used for the treatment of cerebral infarction.

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

Claims (7)

  A pharmaceutical composition for treating cerebral infarction, comprising a thrombolytic agent and a binding inhibitor that inhibits binding between vascular endothelial growth factor (VEGF) and the receptor for VEGF.   The composition according to claim 1, wherein the composition is administered to a patient after an acute stage of cerebral infarction.   The composition according to claim 2, wherein the acute phase of cerebral infarction is within 3 hours from the onset of cerebral infarction.   The composition according to any one of claims 1 to 3, wherein the thrombolytic drug contains tissue-type plasminogen activator (t-PA) or a derivative thereof.   The binding inhibitor includes a polyclonal antibody or a monoclonal antibody that specifically binds to at least one of VEGF and the receptor for VEGF, and has an activity of inhibiting the signal transduction of the VEGF, an antigen-binding fragment of the antibody, The composition according to any one of claims 1 to 4, wherein the composition is selected from the group consisting of a recombinant antibody or chimeric antibody containing the antigen-binding fragment, and derivatives thereof.   The composition according to claim 1, wherein the binding inhibitor binds to VEGF-A.   The composition according to claim 6, wherein the VEGF-specific binding partner is an anti-VEGF-A neutralizing antibody or a derivative thereof.