JP2011046684A - Pharmaceutical composition for treatment of cerebral infarction, containing immunological pharmaceutical preparation - Google Patents
Pharmaceutical composition for treatment of cerebral infarction, containing immunological pharmaceutical preparation Download PDFInfo
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- JP2011046684A JP2011046684A JP2010124374A JP2010124374A JP2011046684A JP 2011046684 A JP2011046684 A JP 2011046684A JP 2010124374 A JP2010124374 A JP 2010124374A JP 2010124374 A JP2010124374 A JP 2010124374A JP 2011046684 A JP2011046684 A JP 2011046684A
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Abstract
Description
本発明は、脳梗塞治療用医薬品組成物に関し、具体的には、血栓溶解薬、及び、血管内皮増殖因子(VEGF)と前記VEGFの受容体との結合阻害剤を含む、脳梗塞の治療用医薬品組成物に関する。 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.
脳梗塞は、脳における局所的な血流の遮断即ち虚血によって生じる。脳梗塞急性期の虚血中心部分は血流を再開しても不可逆的で細胞死に至るが、その周囲には可逆的な不完全虚血領域が存在し、特に、ペナンブラと呼ばれる。前記虚血中心部分は治療を施さない限り拡大し、ペナンブラは徐々に消失する。この結果、病理学的には脳梗塞部分が拡大され、臨床的には機能障害が生じ、最悪の場合には死に至る。脳梗塞急性期の治療目的は、前記ペナンブラでの血流を回復することである。前記回復は、虚血の程度及びその持続時間に依存する。つまり、前記ペナンブラへの血流をいかに迅速に再開させるかが脳梗塞の早期回復を決定する。 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.
組織型プラスミノゲン・アクチベーター(以下、「t−PA」と称することがある。)は、虚血の原因となっている血栓を溶解することによってペナンブラへの血液供給を再開させる血栓溶解療法として有効なので、脳梗塞急性期の治療薬として承認されている。しかし、脳梗塞急性期徒過後の患者へのt−PA投与は有効ではなく、むしろ脳出血の合併症と、予後の増悪とをもたらすので、脳梗塞急性期徒過後、即ち、脳梗塞の発症から3時間以上経過後の患者へのt−PAの投与は禁忌とされている。 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.
したがって、脳梗塞急性期徒過後の患者にも投与できる脳梗塞治療用医薬品組成物の早急な開発が望まれているのが現状である。 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.
本発明は、従来における前記諸問題を解決し、以下の目的を達成することを課題とする。即ち、本発明は、脳梗塞急性期徒過後の患者にも投与できる脳梗塞治療用医薬品組成物を提供することを目的とする。 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.
前記課題を解決するため、本発明者らは鋭意検討した結果、前記血栓溶解薬の脳梗塞急性期徒過後の投与による脳出血の合併症や予後の増悪は、血栓溶解薬の投与により血流が再開されると、血管内皮増殖因子(VEGF)の発現が増加し、これにより、VEGF受容体シグナル伝達系が活性化され、血管壁を構築しているタンパク質の分解が促進されることによるものであることを見出した。
そこで、前記血栓溶解薬、及び、前記VEGFと前記VEGF受容体との結合を阻害する結合阻害剤を併用することで、脳梗塞急性期徒過後、即ち、脳梗塞の発症から3時間以上経過後の患者にも前記血栓溶解薬を投与できることを知見し、本発明の完成に至った。
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.
本発明は、本発明者らによる前記知見に基づくものであり、前記課題を解決するための手段としては、以下の通りである。即ち、
<1> 血栓溶解薬、及び、血管内皮増殖因子(VEGF)と前記VEGFの受容体との結合を阻害する結合阻害剤を含むことを特徴とする、脳梗塞の治療用医薬品組成物である。
<2> 脳梗塞急性期徒過後の患者に投与されることを特徴とする、前記<1>に記載の組成物である。
<3> 前記脳梗塞急性期は脳梗塞の発症から3時間以内であることを特徴とする、前記<2>に記載の組成物である。
<4> 前記血栓溶解薬は組織型プラスミノゲン・アクチベーター(t−PA)又はその誘導体を含むことを特徴とする、前記<1>から<3>のいずれかに記載の組成物である。
<5> 前記結合阻害剤は、VEGF及び前記VEGFの受容体の少なくともいずれかと特異的に結合して、該VEGFのシグナル伝達を阻害する活性を有する、ポリクローナル抗体又はモノクローナル抗体と、該抗体の抗原結合断片と、該抗原結合断片を含む組換え抗体又はキメラ抗体と、これらの誘導体と、からなるグループから選択されることを特徴とする、前記<1>から<4>のいずれかに記載の組成物である。
<6> 前記結合阻害剤はVEGF−Aと結合することを特徴とする、前記<1>から<5>のいずれかに記載の組成物である。
<7> 前記VEGF特異的結合パートナーは抗VEGF−A中和抗体又はその誘導体であることを特徴とする、前記<6>に記載の組成物である。
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.
本発明によれば、従来における前記諸問題を解決し、前記目的を達成することができ、脳梗塞急性期徒過後の患者にも投与できる脳梗塞治療用医薬品組成物を提供することができる。 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.
(脳梗塞の治療用医薬品組成物)
本発明の脳梗塞の治療用医薬品組成物は、血栓溶解薬、及び、血管内皮増殖因子(VEGF)と前記VEGFの受容体との結合阻害剤を少なくとも含み、必要に応じて、更にその他の成分を含有する。
(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.
<血栓溶解薬>
前記血栓溶解薬としては、脳梗塞急性期の血栓溶解に適用することができれば、特に制限はなく、目的に応じて適宜選択することができ、例えば、組織型プラスミノゲン・アクチベーター(t−PA)又はその誘導体、ウロキナーゼ、ストレプトキナーゼ、一本鎖ウロキナーゼ型プラスミノゲン・アクチベーター(u−PA)、デスモテプラーゼなどが挙げられる。これらは、1種単独で使用してもよく、2種以上を併用してもよい。
これらの中でも、前記血栓溶解薬は、組織型プラスミノゲン・アクチベーター(t−PA)又はその誘導体を含むことが、血栓溶解の成功率を高めることができる点で好ましい。
前記血栓溶解薬の製造方法としては、特に制限はなく、前記血栓溶解薬の種類などに応じて適宜選択することができ、例えば、遺伝子組換え法、合成法などが挙げられる。また、市販品を用いてもよい。
前記t−PAの誘導体としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記t−PAに、糖鎖、オリゴヌクレオチド、ポリヌクレオチド、ポリエチレングリコール、その他の医薬品として許容される添加剤や処理剤を結合したものなどが挙げられる。また、t−PAのアミノ酸配列において、1個又は数個のアミノ酸が置換されたものであってもよい。
前記t−PA誘導体の具体的な例としては、モンテプラーゼ、パミテプラーゼ、レテプラーゼ等の前記t−PAのアミノ酸配列において一部のアミノ酸が置換されたt−PA誘導体;テネクテプラーゼ、ラノテプラーゼ等の前記t−PAのアミノ酸配列において一部のアミノ酸が置換され、更に糖鎖が修飾されたt−PA誘導体などが挙げられる。
<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.
前記脳梗塞の治療用医薬品組成物における、前記血清溶解薬の含有量としては、特に制限はなく、前記血清溶解薬の種類などに応じて適宜選択することができる。 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.
本発明における「脳梗塞急性期」とは、脳梗塞の発症の初期で、脳血流量の低下に伴う脳神経機能障害が認められるが、前記血栓溶解薬による迅速な血流再開のみによって回復可能な時期をいう。ここで、脳梗塞急性期は、一般的には脳梗塞の発症から3時間以内をいう。 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.
本発明における「患者」とは、ヒトを含むがヒトに限られない。 The “patient” in the present invention includes, but is not limited to, a human.
<結合阻害剤>
前記結合阻害剤は、血管内皮細胞増殖因子(VEGF)と、前記VEGF受容体との結合を阻害することができれば、特に制限はなく、目的に応じて適宜選択することができるが、前記VEGF及び前記VEGF受容体の少なくともいずれかと特異的に結合するものであることが好ましい。これにより、前記VEGFのシグナル伝達を阻害することができる。
前記結合阻害剤としては、例えば、前記VEGF及び前記VEGF受容体の少なくともいずれかと特異的に結合するレセプター又はリガンドなどが挙げられる。
前記レセプター又はリガンドとしては、特に制限はなく、目的に応じて適宜選択することができ、例えば、タンパク質、炭水化物、核酸、脂質、その他の生体高分子などが挙げられる。
<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.
<<VEGFに特異的に結合する結合阻害剤>>
前記VEGFとは、脈管形成及び血管新生に関与する一群の糖タンパク質である。前記VEGFが、血管内皮細胞表面に存在するVEGF受容体にリガンドとして結合すると、VEGFシグナル伝達系が活性化される。脳梗塞においては、このVEGFシグナル伝達系の活性化により血管壁を構築しているタンパク質の分解が促進され、脳出血の合併症が起こることが本発明において確認された。
前記VEGFファミリーとしては、例えば、VEGF−A、VEGF−B、VEGF−C、VEGF−D、VEGF−E、胎盤増殖因子(PIGF)−1、PIGF−2などが挙げられる。VEGFファミリーのそれぞれのメンバーには、更にいくつかの亜型が存在し、例えば、ヒトのVEGF−Aには、アミノ酸数が121個(VEGF−A121)、165個(VEGF−A165)、189個(VEGF−A189)、206個(VEGF−A206)、145個(VEGF−A145)、183個(VEGF−A183)などが知られている。また、ヒトのVEGF−Bには、アミノ酸数が167個(VEGF−B167)、186個(VEGF−B186)などが知られている。
前記VEGFに特異的に結合する結合阻害剤は、前記VEGFファミリーのいずれに結合するものであってもよい。
<< 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.
前記VEGFに特異的に結合する結合阻害剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記VEGFを認識するポリクローナル抗体又はモノクローナル抗体、該抗体の抗原結合断片、該抗原結合断片を含むキメラ抗体又は組換え抗体(以下、「抗VEGF抗体など」と称することがある。)、及びこれらの誘導体からなるグループから選択される場合がある。これらの中でも、前記VEGFに特異的に結合する結合阻害剤は、モノクローナル抗体が好ましく、抗VEGF−A中和抗体が、血管新生時の血管の破綻に関与するVEGF−Aの、VEGF受容体への結合を効率よく阻害できる点でより好ましい。 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.
前記VEGFに特異的に結合する結合阻害剤の製造方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、遺伝子組換え法、合成法などが挙げられる。また、市販品を用いてもよい。
また、前記VEGFに特異的に結合する結合阻害剤は、前記抗VEGF抗体など、及びこれらの誘導体の少なくともいずれかそのものであってもよく、ポリエチレングリコール、その他の医薬品として許容される添加剤や処理剤等のその他の成分を結合又は添加してもかまわない。前記VEGFに特異的に結合する結合阻害剤における、その他の成分の含有量としては、特に制限はなく、目的に応じて適宜選択することができる。
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.
−ポリクローナル抗体−
前記ポリクローナル抗体は、前記VEGFやこれらの断片を免疫原として、ほ乳類(例えば、マウス、ラット、ウサギ、ヒツジ又はヤギ)又は鳥類(例えば、ニワトリ)のいずれかの動物宿主に注射される。VEGFの断片を免疫原とする場合には、ウシ血清アルブミン又はスカシ貝ヘモシアニン(keyhole limpet hemocyanine)のような担体タンパク質と連結される場合に優れた免疫応答が誘発される場合がある。
前記免疫原は、1回又は2回以上のブースター免疫を取り込んだ予め定められたスケジュールに従って、前記動物宿主に注射されることが好ましい。
前記免疫原は、完全又は不完全フロイントアジュバントその他の免疫増強剤に混合して前記動物宿主に注射される場合がある。
前記ポリクローナル抗体は、かかる抗血清から、例えば適当な固体支持体に結合されたVEGFやこれらの断片を用いるアフィニティクロマトグラフィーによって精製され、VEGFと、VEGF受容体との結合が阻害されることや、この結合阻害によりVEGFシグナル伝達を阻害できることを確認されたものの場合がある。
前記ポリクローナル抗体としては、例えば、ヒト組換えVEGF165を免疫源として作製したウサギ抗ラットVEGF抗体IgG(RB−222、19kDa〜22kDa)などが挙げられる。なお、前記RB−222は、VEGF165及びVEGF121を認識することができる。
-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.
−モノクローナル抗体−
前記モノクローナル抗体は、Kohler及びMilstein(Eur.J.Immunol.6:511−519(1976))の技術と、その改良技術を用いて調製される場合がある。これらの方法は、所望の特異性を有する抗体を産生できる不死性細胞株の調製を伴う。
前記不死性細胞株は、前記ポリクローナル抗体の製造方法と同様の方法で免疫された動物宿主由来の脾臓細胞から作製される場合がある。前記脾臓細胞は、様々な方法で不死化され、抗体産生能を有する不死化細胞株が調製される。
前記脾臓細胞は、例えば、前記免疫された動物と同種かあるいは異種の動物由来のミエローマ細胞との融合によって不死化される。当業者に周知の様々な融合技術を用いる場合がある。
-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.
例えば、前記脾臓細胞とミエローマ細胞とは、非イオン性界面活性剤と数分間混合され、それから、ハイブリッド細胞の増殖は支持するがミエローマ細胞の増殖は支持しない選択培地に低濃度でプレートされる。好ましい選択技術は、HAT(ヒポキサンチン、アミノプテリン、チミジン)選択を用いる。通常約1週間〜2週間の十分な時間の後、ハイブリッドのコロニーが観察される。シングルコロニーが選択され、該シングルコロニーは、HAT(ヒポキサンチン、アミノプテリン、チミジン添加培地)等の培地で培養され、その培養上清が、前記VEGFやこれらの断片に対する結合活性についてテストされ、更に、前記VEGFと、VEGF受容体との結合が阻害されることや、この結合阻害によりVEGFシグナル伝達を阻害する活性についてもテストされる。反応性及び特異性が高いハイブリドーマが好ましい。限界希釈法によるクローニングを繰り返すことにより、反応性及び特異的が高い抗体を安定的に大量に産生するハイブリドーマのクローンが選択される。モノクローナル抗体は増殖中の選択されたハイブリドーマクローン由来の細胞株のコロニーの上清から単離される場合がある。
更に、マウスのような適当な脊椎動物宿主の腹腔内に前記ハイブリドーマ細胞株を注射するような、収率を向上させるための様々な技術が用いられる場合がある。
前記モノクローナル抗体は、前記ハイブリドーマ細胞腹水又は血液から回収される場合がある。細胞屑由来の不純タンパク質等の汚染物は、クロマトグラフィー、ゲルろ過、沈殿及び抽出のような従来技術によって前記抗体から除去される場合がある。
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.
また、前記モノクローナル抗体は、例えば、前記VEGFに対するマウスモノクローナル抗体を遺伝子組み換えによってヒト化したベバシズマブ(Bevacizumab)、前記ベバシズマブのFabフラグメントであり、前記VEGFとの結合が更に強くなるように遺伝子改変が行われたラニビズマブ(Ranibizumab)等のモノクローナル抗体製剤の抗VEGF−A中和抗体などが挙げられる。前記モノクローナル抗体製剤は、既に悪性腫瘍に対して臨床応用され、ヒトに対する安全性が確認されている。 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.
−抗原結合断片−
前記抗体の抗原結合断片は、抗原結合に関与する抗体の部分を指す。前記抗原結合部位は、重(H)鎖及び軽(L)鎖のN末端の可変(V)領域のアミノ酸残基によって形成される。
前記抗体の抗原結合断片は、それぞれタンパク質分解酵素パパイン又はペプシンでインタクトなポリクローナル抗体又はモノクローナル抗体を分解して得られるFab断片又はF(ab’)2断片の他、天然抗体分子の抗原認識能及び結合能の多くを保持する抗原結合部位を含む非共有結合的なVH及びVL領域のヘテロ2量体を含むFv断片を含む。
-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.
−組換え抗体−
前記組換え抗体は、適当な細菌宿主への形質転換や、適当なほ乳類細胞宿主へのトランスフェクションなどを含む抗体遺伝子の発現クローニングによって調製される場合がある。
また、前記組換え抗体は、例えば、原核生物及び真核生物由来の遺伝子発現システムを用いて大量に調製することができる。
-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.
−キメラ抗体−
前記キメラ抗体は、前記組換え抗体の抗原結合部位がVEGFと特異的に結合できるように同種又は異種の抗体の定常ドメインによって支持された融合タンパク質である。
前記キメラ抗体には、抗体軽鎖可変領域(VL)に操作可能に連結された抗体重鎖可変領域(VH)を含む短鎖可変部抗体(scFv)と、ラクダ科(Camelidae、ラクダ、ヒトコブラクダ、ラマを含む)の動物が産生する軽鎖がないIgGのクラスであるラクダ重鎖抗体(HCAb)又はその重鎖可変部ドメイン(VHH)とを含む。
-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).
−誘導体−
前記VEGFと前記VEGF受容体との結合阻害活性を有する前記抗VEGF抗体などの誘導体としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記抗VEGF抗体などに、糖鎖、オリゴヌクレオチド、ポリヌクレオチド、ポリエチレングリコール、その他の医薬品として許容される添加剤や処理剤等を結合したものなどが挙げられる。
-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.
前記抗VEGF抗体などの誘導体の具体的な例としては、前記VEGF遺伝子のエクソン7部分に結合し、前記VEGFの生成を阻害するRNAアプタマーのペガプタニブなどが挙げられる。 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.
また、前記抗VEGF抗体などに、糖鎖、オリゴヌクレオチド、ポリヌクレオチド、ポリエチレングリコール、その他の医薬品として許容される添加剤や処理剤を添加したものであってもよい。
これらの糖鎖、オリゴヌクレオチド、ポリヌクレオチド、ポリエチレングリコール、添加剤や処理剤としては、特に制限はなく、目的に応じて適宜選択することができる。
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.
<<VEGF受容体に特異的に結合する結合阻害剤>>
前記VEGF受容体(VEGFR)とは、受容体型チロシンキナーゼの一種であり、リガンドである前記VEGFによる血管内皮細胞の増殖や遊走の促進などの作用の発現に関与している。
VEGF受容体には、VEGFR−1(Flt−1と称することがある。)、VEGFR−2(KDR、Flk−1と称することがある。)、VEGFR−3(Flt−4と称することがある。)、可溶性VEGFR−1、可溶性VEGFR−2、可溶性VEGFR−3などが知られている。前記VEGFファミリーは、それぞれ決まった受容体に結合し、VEGF−AはVEGFR−1及びVEGFR−2に、VEGF−B、PlGF−1、及びPlGF−2はVEGFR1に、VEGF−C及びVEGF−DはVEGFR−2及びVEGFR−3に、VEGF−EはVEGFR2に結合する。
前記VEGF受容体に特異的に結合する結合阻害剤は、前記VEGF受容体のいずれに結合するものであってもよい。
<< 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.
前記VEGF受容体に特異的に結合する結合阻害剤としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記VEGFのアナログ、VEGFの拮抗阻害剤、VEGF受容体を認識するポリクローナル抗体又はモノクローナル抗体、該抗体の抗原結合断片、該抗原結合断片を含むキメラ抗体又は組換え抗体(以下、「抗VEGFR抗体など」と称することがある。)、及びこれらの誘導体からなるグループから選択される場合がある。これらの中でも、前記VEGF受容体に特異的に結合する結合阻害剤は、モノクローナル抗体が好ましく、抗VEGFR−1中和抗体、抗VEGFR−2抗体がより好ましい。 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.
前記VEGF受容体に特異的に結合する結合阻害剤の製造方法としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、遺伝子組換え法、合成法などが挙げられる。また、市販品を用いてもよい。
また、前記VEGF受容体に特異的に結合する結合阻害剤は、前記抗VEGFR抗体など、及びこれらの誘導体の少なくともいずれかそのものであってもよく、ポリエチレングリコール、その他の医薬品として許容される添加剤や処理剤等のその他の成分を結合又は添加してもかまわない。前記VEGF受容体に特異的に結合する結合阻害剤における、その他の成分の含有量としては、特に制限はなく、目的に応じて適宜選択することができる。
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.
−ポリクローナル抗体、モノクローナル抗体、抗原結合断片−
前記ポリクローナル抗体、モノクローナル抗体、及び抗原結合断片は、VEGF受容体や、これらの断片を免疫原として、前記VEGFを認識するポリクローナル抗体、モノクローナル抗体、及び抗原結合断片と同様の方法で製造することができる。
-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.
−組換え抗体−
前記組換え抗体は、前記VEGFを認識する組換え抗体と同様の方法で製造することができる。
-Recombinant antibody-
The recombinant antibody can be produced in the same manner as the recombinant antibody recognizing VEGF.
−キメラ抗体−
前記キメラ抗体は、前記組換え抗体の抗原結合部位がVEGF受容体と特異的に結合できるように同種又は異種の抗体の定常ドメインによって支持された融合タンパク質であること以外は、前記VEGFを認識するキメラ抗体と同様の抗体などが挙げられる。
-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.
−誘導体−
前記VEGFと前記VEGF受容体との結合阻害活性を有する前記抗VEGFR抗体などの誘導体としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、前記抗VEGF抗体などに、糖鎖、オリゴヌクレオチド、ポリヌクレオチド、ポリエチレングリコール、その他の医薬品として許容される添加剤や処理剤等を結合したものなどが挙げられる。
-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.
また、前記抗VEGF抗体などに、糖鎖、オリゴヌクレオチド、ポリヌクレオチド、ポリエチレングリコール、その他の医薬品として許容される添加剤や処理剤を添加したものであってもよい。
前記添加剤、処理剤、糖鎖、オリゴヌクレオチド、ポリヌクレオチド、ポリエチレングリコールなどは、前記抗VEGF抗体などと同様のものなどが挙げられる。
これらの糖鎖、オリゴヌクレオチド、ポリヌクレオチド、ポリエチレングリコール、添加剤や処理剤としては、特に制限はなく、目的に応じて適宜選択することができる。
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.
前記脳梗塞の治療用医薬品組成物における、前記結合阻害剤の含有量としては、特に制限はなく、前記結合阻害剤の種類などに応じて適宜選択することができる。 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.
<その他の成分>
前記その他の成分としては、特に制限はなく、薬理学上許容される担体の中から投与方法や剤型などに応じて適宜選択することができる。
例えば、前記脳梗塞の治療用組成物が、経口固形剤として用いられる場合、乳糖、白糖、塩化ナトリウム、ブドウ糖、デンプン、炭酸カルシウム、カオリン、微結晶セルロース、珪酸等の賦形剤;水、エタノール、プロパノール、単シロップ、ブドウ糖液、デンプン液、ゼラチン液、カルボキシメチルセルロース、ヒドロキシプロピルセルロース、ヒドロキシプロピルスターチ、メチルセルロース、エチルセルロース、シェラック、リン酸カルシウム、ポリビニルピロリドン等の結合剤;乾燥デンプン、アルギン酸ナトリウム、カンテン末、炭酸水素ナトリウム、炭酸カルシウム、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド、乳糖等の崩壊剤;精製タルク、ステアリン酸塩、ホウ砂、ポリエチレングリコール等の滑沢剤;酸化チタン、酸化鉄等の着色剤;白糖、橙皮、クエン酸、酒石酸等の矯味/矯臭剤などが挙げられる。
例えば、前記脳梗塞の治療用組成物が、経口液剤として用いられる場合、白糖、橙皮、クエン酸、酒石酸等の矯味/矯臭剤;クエン酸ナトリウム等の緩衝剤;トラガント、アラビアゴム、ゼラチン等の安定化剤などが挙げられる。
例えば、前記脳梗塞の治療用組成物が、注射剤として用いられる場合、クエン酸ナトリウム、酢酸ナトリウム、リン酸ナトリウム等のpH調節剤及び緩衝剤;ピロ亜硫酸ナトリウム、EDTA、チオグリコール酸、チオ乳酸等の安定化剤;塩化ナトリウム、ブドウ糖等の等張化剤;塩酸プロカイン、塩酸リドカイン等の局所麻酔剤;DMSO(ジメチルスルホキシド)、ポリエチレングリコール等の界面活性剤などが挙げられる。
また、前記脳梗塞の治療用組成物は、糖鎖、オリゴヌクレオチド、ポリヌクレオチドなどを含有していてもよい。これらは、前記結合阻害剤と同様のものなどを用いることができる。これらの糖鎖、オリゴヌクレオチド、ポリヌクレオチド、ポリエチレングリコール、添加剤や処理剤としては、特に制限はなく、目的に応じて適宜選択することができる。
前記脳梗塞の治療用医薬品組成物における前記その他の成分の含有量としても、特に制限はなく、目的に応じて適宜選択することができる。
<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.
<投与>
前記脳梗塞の治療用医薬品組成物の投与時期としては、特に制限はなく、目的に応じて適宜選択することができるが、脳梗塞発症後3時間以降が好ましく、3時間〜6時間がより好ましい。前記脳梗塞の治療用医薬品組成物は、脳梗塞急性期徒過後の患者に対しても投与でき、更に前記血栓溶解薬の投与による脳出血の合併症や予後の増悪を改善できる点で有利である。
前記脳梗塞の治療用医薬品組成物の投与方法としては、特に制限はなく、該脳梗塞の治療用医薬品組成物における、前記血栓溶解薬や前記結合阻害剤の種類や含有量などに応じて適宜選択することができ、例えば、経口投与法、注射による方法、吸入による方法などが挙げられる。
前記脳梗塞の治療用医薬品組成物の投与量としても、特に制限はなく、投与対象個体の年齢、体重、体質、症状、他の成分を有効成分とする医薬の投与の有無など、様々な要因を考慮して適宜選択することができる。
前記投与対象となる動物種としては、特に制限はなく、目的に応じて適宜選択することができ、例えば、ヒト、サル、ブタ、ウシ、ヒツジ、ヤギ、イヌ、ネコ、マウス、ラット、トリなどが挙げられるが、これらの中でもヒトに好適に用いられる。
<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.
前記脳梗塞の治療用医薬品組成物における、前記血栓溶解薬と、前記結合阻害剤とは、同時に併用して投与されてもよく、別々に投与されてもよい。
前記血栓溶解薬がt−PAである場合、該t−PAによって活性化されるプラスミンがVEGFのプロセッシングに関与するため、t−PAの投与に先立って結合阻害剤を脳内に送達しておくことは、前記VEGFと前記VEGF受容体との結合をより強く阻害すること、これにより、前記VEGFのシグナル伝達をより強く阻害することにつながる。したがって、結合阻害剤を投与した後にt−PAを投与する場合もある。
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.
前記血栓溶解薬の投与量及び投与方法としては、特に制限はなく、目的に応じて適宜選択することができるが、各医薬製造メーカーの指示に従った投与量及び投与方法が好ましい。
例えば、前記血栓溶解薬が、前記t−PA製剤の1つであるアルテプラーゼである場合、その投与量及び投与方法としては、特に制限はなく、目的に応じて適宜選択することができるが、0.6mg/kg〜0.9mg/kgであり、上限としては、1個体当たり60mg〜90mgを、静脈内投与する方法などが挙げられる。具体的には、全投与量の10%を1分間〜2分間のボーラス投与で、残り90%を1時間の点滴投与で静脈内注射する方法などが挙げられる。
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.
前記結合阻害剤の投与量及び投与方法としては、特に制限はなく、目的に応じて適宜選択することができるが、各医薬製造メーカーの指示に従った投与量及び投与方法が好ましい。
例えば、前記結合阻害剤が、前記抗VEGF−A中和抗体又はその誘導体である場合、5mg/kg〜10mg/kgを静脈内投与する方法が好ましい。
また、前記抗VEGF−A中和抗体が、ベバシズマブである場合、5mg/kg〜10mg/kgを生理食塩水100mLに溶解し、90分間かけて静注投与することが好ましい。
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.
前記脳梗塞の治療用医薬品組成物における、前記血栓溶解薬と、前記結合阻害剤とが、同時に投与される場合、前記組成物の投与量及び投与方法としては、特に制限はなく、目的に応じて適宜選択することができ、前記組成物における前記血栓溶解薬及び前記結合阻害剤の種類、含有量などに応じて、適宜選択することができる。 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.
<用途>
前記脳梗塞の治療用医薬品組成物は、脳梗塞急性期徒過後の患者にも投与でき、脳出血の合併症や予後の増悪を改善できるため、脳梗塞の治療に好適に利用可能である。
前記脳梗塞の治療においては、血栓溶解薬を投与するステップと、該血栓溶解薬を投与するステップと同時に、あるいは、先だって、前記結合阻害剤を投与するステップとを含む治療方法を用いることが好ましい。
<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. .
以下に本発明の実施例を挙げて本発明を具体的に説明するが、本発明はこれらの実施例に何ら限定されるものではない。なお、以下の実施例は、新潟大学動物実験倫理委員会によって承認された後に実施された。 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.
(実施例1:ラット脳梗塞モデルの作製)
<実験動物>
ラット脳梗塞モデルを作製するためにスプラーグ−ドーリーラット(オス、8週齢、日本チャールス・リバー株式会社より入手)を用いた。
(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.
<ラット脳梗塞モデルの作製>
図1A及び図1Bを参照して、本願発明のラット脳梗塞モデルの作製方法を説明する。
従来の中大脳動脈閉塞モデルでは、外頸動脈(ECA)1と総頸動脈(CCA)3の分岐部、若しくは外頚動脈(ECA)1から中大脳動脈(MCA)2起始部にナイロン糸を侵入させて中大脳動脈を閉塞させていた(図1A)。
しかし、血栓溶解療法の治療可能時間を超えて血栓溶解薬を投与することによる脳出血併発を再現させるために、本実施例では、図1Bに示すラット脳塞栓モデルを作製した。血栓は、ラットの自家血液及びトロンビンを、直径0.35mmのポリエチレンチューブカテーテル(PE−50、ベクトン・ディクティンソン社製)中でゲルとして凝固させ、終夜放置後、1mmの長さに切断された。前記血栓は、前記カテーテルを用いて、1質量%〜1.5質量%のハロタン麻酔下でラットの外頸動脈(ECA)1から中大脳動脈(MCA)2に注入された。その後、血栓注入前と、血栓注入の30分間後又は24時間後に、レーザードップラー血流計(AFL21、株式会社アドバンス製、東京)を用いて脳表血流値(CBF)が測定された。脳表血流値が血栓注入前と比べて50%未満の動物を以下の実験でラット脳梗塞モデル動物として用いた。
<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.
<栓溶解療法>
ラット脳梗塞モデルに対する血栓溶解療法には、血栓溶解薬であるt−PA(アルテプラーゼ、田辺三菱製薬株式会社製)が、血栓注入の1時間又は4時間後に大腿静脈に30分間静注された(10mg/kg、10%ボーラス投与及び90%点滴投与)。
<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染色>
血栓注入の24時間後にハロタン過剰投与で安楽死させたラットにPBSを潅流して、非固定の脳冠状切片が作製された。前記脳冠状切片は、37℃で15分間、2質量%トリフェニルテトラゾリウム塩(TTC)を含むPBS(pH7.4)中でTTC染色され、スキャナー(CanoScaner、Canon社製)を用いて走査された。
脳梗塞及び浮腫の体積は、Swanson、R.A.ら(J. Cereb. Blood Flow Metab.、10:290−293(1990))に基づいて算出された。
<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)).
<結果>
図2A〜図2Cは、t−PA投与の脳梗塞軽減効果と、脳出血惹起効果とを示す脳冠状切片の写真である。黒色部分は、健常組織を示し、白色部分は、脳梗塞部分を示す。
血栓注入後t−PAを投与せずに24時間経過すると、術側大脳に広範な脳梗塞が観察された(図2A)。
血栓注入の1時間後にt−PAを投与すると、t−PA非投与動物と比較して脳梗塞部分の縮小が観察された(図2B)。
しかし、血栓注入の4時間後にt−PAを投与すると、1時間後にt−PAを投与した動物と比較して、脳梗塞部分の拡大と、前記部分での出血とが観察された(図2C)。
以上の結果から、前記ラット脳梗塞モデルは、ヒトにおける脳梗塞急性期徒過後のt−PA投与に伴う、脳出血合併症と脳梗塞の増悪とを再現できることが示された。
<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.
(実施例2:抗VEGF抗体を用いたVEGFの発現抑制)
ヒトにおける脳梗塞急性期徒過後のt−PA投与に伴う、脳出血合併症と、脳梗塞の増悪とを抑制又は軽減するために、100μgのウサギ抗ラットVEGF抗体IgG(RB−222、Lab Vision−Neomarkers社製、以下、「抗VEGF抗体」と称することがある。)がt−PAとともにボーラス投与された。対照実験では、100μgのウサギ抗ヒトIgG(R5G10−048、OEM Concepts社製、以下、「対照抗体」と称することがある。)がt−PAとともにボーラス投与された。
(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.
<ウエスタン・ブロット法>
ウエスタン・ブロット法は、全細胞抽出液をサンプルとして用いてShimohata、 T.ら(J. Cereb. Blood Flow Metab.,27:1463−1475 (2007))に記載の方法に従って実施された。
VEGFの検出には、1次抗体として抗VEGF抗体(SC−152、Santa Cruz Biotechnologies社製、希釈比1:200)が、2次抗体としてペルオキダーゼ・コンジュゲート抗ウサギIgG抗体(希釈比1:10,000)が用いられた。
また、適用されたタンパク質の量がどのサンプルでも均一であることを確認するために、前記1次抗体及び前記2次抗体を除去した後のブロッティング膜に、抗β−アクチン抗体(SC−1616、Santa Cruz Biotechnologies社製、希釈比1:2,000)及び前記2次抗体を反応させてβ−アクチンが検出された。
<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.
<結果>
図3は、t−PA及び抗VEGF抗体の併用投与後に、VEGFの発現が抑制されたことを示すウエスタン・ブロット図である。
レーン1は、血栓注入による脳梗塞発症を行わなかった動物のサンプルを示し、レーン2は、血栓注入による脳梗塞発症を行わないでt−PA及び対照抗体を投与した動物のサンプルを示し、レーン3は、血栓注入による脳梗塞発症の1時間後に対照抗体のみを投与した動物のサンプルを示し、レーン4は、血栓注入による脳梗塞発症の1時間後にt−PA及び対照抗体を投与した動物のサンプルを示し、レーン5は、血栓注入による脳梗塞発症の1時間後にt−PA及び抗VEGF抗体を併用投与した動物のサンプルを示し、レーン6は、血栓注入による脳梗塞発症の4時間後にt−PA及び対照抗体を投与した動物のサンプルを示し、レーン7は、血栓注入による脳梗塞発症の4時間後にt−PA及び抗VEGF抗体を併用投与した動物のサンプルを示す。
<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.
レーン3及びレーン4では、VEGFの発現が観察された。レーン6では、非常に多くのVEGFの発現が観察された。一方、レーン1、レーン2、レーン5、及びレーン7では、ほとんどVEGFの発現は観察されなかった。
これらのレーンで検出されたVEGF量の相違は、β−アクチンの量の相違とは全く相関しなかった。レーン3、レーン4、及びレーン5の比較から、血栓注入による脳梗塞発症の4時間後にt−PAを投与すると、VEGFの発現量が非常に増大したことがわかった。また、レーン4とレーン5との比較、並びに、レーン6とレーン7との比較によって、t−PA及び抗VEGF抗体の併用投与は、VEGFの発現を顕著に抑制したことがわかった。
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.
虚血性血管内皮細胞障害と、その後の脳血液関門の機能不全とがt−PA投与後の脳出血に関係することが知られている。また、VEGFは、MMP−9を活性化し、活性化されたMMP−9は、ゾナオクルデンス−1や基底膜IV型コラーゲンのような脳血液関門に関与するタンパク質を分解することが知られている。したがって、理論的に拘泥するわけではないが、t−PA及び抗VEGF抗体の併用投与の作用機序は、脳梗塞急性期徒過後のt−PA投与によるVEGFの増加を抑制することによって、MMP−9活性化のような脳血液関門の機能不全を防止して、脳出血を予防することで説明できる可能性がある。 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.
(実施例3:t−PA及び抗VEGF抗体の併用投与の影響評価)
t−PA及び抗VEGF抗体の併用投与は、実施例2で説明されたとおり実施された。血栓注入による脳梗塞発症から4時間後のt−PA及び抗VEGF抗体の併用投与の効果は、血栓注入による脳梗塞発症の24時間後のTTC染色脳冠状切片の、脳梗塞の体積、浮腫の体積、脳出血量、及び運動機能スケールを測定して評価された。
前記TTC染色脳冠状切片の、脳梗塞の体積及び浮腫の体積は、Swanson、R.A.ら(J. Cereb. Blood Flow Metab.、10:290−293(1990))に基づいて算出され、統計的有意性は、ANOVA(分散分析)にて検証され、事後比較(post hoc比較)は、Tukey法で行った。
脳出血量は、分光光度計で術側脳組織1dL当たりのヘモグロビン濃度(単位:g/dL)が測定された。
運動機能スケールは、Andersen、M.ら(Stroke、30: 1464−1471(1999))に基づいて5段階で評価された(段階0:運動障害なし、段階1:術側と反対側の前肢の屈曲、段階2:麻痺側へ身体を押し動かすことへの抵抗力の減少、段階3:麻痺側への自発的な回転、段階4:死亡)。運動機能スケールを比較する際の統計的有意性は、ANOVA(分散分析)にて検証され、事後比較(post hoc比較)はTukey法で行った。
(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.
<脳梗塞及び浮腫の体積と、脳出血量との結果>
図4A〜図4Cは、それぞれ、血栓注入による脳梗塞発症の24時間後のTTC染色脳冠状切片の、脳梗塞の体積、浮腫の体積、及び脳出血量を示す棒グラフである。白色の棒は、血栓注入による脳梗塞発症の4時間後に対照抗体のみを投与した群で、黒色の棒は、血栓注入による脳梗塞発症の4時間後にt−PA及び対照抗体を投与した群で、灰色の棒は、血栓注入による脳梗塞発症の4時間後にt−PA及び抗VEGF抗体を投与した群である。各群の個体数は6であった。
これらの結果から、t−PA及び抗VEGF抗体の併合投与は、t−PA及び抗VEGF抗体を投与した場合に比べて脳梗塞及び浮腫の体積を低減することはできなかったが、脳出血量を低減することはできた(P=0.013)。
<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).
<運動機能スケール評価結果>
図4Dは、血栓注入による脳梗塞発症の24時間後の運動機能スケールを示す帯グラフである。帯の異なる色の部分は、5段階のそれぞれの個体数を表す。左側の帯は、血栓注入による脳梗塞発症の4時間後に対照抗体のみを投与した群(個体数23)を示し、中央の帯は、血栓注入による脳梗塞発症の4時間後にt−PA及び対照抗体を投与した群(個体数20)を示し、右側の帯は、血栓注入による脳梗塞発症の4時間後にt−PA及び抗VEGF抗体を投与した群(個体数12)を示す。
左側の帯と中央の帯との比較から、脳梗塞発症の4時間後にt−PA及び対照抗体を投与した群は、対照抗体のみを投与した群より予後が悪かった。この結果から、前記ラット脳梗塞モデルが、ヒトにおける脳梗塞急性期徒過後のt−PA投与に伴う、脳出血合併症と脳梗塞の増悪とを再現することを確認できた。
中央の帯と右側の帯との比較から、t−PA及び抗VEGF抗体の併合投与は、t−PA及び対照抗体の併合投与より予後が改善された(P=0.0001)。更に、左側の帯と右側の帯との比較から、t−PA及び抗VEGF抗体の併合投与は、対照抗体のみの投与よりも予後が改善された(P=0.045)。
なお、t−PA及び抗VEGF抗体を併合投与されたラット個体を病理学的に剖検したところ、抗原抗体複合体の存在は肝臓、膵臓及び腎臓に認められなかった。
<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.
以上の実験結果から、t−PA及び抗VEGF抗体の併用投与は、脳梗塞を発症した患者において、t−PAを投与するまでの時間を従来よりも延長することができ、かつ、脳出血合併症を予防しつつ運動機能及び生存割合を改善できることが示された。 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.
本発明の脳梗塞の治療用医薬品組成物は、脳梗塞急性期徒過後の患者にも投与できるため、脳梗塞の治療に好適に利用可能である。 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 外頸動脈(ECA)
2 中大脳動脈(MCA)
3 総頸動脈(CCA)
1 External carotid artery (ECA)
2 Middle cerebral artery (MCA)
3 Common carotid artery (CCA)
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JP5823671B2 (en) * | 2009-07-27 | 2015-11-25 | 国立大学法人 新潟大学 | Pharmaceutical composition for the treatment of cerebral infarction comprising immunological preparation |
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JP2002534359A (en) * | 1998-12-22 | 2002-10-15 | ジェネンテック・インコーポレーテッド | Vascular endothelial cell growth factor antagonist and its use |
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Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2018158948A (en) * | 2009-07-27 | 2018-10-11 | 国立大学法人 新潟大学 | Pharmaceutical compositions comprising immunological formulations for treating cerebral infarction |
KR20170082631A (en) * | 2014-11-17 | 2017-07-14 | 씨에스엘 리미티드 | Method of Treating or Preventing Stroke |
KR102560025B1 (en) * | 2014-11-17 | 2023-07-26 | 씨에스엘 리미티드 | Method of Treating or Preventing Stroke |
CN110248653A (en) * | 2017-01-05 | 2019-09-17 | 希莫加尼有限责任公司 | For treating the pharmaceutical admixtures of cerebral ischemia |
JP2020504181A (en) * | 2017-01-05 | 2020-02-06 | シモジャニ リミテッド ライアビリティー カンパニーShimojani, Llc | Drug regimen for treatment of cerebral ischemia |
JP7263249B2 (en) | 2017-01-05 | 2023-04-24 | シモジャニ リミテッド ライアビリティー カンパニー | Drug Regimens for Treatment of Cerebral Ischemia |
US12109265B2 (en) | 2017-01-05 | 2024-10-08 | Shimojani, LLC | Drug regimen for treatment of cerebral ischemia |
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JP2018158948A (en) | 2018-10-11 |
JP5823671B2 (en) | 2015-11-25 |
JP2015098474A (en) | 2015-05-28 |
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