JP6353654B2 - 抗血管新生剤及びこのような薬剤の使用方法 - Google Patents
抗血管新生剤及びこのような薬剤の使用方法 Download PDFInfo
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- JP6353654B2 JP6353654B2 JP2013519804A JP2013519804A JP6353654B2 JP 6353654 B2 JP6353654 B2 JP 6353654B2 JP 2013519804 A JP2013519804 A JP 2013519804A JP 2013519804 A JP2013519804 A JP 2013519804A JP 6353654 B2 JP6353654 B2 JP 6353654B2
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Classifications
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
- A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Landscapes
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Description
本出願は、2010年7月13日に出願された米国仮特許出願第61/363,933号の優先権を主張するものであり、この出願は、参照により本明細書に組み込まれる。
本開示は、血管増殖を特徴とする又は血管増殖に依存する状態である血管新生依存性状態を制御又は治療する手段としての、血管新生の阻害又は防止に関する。本開示は更に、抗血管新生剤と化学療法剤との併用に関する。
以下の定義は、本明細書の全体を通じて使用する特定の用語の理解を容易にするために示す。
本開示は、抗血管新生剤、及び、著しい毒性を生じさせずに状態の退縮又は阻止をもたらす治療有効量及び頻度で哺乳動物に抗血管新生剤を投与することによって哺乳動物の依存性状態を治療する、血管新生の阻害方法を提供する。血管新生依存性状態は、新生物、例えば、固形腫瘍新生物、例えば、乳がん、肺がん、前立腺がん、結腸がん、前立腺がん、卵巣がん、神経芽細胞腫、中枢神経系腫瘍、神経芽細胞腫、多形性神経膠芽細胞腫又は黒色腫からなる群から選択され得る。リストは完全とはいえないが、この抗血管新生剤は、全部ではないがほとんどの固形腫瘍の退縮又は阻止をもたらすのに使用できる。治療を受ける哺乳動物は、ヒトであり得る。
〔実施例1〕
抗血管新生剤を、細菌E.coliから発現させ、精製した。設計したポリペプチドが依然として適切に折り畳まれることを確認するのを助けるために、1H−NMR分析で構造を確認した。抗血管新生剤(120μM)とCD2−D1(120μM)のNMRスペクトルを比較した。図2に示すように、抗血管新生剤のNMRスペクトルはCD2−D1(上部)のNMRスペクトルとほぼ同一であったが、ほどかれた(折り畳まれていない)ポリペプチド(有機溶媒によって)は完全に異なるスペクトル(下部)を示した。得られたポリペプチドは、宿主タンパク質及び開発されたタンパク質の両者の1H−NMR、CD及び蛍光スペクトルの類似性によって示されるように、非常に類似した構造特性を示した。
〔実施例2〕
内皮細胞に対する抗血管新生剤の効果を判定するために、HUVEC細胞を用いて細胞生存率アッセイを行った。種々の濃度の開発した抗血管新生剤の一例、anginex、及びその抗血管新生剤が由来する宿主ポリペプチドで、細胞を処理した。図3Aに示すように、抗血管新生剤は、HUVEC細胞のアポトーシス誘導においてより効果的であった(図2A)。我々は更に、効果が内皮細胞に特異的か否か試験した。このために、5μM又は10μMの抗血管新生剤、Anginex及び宿主タンパク質の存在下でHUVEC細胞、M4A4細胞を用いて、細胞増殖アッセイを行った。図3B及び9に示すように、HUVEC細胞の場合には薬剤によって細胞増殖の強い阻害が観察されるが、上皮M4A4細胞の場合には効果が観察されないことが明白であった。これらの観察から、抗血管新生剤の効果が内皮細胞に特異的であることが示された。
〔実施例3〕
HUVEC細胞に対する成長阻害及びアポトーシス誘導において、抗血管新生剤の活性が強いことは明らかであった。PC−3細胞の異種移植片モデルは、免疫不全マウスを用いて調製した。担腫瘍マウス(マウス6匹/群)を、抗血管新生剤(10mg/kg)、PEG化抗血管新生剤(10mg/kg)、宿主タンパク質(10mg/kg)及び緩衝生理食塩水を用いて一日量によって2週間治療した。治療は、腫瘍接種の7日後に開始した。腫瘍は、体積によって又は腫瘍細胞の生物発光によって測定した。
〔実施例4〕
図7は、治療マウスから採取した腫瘍組織切片の免疫蛍光染色を用いて観察した血管密度が、抗血管新生剤とPEG化抗血管新生剤による治療後にはPEG化宿主タンパク質及び緩衝液によって治療した群と比較して劇的に減少したことを示している。腫瘍血管に対する抗血管新生剤治療の効果が実際にあったかどうかを判定するために、治療後の腫瘍を収集した。収集した腫瘍から、組織スライドを調製した。スライドを、内皮細胞に特異的な分子マーカーであるCD31に対する抗体で免疫染色した。腫瘍組織スライドの免疫染色を、共焦点顕微鏡によって可視化した。結果から、抗血管新生剤は腫瘍血管新生に特異的な影響を及ぼすことが示された。
〔実施例5〕
CD2のドメイン1の親タンパク質の毒性は、以前に分析され、マウスでは毒性がなかった。新しい設計がタンパク質の毒性を変えなかったことを確認するのを助けるために、抗血管新生ポリペプチドの毒性を、CD−1マウスを用いて検討した。まず、担腫瘍ヌードマウスの体重を、14日間の治療過程において慎重に測定した。図8に示すように、どの治療群でも、マウス体重に有意な変化は観察されなかった。更に、毒性を、正常CD−1マウスで試験した。3つの群のマウス(マウス7匹/群)に、各注射間の間隔を3日間として、100μlのポリペプチド(100mg/kg、使用投与量の20倍)を静脈内に1用量、2用量及び3用量注射した。動物を30日間、それらのケージに戻した。試験したマウスに、死亡は観察されなかった。全ての動物は、正常な挙動を示した(食餌習性に変化はなく、異常な重量増加又は減少はなく、毛皮に異常外観はなかった)。
〔実施例6〕
配列番号11で示されるポリペプチドを、ピキア・パストリス(Pichia pastoris)発現系から発現させ、精製した。酵母ピキア・パストリスにおけるこのポリペプチドの発現は、細胞内発現及び分泌発現の両方によって行った。このポリペプチドを、イオン交換カラムを用いて更に精製した。ポリペプチドは、Hisタグポリペプチドとして発現された。Hisタグを、トロンビン切断によって除去した。酵母ピキア・パストリスから発現させ、精製したグリコシル化タンパク質は、抗血管新生剤であり、N−結合グリコシル化されていることがわかった。図13に示すように、抗血管新生剤の種々の例によって治療された細胞は、強い生存能を示した。
〔実施例7〕
配列番号1は、ラット由来のCD2のドメイン1(WTラットCD2−D1)のアミノ酸配列である。
RDSGTVWGAL GHGINLNIPN FQMTDDIDEV RWERGSTLVA EFKRKMKPFL KSGAFEILAN GDLKIKNLTR DDSGTYNVTV YSTNGTRILN KALDLRILE
KEITNALETWGALGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQER
RDSGTVQMKL GHGINLNIPN FQMTDDIDEV RWERGSTLVA EFKRKMKPFL KSGAFEILAN GDLKIKNLTR DDSGTYNVTV YSTNGTRILN KALNLKVII
RDSGTVKWKA GHGINLNIPN FQMTDDIDEV RWERGSTLVA EFKRKMKPFL KSGAFEILAN GDLKIKNLTR DDSGTYNVTV YSTNGTRILN KALSLDVNI
RDSGTEVIKA GHGINLNIPN FQMTDDIDEV RWERGSTLVA EFKRKMKPFL KSGAFEILAN GDLKIKNLTR DDSGTYNVTV YSTNGTRILN KALKLTAIL
RDSGTVQMKL GHGINLNIPN FQCTDDIDEV RWERGSTLVA EFKRKMKPFL KSGAFEILAN GDLKIKNLTR DDSGTYNVTV YSTNGTRILN KALNLKVII
RDSGTVWGAL GHGINLNIPN FQMTDDIDEV RWERGSTLVA IFVRLGSVKM KPLLKSGAFE ILANGDLKIK NLTRDDSGTY NVTVYSTNGT RILNKALDLR ILE
RDSGTVWGALGHGINLNIPNFQMTDDIDEVRWERGSTLVANFKRKQKPSL KSGAFEILANGDLKIKNLTRDDSGTYNVTVYSTNGTRILNKALDLRILE
KEITNALSVQMKLGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYELLKNGALKIKHLKTDDQDIYKVSIADTKGKNVLEKIFNLKVII
KEITNALSVQMKLGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYELLKNGALKIKHLKTDDQDIYKVSIADTKGKNVLEKIFNLKVII
KEITNALSVQMKLGQDINLDIPSFQMSDDIDDIKWEKTSDKKKIAQFRKEKETFKEKDTYKLFKNGTLKIKHLKTDDQDIYKVSIADTKGKNVLEKIFNLKVII
〔実施例7〕
図10は、14日間以上の治療期間における腫瘍の増殖曲線を示す。結果は、配列番号10をコードするポリペプチドから開発されたhProAgio又はAngio4が、腫瘍成長の抑制において効果的であったことを示している。実験は、PC−3異種移植片を使用して、hProAgio又はAngio4(10mg/kg、一日量)及び対照としての緩衝生理食塩水を用いて行った。治療は、腫瘍接種の8日後に開始した。
〔実施例8〕
抗血管新生ポリペプチドの有効性を更に試験するために、PC−3異種移植片に関してrProAgio−PEG又はAngio2(20mg/kg、一日量)及びAvastin(20mg/kg、2日毎に1用量)を用いて実験を実施し、分析した。治療は、腫瘍接種の21日後に開始した。図11は、Avastin(登録商標)及びrProAgio−PEG又はAngio2の腫瘍成長曲線である。図12は、14日間の治療過程の終了時における腫瘍重量のグラフを示す。各治療群の腫瘍を摘出し、秤量した。Angio2及びAvastin(登録商標)で治療された動物群の腫瘍の重量及び成長には、有意差が認められた。
Claims (11)
- 個体における血管新生を低減するための、ヒト又はラット由来の改変されたCD2ポリペプチドのドメイン1のバリアントの有効用量を含有する医薬組成物であって、前記改変されたCD2ポリペプチドのドメイン1のバリアントが、配列番号3、配列番号6、配列番号9、配列番号10及び配列番号11からなる群から選択される、医薬組成物。
- 前記バリアントが、腫瘍成長、アテローム性動脈硬化症、糖尿病性網膜症、加齢性黄斑変性症及び水晶体後線維増殖症から選択される障害に関連する血管新生を低減する、請求項1に記載の医薬組成物。
- 静脈内、固形腫瘍の内部又は周囲、全身、動脈内、眼内、腹膜内及び局所から選択される経路により投与される、請求項1に記載の医薬組成物。
- 内皮細胞のアポトーシスを誘導する、請求項1に記載の医薬組成物。
- 治療有効量又は予防量の化学療法剤を更に含む、請求項1に記載の医薬組成物。
- 放射線療法、治療有効量又は予防量の第2の薬剤を更に含む、請求項1に記載の医薬組成物。
- 改変されたCD2ポリペプチドのドメイン1のバリアントからなる抗血管新生ポリペプチドであって、前記改変されたCD2ポリペプチドのドメイン1のバリアントが、配列番号3、配列番号6、配列番号9、配列番号10及び配列番号11からなる群から選択される、ポリペプチド。
- ポリエチレングリコール(PEG)部分を更に含む、請求項7に記載のポリペプチド。
- グリカン部分を更に含む、請求項7に記載のポリペプチド。
- 請求項7に記載のポリペプチドをコードする核酸。
- 請求項7に記載のポリペプチドをコードする核酸を酵母中において発現させるステップを含み、発現の際に前記ポリペプチドがグリコシル化される、改変されたCD2のドメイン1のバリアントからなる抗血管新生ポリペプチドの調製方法。
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