JP6692816B2 - 炎症を処置および予防するための組成物および方法 - Google Patents
炎症を処置および予防するための組成物および方法 Download PDFInfo
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Description
本出願は、2014年9月5日に出願された米国特許仮出願第62/046,459号の優先性を主張する。この特許仮出願は参照によりその全体が本明細書に組み込まれる。
本出願は、「12810_513_ST25」という名称で2015年9月8日に作成された580キロバイトのサイズを有するコンピュータ可読形式の配列表を含む。このコンピュータ可読形式配列表は、参照により本明細書に組み込まれる。
該当なし。
本発明は、炎症に関する。より具体的には、本発明は、炎症の予防または処置のためのNRP1経路の阻害に関する。
本発明者らは、NRP1依存性細胞内シグナル伝達(および特に、SEMA3A媒介細胞内シグナル伝達)の阻害により、自然免疫応答の過剰活性化を伴うものなどの炎症性疾患および状態から保護(予防または処置)することが可能であることを見出した。特に、NRP1媒介細胞内シグナル伝達の阻害は、単核貪食細胞(MP、例えば、ミクログリア、マクロファージ)の望ましくない(病理学的)動員および組織損傷(例えば、増加した血管変性、病的な血管新生、細胞死または細胞損傷)、炎症および浮腫の一因となる炎症促進性サイトカインの産生/分泌を減らす。
NRP1媒介細胞内シグナル伝達の阻害は、本発明のNRP1トラップを使って達成することができる。本明細書で使用される場合、「NRP1トラップ」または「NRP1ポリペプチドトラップ」という用語は、天然の可溶性NRP1ポリペプチド(例えば、NRP1分泌アイソフォームb(図22、配列番号65))、および合成(例えば、組換えにより産生)NRP1ポリペプチドトラップを包含し、これらには、内在性NRP1とリガンド結合に関し競合する任意のNRP1の機能性可溶断片(例えば、NRP1アイソフォーム1または2)または任意のNRP1の機能性変異体が含まれる。一実施形態では、本発明のNRP1トラップは、自然界に存在しない(すなわち、天然ではない)が、天然NRP1ポリペプチドから「誘導」される(すなわち、それらは合成であり、例えば、NRP1アイソフォーム1またはその断片もしくは変異体の細胞外ドメインを含む)。本発明のNRP1トラップは、最初は、そのN末端にシグナルペプチドを含み(例えば、図26に示すNRP1(例えば、配列番号69)のアミノ酸1〜21(配列番号70))、これは細胞による分泌時に切断される。したがって、本発明のNRP1ポリペプチドトラップは、精製ポリペプチドとして投与される場合、または精製されたまたは実質的に純粋な形態を含む医薬組成物として調製される場合、アミノ酸1〜21を欠いている。本発明のNRP1トラップをコードする核酸(例えば、配列番号2、4、6、8、10、12、14、16、18、20、22、24、26、28、34、32、34、36、39、41、43、45、47、など。表1も参照されたい)は、NRP1トラップが細胞により合成され、分泌されるのを可能とするシグナルペプチド(N末端の最初の21アミノ酸をコードする最初の63ヌクレオチド)をコードするポリヌクレオチド配列を5’に含む。特定の実施形態では、シグナルペプチドは、配列番号65(図22)または配列番号69(図26)で示されるNRP1ポリペプチドの最初の20アミノ酸に対応する。本発明のNRP1トラップは、対応する「野性型」NRP1ポリペプチドまたはその断片(例えば、集団中で天然に見つかる多形変異体)の機能性変異体を包含する。
NRP1細胞活性は、NRP1の1つまたは複数のそのリガンド(例えば、SEMA3A、VEGFおよび/またはTGF−β)との結合を遮断する薬剤を使用して阻害することができる。このような薬剤の一例は、NRP1に結合し、NRP1のSEMA3A、VEGFおよび/またはTGF−βとの結合を遮断する抗体である。
NRP1またはそのリガンド(例えば、SEMA3A、VEGFまたはTGF−β)の発現を低下させて(mRNAレベルまたはタンパク質レベルで)、NRP1媒介細胞内シグナル伝達を阻害し、それにより、炎症および自然免疫応答の過剰活性化(すなわち、i)炎症促進性サイトカインの産生および/または分泌;ii)単核貪食細胞(MP)の動員;iii)血管透過性亢進化;および/またはiv)浮腫;v)神経損傷、脈絡膜新生血管など)を減らすための種々の手法を利用可能である。非限定的な例には、NRP1、SEMA3A、VEGFまたはTgf−βプロモーターなどを標的にする低分子ヘアピンshRNA(RNAi)、アンチセンス、リボザイム、TALエフェクタの使用が挙げられる。
本発明のNRP1依存性細胞内シグナル伝達を阻害する薬剤(すなわち、NRP1阻害剤)は、単独でまたは医薬組成物として、ヒト対象に投与することができる。医薬組成物の場合、標的疾患または状態を処置または予防するまたは所望の細胞の反応を生じる用量で、適切なキャリアまたは賦形剤(単一または複数)と、それらの薬剤を混合する。
好適な投与経路には、例えば、全身、経口および経眼(点眼薬または眼内注射)を含めてよい。好ましい投与経路としては、眼疾患に対しては点眼薬および眼内注射、慢性炎症状態に対しては経口投与、および敗血症および脳卒中などの特定の神経細胞疾患に対しては全身投与を含む。製剤はまた、持続放出製剤の形態であってもよい。
本発明での使用に適する医薬組成物には、活性成分がその使用目的を達成するための有効量で含まれる組成物が挙げられる。より具体的には、治療有効量は、治療対象の既存の症状の進行防止または緩和に対する有効量を意味する。有効量の決定は、当業者の能力の範囲内で適切に行うことができる。
分かりやすくするために、本発明において、次の用語の定義が適用される。
材料と方法(実施例2〜9および12)
LyzM−cre/Nrpfl/flマウスの生成。C57Bl/6野生型(WT)マウスをThe Jackson Laboratoryから購入した。LyzM−Cre(Lyz2tm1(cre)Ifo/J;no.004781)およびNRP1 floxedマウス(Nrp1tm2Ddg/J;no.005247)を、The Jackson Laboratoryから購入し、交配して、NRP1欠損骨髄細胞を有するLyzM−cre/Nrpfl/flマウスを得た。
NRP1が血管傷害に続いて動員される単核貪食細胞(MP)集団を特定する
ミクログリアまたはマクロファージなどのMP(単核貪食細胞)が増殖網膜症に関連する血管病変形成に加わるかどうかを判定するために、マウス全網膜に対し最初にFACS分析を行い、酸素誘導網膜症の進展全体(OIR、図1A、P7〜P12(生後7〜12日)に75%酸素下で血管喪失を誘導し、P17まで室内気下で最大網膜前血管新生を得る(26、33))(図1B、E、H)にわたるマクロファージ/ミクログリア蓄積の動力学を明らかにした。結果から、OIR中の解析した全時点で、P10(P=0.0004)(図1C)での血管喪失期中の36%の増加、P14での新生血管期中の63%の上昇(P<0.0001)(図1F)およびP17での最大血管新生中の172%の急増(P=0.0006)(図1I)を含む著しく高い数の網膜マクロファージ/ミクログリア細胞(Gr−1−、F4/80+、CD11b+細胞、データは示さず)が明らかになった。
NRP1+骨髄細胞は網膜中で病理学的血管新生部位に局在化する
OIR中のNRP1+マクロファージ/ミクログリアの顕著な流入を考慮して、疾患の進行中のこれらの細胞の局在化を次に測定した。網膜フラットマウントへの免疫蛍光法により、NRP1陽性マクロファージ/ミクログリア(IBA1およびNRP1で同時標識)は、OIRのP14での発生期病理学的タフト(図2A〜C)ならびにOIRのP17での成熟タフト(図2D〜F)と完全に結合することが明らかになった。図2Bおよび2E白色矢印は、網膜前タフトと結合したNRP1陽性MPを指す。以前に報告したように(21)、NRP1はまた、新生血管タフトの内皮上の内皮細胞にも発現した。図1に示すデータと一致して、LysM−Cre/Nrp1fl/flマウスは、より小さい数のマクロファージ/ミクログリア、およびそれほど多くない血管新生を有した(全体の定量化については下記を参照されたい)(図2G〜K)。
SEMA3Aは活動性増殖糖尿病網膜症に罹患している患者の硝子体中で上昇している
我々の知見と、網膜症でのMP走化性におけるNRP1の必須の役割との臨床的関連性を確かめるために、活動性PDRに罹患している患者の硝子体の内部のSEMA3Aの濃度をじかに決定した。17個の無希釈硝子体試料をPDRに罹患している患者から、および17個を非血管病変を有する対照患者から採取した。患者の詳細特徴は表1(実施例1)に含まれている。対照患者(20)は、非血管病変を呈し、線維性組織および斑状隆起(図3C)に続発する脈管構造への牽引張力(図3A、B(白色矢印))などの糖尿病に関連しない網膜損傷の徴候を示す。対照的に、PDR患者由来の全ての網膜は、乳頭(図3D)または網膜前血管新生(図3F)の徴候を示し、高度に透過性微小血管(蛍光染料の漏出)(図3D、G挿入図)、毛細血管瘤(図3D〜G)および繊維状瘢痕組織を有し、進行した網膜症を示す(図3G)。さらに、患者は、血管外漏出液の局所的合体による嚢胞形成(白色矢頭)を含む、損なわれた血管障壁機能が原因の黄斑浮腫のいくつかの証拠を示す(図3H)。
NRP1のリガンドは、OIR中に、網膜神経節細胞層中で誘導される
増殖網膜症での2つの主要なNRP1リガンド発現の正確な動力学的プロファイルを得るために、OIRマウスモデルにおけるSEMA3AおよびVEGFメッセージのレベルを測定した。全網膜に対するリアルタイム定量的PCR(RT−qPCR)は、SEMA3Aが、P10での高酸素圧(血管退行性)期およびP12〜P17での虚血/新生血管段階の両方の間に、OIRにおいて確実に誘導されたことを明らかにした(図4A)。観察された誘導は、野性型およびLysM−Cre/Nrp1fl/fl網膜の両方で発生した。逆に、予測したように、VEGF転写物は、P12〜P17のOIRの虚血性期においてのみ上昇した(図4B)。重要なのは、野性型網膜に比べて、VEGFのLysM−Cre/Nrp1fl/flにおける誘導が著しく少く、P12でわずかに増加し(P=0.0451)、P14で野性型OIRに比べて約55%低く(P=0.0003)(図4B)、これは、より健康な網膜であることを示すことである。
単核貪食細胞(MP)は血管傷害後、網膜中で増殖しない
NRP1+ MPの顕著な上昇が体循環からの流入によるのか、または網膜内のMP増殖の増加によるのかを明らかにするために、これらの細胞の局所的網膜増殖を調査した。マウスに、P13でBrdUを全身注入し(屠殺の24時間前)、網膜(図5A)および脾臓(図5B)のFACS分析を行った。網膜内で、Gr1−/CD11b+/F4/80+ MPは、顕著な増殖を示さなかった(P=0.4708)。脾臓中では、かなり多い増殖が観察された。正常酸素圧とOIRとの間では大きな差異は観察されなかった(図5C)。網膜中でのMPの増殖の欠除は、網膜症の間の顕著な付着物NRP1+ MPが、全身起原を有することを示唆している。
SEMA3AおよびVEGF165はNRP1を介してMPを動員する
骨髄細胞の血管病変の部位への動員(図1)のためのNRP1の要件ならびに網膜症におけるNRP1の主なリガンドの誘導(図3〜4)およびこれらの細胞の考えられる全身起原(図5)を考慮して、MP走化性を誘発するこれらのキューの性質を明確にした。初代マクロファージ培養物を野性型マウスから単離し、トランスウェルボイデンチェンバ遊走アッセイに供した。SEMA3A(100ng/ml)(p<0.0001)およびVEGF165(50ng/ml)(P=0.0027)の両方は、陽性対照MCP−1(100ng/ml)(p<0.0001)と類似の程度にマクロファージ走化性を誘発した(図6A、B)。これらのデータを、Y−27632:選択的阻害剤ROCK(Rho結合コイルドコイル形成タンパク質セリン/トレオニンキナーゼ)がそれらの走化特性を消滅させることを示して検証した。ROCKは、NRP1シグナル伝達の下流にあり(40)、単球遊走を媒介することが知られている(41)。VEGF遊走を、部分的に減少させ、それほど著しく減少させてはいないことは、最近報告されたVEGFR1などの別の受容体からの寄与を示唆している(33)。SEMA3AおよびVEGF媒介走化性におけるNRP1に対する役割と一致して、LysM−Cre/Nrp1fl/flマウス由来のマクロファージはMCP−1に特異的に応答し、SEMA3AまたはVEGFによっては動員されない(図6C)。
NRP1+マクロファージは、エクスビボ毛細血管新芽形成を増強する
NRP1発現マクロファージの毛細血管の血管新生に対する影響を調査するために、LysM−Cre/Nrp1+/+マウスまたはLysM−Cre/Nrp1fl/flマウス由来の脈絡膜層組織を単離し、マトリゲル(商標)中で増殖させて、毛細血管の新芽形成を評価した。LysM−Cre/Nrp1fl/flマウス由来の脈絡膜層は、LysM−Cre/Nrp1+/+マウスと比較して、約20%少ない微小血管を発芽する(P=0.018)(図7A)。毛細血管の新芽形成の促進におけるNRP1+マクロファージの役割を調査するために、クロドロン酸リポソームを使って、内在性マクロファージを単離脈絡膜層組織から除去した。LysM−Cre/Nrp1fl/flおよびLysM−Cre/Nrp1+/+マウスの両方由来の外植片では、リポソーム含有PBS(すなわち、ビークル対照)は、血管の新芽形成に対し影響を与えなかったが、クロドロン酸リポソームは、毛細血管の新芽形成を約60%低減させた(LysM−Cre/Nrp1+/+脈絡膜層ではP=0.0114、LysM−Cre/Nrp1fl/fl脈絡膜層ではP=0.0007)(図7B〜E)。NRP1+マクロファージが血管新生を促進する性質を有するかどうかを検証するために、腹膜マクロファージをLysM−Cre/Nrp1+/+またはLysM−Cre/Nrp1fl/flマウスから抽出し、前もってクロドロン酸リポソームで処理し、洗浄した脈絡膜層外植片培養液中に導入した。LysM−Cre/Nrp1+/+マクロファージは、LysM−Cre/Nrp1fl/flマウス由来のマクロファージに比べて、毛細血管の新芽形成を50〜100%確実に増強し(LysM−Cre/Nrp1+/+脈絡膜層ではP=0.0068、LysM−Cre/Nrp1fl/fl脈絡膜層ではP=0.0491)(図7DおよびE)、脈絡膜外植片の遺伝子型には無関係であった。
骨髄常在性NRP1の欠損は、網膜症における血管変性および病理学的血管新生を減らす
OIRの初期段階中のMP浸潤におけるNRP1細胞内シグナル伝達に必須の役割(図1)を考慮して、NRP1の骨髄細胞特異的除去の疾患の進行に与える影響を次に明確にした。P12で、75%O2から出す際に、LysM−Cre/Nrp1fl/flマウスは、野性型(P=0.0011)およびLysM−Cre/Nrp1+l+(P<0.0001)対照に比べて、著しく低レベルの網膜血管喪失を示した(8A、B)。これは、LysM−Cre/Nrp1fl/flマウスの網膜中に存在する、より低いレベルのIL−1βが原因である可能性がある(データは示さず)。重要なのは、P17で、病理学的血管新生ピークの場合(26)、骨髄常在性NRP1の欠失が無血管領域を大きく低減させたことである(野性型と比較した場合に約35%(P<0.0001)およびLysM−Cre/Nrp1+l+マウスと比較した場合に約30%(P=0.0008))(図8C、D)。さらには、虚血性網膜症と関連する有害な網膜前血管新生の大きな低減が観察された(野性型と比較した場合に約36%(P=0.0008)およびLysM−Cre/Nrp1+l+マウスと比較した場合に約34%(P=0.0013))(図8E、F)。
可溶性SEMA3A中和トラップの調製
SEMA3Aを阻害/中和するための高親和性トラップを生成した。これらのトラップは、ニューロピリン1(NRP1)から誘導し、任意選択で、6XヒスチジンタグまたはFCタンパク質と結合した(図19、20および27、および表1を参照)。SEMA3A結合を維持することができる全NRP1細胞外ドメインまたは機能性変異体を含む種々の変異体を生成した。b1ドメイン(これは、VEGFに結合する)を含み、中和VEGF165変異を含むトラップを生成した。トラップは高度に発現し、形質転換ヒト細胞中で分泌されることが示された。簡単精製および処方プロトコルを開発し、後ほど行うSARおよびインビボ効力調査用のトラップ試料を産生した。
方法
SEMA3AおよびVEGFに対するトラップの親和性
AP−VEGF165の産生。ヒトVEGF165変異体1(NM_001025366)のコード配列を、pAPtag5ベクター(GenHunter)に、アルカリフォスファターゼドメインとインフレームで、サブクローニングした(AP−VEGF165)。ポリエチレンイミン(PEI)遺伝子導入法を使って、HEK293T細胞にAP−VEGF165構築物を遺伝子導入した。一晩の形質導入段階後、無血清培地(In vitrogen)中で細胞をさらに60時間培養した。細胞培地を集め、PES装置(Pierce)で濃縮した。濃縮したAP−VEGF165リガンドをPAGE−SDSで分析し、SimplyBlue safe stain(Life technologies)を使って定量化した。
硝子体内への可溶性NRP1の治療投与が、網膜症におけるMP浸潤および病理学的血管新生を減らす
上記知見の技術移転の可能性を明確にするために、可溶性組換えマウス(rm)NRP1 mTrap1ポリペプチド(配列番号25のドメインa1、a2、b1、b2およびcを含む図19Cおよび20R)をOIR誘導NRP1リガンドを捕捉するためのトラップとして、次に採用した。P12でのrmNRP1の単回の硝子体内注射により、P14でのOIRに晒された網膜中に存在するミクログリアの数の30%の低減(P=0.0282)がもたらされる(図9A)。この知見は、可溶性NRP1(1μlの50μg/ml)のミクログリア動員を損なう効力を立証する。可溶性NRP1の硝子体内投与が、ビークル注射対照に比べて、病理学的網膜前血管新生の約40%の顕著な減少を誘発した(P=0.0025)(図9B、C)。上記の結果は共に、これらのデータが、NRP1のリガンドの中和が網膜症における有害な血管新生の低減に効果的な戦略であることを示唆している。
敗血症モデル−実施例14〜19に対する材料と方法
敗血症マウスモデル。研究は、カナダ動物管理協会による研究における動物使用に関するカナダのガイドラインによる規制に従って行われた。LPS注射を6〜8週齡のC57BL/6マウスの腹腔内(i.p)に行った。
セマフォリン3Aは、敗血性ショック中にいくつかの器官で発現上昇している
OIRにおけるSEMA3A、NRP1および自然免疫応答の間の関連(上記実施例2〜9で示すように)を考慮して、全身性炎症におけるNRP1依存性細胞応答の意味を次に評価した。これは、最初、敗血性ショック中のSEMA3A発現の動力学を測定することにより調査した。
SEMA3Aは骨髄細胞中でNRP1を介して炎症促進性サイトカインの分泌を誘導する
単球および骨髄細胞の急性炎症反応に対する寄与および骨髄細胞上のNRP1の存在を考慮して、炎症性サイトカインの産生におけるSEMA3Aおよび骨髄常在性NRP1の寄与を明らかにした。
骨髄常在性NRP1の欠損は、敗血症における炎症促進性サイトカインのインビボ産生を減らす
骨髄常在性NRP1はIL−6およびTNF−αなどのインビトロでの炎症促進性サイトカインの放出にとって重要であったので、インビボでのその寄与を次に調査した。LyzM/NRP1fl/flおよび対照野性型マウスにビークルまたはLPS(15mg/kg)を投与し、脳および肝臓をLPS注射の6時間後に集めた。TNF−α(図14A、C)およびIL−1b(図14B、D)レベルのリアルタイムPCR分析は、LyzM/NRP1fl/fl中のこれらのサイトカインの比較的大きい低下を示した。これらの結果は、敗血症のインビボ発生に対するNRP1およびそのリガンドの大きな寄与を明確に示す。
NRP1シグナル伝達の阻害が、敗血症誘導バリア機能破壊を防ぐ
しかし、臓器不全の一因となると思われる、重篤な敗血性ショックの病理学的機構の1つは、血液関門(肺の血液と空気、腎臓の血液と尿、肝臓の血液と胆汁、および脳中の体液分子)の損傷である。血液網膜関門の破壊におけるSEMA3Aの役割(46)および敗血症中のSEMA3Aの発現に関する本件の新規データを考慮して、ヒトNRP1の細胞外ドメイン由来のトラップによるSEMA3Aの中和の効果を評価した(トラップ−1(FC含まず)、図19B、配列番号83)。Evans Blue透過性(EBP)アッセイを使って、我々は、マウスが4ugのNRP1由来トラップ(0.2mg/kg、i.v.)で処置された場合、調査した全器官、すなわち、脳(図15A)、腎臓(図15B)および肝臓(図15C)において、LPS誘導バリア機能破壊の顕著な減少が観察されることを見出した。これらの結果は、可溶性NRP1およびその誘導体のトラップが敗血症を阻止するための注目すべき治療薬であることを強く示唆している。
NRP1由来トラップは、敗血症から保護する
敗血症中のNRP1リガンドの中和またはNRP1阻害の治療効果を明らかにするために、生存調査を行った。高用量のLPS(25mg/kg)をマウスに投与した。その後、マウスをモニターし、適切な終了点に到達すると、倫理的に屠殺した。第2の群では、マウスに4ugの組換えトラップ−1(FC含まず)(0.2mg/kg、図19Bおよび20A、配列番号83)をi.v.注射し、続いて、LPS腹腔内の注射を行った。対照群では、LPS注射後の最初の30時間以内に、5/5マウス(100%)が死亡した(図16A)。逆に、トラップで処置した全てのマウスが、30時間後でもまだ生存し、大きく改善された60時間後の生存率(3/5)を示した。したがって、死亡率は、30時間後の100%(対照群の)から、60時間後の40%(図16A)まで低下した。さらに、40%のトラップ処置マウスは、LPS注射の80時間後に生存したままであった。したがって、生存時間は、NRP1を通る細胞内シグナル伝達を阻害した場合、60%の場合で少なくとも2倍、40%の場合でほぼ3倍であった。
NRP1由来トラップは、敗血性ショックにおける炎症性サイトカインの産生を低下させる
NRP1−トラップの敗血性ショックにおける生存率に対する治療効果を考慮して、敗血性ショック中の炎症性サイトカインの産生に与えるNRP1リガンドの中和の影響を、次に明らかにした。野性型マウスに、i)ビークル(n=3)、ii)LPS(15mg/kg、n=3)またはiii)LPSおよびNRP1マウストラップ1(FC不含、図19C、配列番号25(FC領域不含))を投与し、LPS注射6時間後に脳を採集した。NRP1トラップ−1の注射は、TNF−α(図17A)およびIL−6(図17B)の産生を顕著に低減させた。同様に、NRP1欠損骨髄細胞(LyzM−Cre/Nrpfl/fl)を有するマウス(n=3)は、大幅に少ないTNF−αおよびIL−6を産生し、この細胞経路の敗血性ショックの進行に対する寄与を明らかにした。
実施例21に記載の脳虚血/脳卒中モデルの材料と方法
この調査で使用したマウスは、2〜3月齢の雄C57Bl/6マウス(22〜28g)であった。
NRP1−トラップは、脳虚血および脳卒中から保護する
SEMA3A中和が脳虚血または脳卒中に与える結果を評価するために、成体(8〜12週齢)マウスを一時的な中大脳動脈閉塞(MCAO)モデルとした。実験の詳細は、実施例20で提供される。簡単に説明すると、MCAOの終了後、マウスNRP1−トラップ(トラップ−1(FC不含))の125ulのPBS中の0.4ug(図19C、図20R、配列番号25)を尾静脈に投与した(再灌流開始の約15分後)。MCAOにより誘導された脳損傷を可視化するために、冠状大脳切片をクレシルバイオレットで染色した。それぞれの切片上で、未染色領域は、脳の虚血領域に対応する(図18A)。連続冠状大脳切片のこれらの領域の測定により、MCAOの24時間後に、梗塞ゾーンは、脳が損傷されていないシャム手術動物に比べて、閉塞マウスにおける同側半球の48%になることが明らかになった。NRP1処置は、脳損傷を低減した;同側半球の梗塞体積を80%低減した(図18B、C)。
糖尿病性網膜症および未熟児網膜症のマウスモデルで、虚血性網膜におけるニューロピリン由来トラップは、血管再生を強化し、病理学的血管新生を防ぐ
酸素誘導された増殖網膜症(OIR)のよく確立されたマウスモデル(Smith et al.,1994)を使って病理学的血管変性ならびに網膜前血管増殖を調査した。このモデルは、未熟児網膜症(ROP)を基準にしており、増殖性(血管新生期)の糖尿病性網膜症およびROPの代用として、度々使われる。
ニューロピリン由来トラップは、糖尿病性網膜における血管漏出を減らす
糖尿病性網膜症におけるトラップの血管漏出/透過性に対する効果は、1型糖尿病のストレプトゾトシン(STZ)モデルにおいても調査された。STZ(55mg/kg)を約6週齢のC57BL/6Jマウスに連続5日間にわたって投与し、血糖をモニターした。マウスは、非絶食状態での血糖が17mM(300mg/dL)より高い場合に糖尿病とみなした。マウスに、0.5ug(0.5ug/ul)のトラップG(配列番号38)またはトラップM(配列番号42)を、またはマウス抗VEGF抗体(AF−493−NA、R&Dから入手)を、STZ投与の6および7週後に、硝子体内に投与した。あるいは、STZ投与後12および13週目にマウスの硝子体内に注射し、血管透過性を14週目に評価した。マウスは、SEMAトラップ(図24A参照)または抗VEGF抗体の硝子体内注射の少なくとも3週前には、高血糖性/糖尿病性であった。STZ注射の8週後に、Evans Blueアッセイにより網膜血管漏出を以下のように測定した。網膜Evans Blue(EB)透過性を、読み当たり3個の網膜を使って実施した。45mg/kgのEvan Blueを静脈内に注射し、網膜抽出の前に、2時間にわたり循環させた。網膜におけるEvans Blue透過性を、TECAN Infinite(登録商標)M1000PROを使って、蛍光定量法(620nm最大吸光度−740nm最小吸光度(バックグラウンド))により定量化した。Evan Blue透過性(EBP)[uL/(グラム*時間)として測定]を次のように計算した:[EB(ug)/湿潤網膜重量(g)]/[血漿EB(ug/uL)*循環時間(時間)]。Evans Blue透過性を対照と比較して示した。
ニューロピリン由来トラップは、加齢黄斑変性モデルの脈絡膜新生血管を低減する
NRP1トラップG(配列番号38)の脈絡膜新生血管(CNV)に対する効果を、加齢黄斑変性(AMD)のマウスモデルで測定した。マウスのCNV、したがって、模倣湿潤AMDを誘導するために、6〜8週齡のマウス(1〜2乳頭直径)に、コヒーレントスリットランプ(400mW、50msおよび50μm)上にマウントしたアルゴンレーザー(532nm)を使って乳頭からレーザー凝固術を行った(Combadiere et al.,2007)。レーザー照射後、処置マウスに0.5ugのトラップGを硝子体内に注射した。14日(P14)後、脈絡膜層を半径方向に切開し、フラットマウントし、内皮細胞マーカーの蛍光標識イソレクチンB4で染色し(また、動物を任意選択で、フルオレセインデキストランで潅流し、管腔内血管を可視化した)、レーザー走査型共焦点顕微鏡によりCNVの体積を測定した(Takeda et al.,2009)。
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Claims (10)
- 対象における、敗血症を予防または処置するための組成物であって、
前記組成物は、セマフォリン3A(SEMA3A)へのニューロピリン−1(NRP1)の結合を阻害する物質を含んでなり、当該物質は;
(i)NRP1トラップであり、このNRP1トラップは、ヒトNRP1のドメインa1及びa2のアミノ酸配列(配列番号:66のアミノ酸22〜275)を含むNRP1ポリペプチドであるか、又は当該NRP1ポリペプチドの機能性断片若しくは機能性変異体であって前記アミノ酸配列に対して少なくとも90%の同一性を有するアミノ酸配列を含むものであり、或いは
(ii)抗−NRP1抗体である、
前記組成物。 - 前記NRP1トラップが、
(a)ヒトNRP1のドメインa1、a2、b1、b2及びcのアミノ酸配列(配列番号:66のアミノ酸22〜859)を含むNRP1ポリペプチドであるか、又は当該NRP1ポリペプチドの機能性断片若しくは機能性変異体であって前記アミノ酸配列に対して少なくとも90%の同一性を有するアミノ酸配列を含むもの;
(b)ヒトNRP1のドメインa1、a2、b1及びb2のアミノ酸配列(配列番号:66のアミノ酸22〜589)を含むNRP1ポリペプチドであるか、又は当該NRP1ポリペプチドの機能性断片若しくは機能性変異体であって前記アミノ酸配列に対して少なくとも90%の同一性を有するアミノ酸配列を含むもの;又は
(c)ヒトNRP1のドメインa1、a2及びb1のアミノ酸配列(配列番号:66のアミノ酸22〜428)を含むNRP1ポリペプチドであるか、又は当該NRP1ポリペプチドの機能性断片若しくは機能性変異体であって前記アミノ酸配列に対して少なくとも90%の同一性を有するアミノ酸配列を含むもの;
を含んでなる、請求項1に記載の組成物。 - 前記NRP1トラップが、ヒトNRP1のドメインa1、a2及びb1のアミノ酸配列(配列番号:66のアミノ酸22〜428)を含むNRP1ポリペプチドであるか、又は当該NRP1ポリペプチドの機能性断片若しくは機能性変異体であって前記アミノ酸配列に対して少なくとも90%の同一性を有するアミノ酸配列を含むものである、請求項2に記載の組成物。
- 前記NRP1トラップが、ヒトNRP1のドメインa1、a2、b1及びb2のアミノ酸配列(配列番号:66のアミノ酸22〜589)を含むNRP1ポリペプチドであるか、又は当該NRP1ポリペプチドの機能性断片若しくは機能性変異体であって前記アミノ酸配列に対して少なくとも90%の同一性を有するアミノ酸配列を含むものである、請求項2に記載の組成物。
- 前記NRP1ポリペプチドが、完全に又は部分的にヒトNRP1のドメインb2(配列番号:66のアミノ酸429〜589)を欠く、請求項2に記載の組成物。
- 前記NRP1ポリペプチドが、完全に又は部分的にヒトNRP1のドメインc(配列番号66:のアミノ酸590〜859)を欠く、請求項5に記載の組成物。
- 前記NRP1トラップが、ヒトNRP1のドメインa1、a2及びb1のアミノ酸配列(配列番号66:のアミノ酸22〜428)を含むNRP1ポリペプチドを含んでなる、請求項1〜6のいずれか1項に記載の組成物。
- 前記NRP1トラップがFcドメインをさらに含む、請求項1〜7のいずれか1項に記載の組成物。
- 前記Fcドメインは、配列番号37のアミノ酸配列を含む、請求項8に記載の組成物。
- 対象の障害部位における、IL-6、IL-1βおよびTNFαの分泌を低減し、および/または単核貪食細胞(MP)の動員を低減する、請求項1〜9のいずれか1項に記載の組成物。
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