JP2011512364A - 治療用ペプチド - Google Patents
治療用ペプチド Download PDFInfo
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- JP2011512364A JP2011512364A JP2010547006A JP2010547006A JP2011512364A JP 2011512364 A JP2011512364 A JP 2011512364A JP 2010547006 A JP2010547006 A JP 2010547006A JP 2010547006 A JP2010547006 A JP 2010547006A JP 2011512364 A JP2011512364 A JP 2011512364A
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Abstract
Description
1.1 細胞株および培養
HT29細胞(結腸直腸腺癌)、DU145細胞(前立腺癌腫)、MCF-7細胞(乳腺癌)を、American Type Culture Collection(ATCC,Manassas,VA,United States)から入手した。MKN45(胃癌腫)を、Cancer Research Laboratory,University of New South Wales,Sydney,Australiaから入手した。全ての細胞株を、10%(v/v)濾過ウシ胎仔血清(FCS;Invitrogen)および20mM Hepes(ThermoTrace,Melbourne,Vic,Australia)を含むDMEM培地(Invitrogen,Carlsbad,CA,United Sates)中に維持した。細胞株を、37℃にて、5%CO2を含む湿潤雰囲気中に維持した。細胞を、0.05%トリプシンおよび0.5mM EDTA(Invitrogen)を含む溶液を用い、コンフルエントになる前の密度で継代培養した。
トリプシン処理した生細胞の単細胞懸濁液を、ウシ胎仔血清(FCS)を含む100μLの容量の適切な培地中(例えば、10%(v/v)FCS、グルタミン、Hepesおよび抗生物質を含むDulbecco’s Modified Eagles Medium(DMEM))、1ウェル当たり2×103細胞の密度で96ウェル組織培養プレートに播種した。試験することになるペプチドデンドリマーの各濃度に対し、3つで1組のウェルを調製した。非処理細胞または培地のみを含む追加のサンプルウェルを各処理プレートに設け、参照コントロールとして並行して処理した。処理していない細胞および培地のみのウェルのゼロ時のプレートを同時に調製し、処理プレートへの試験ペプチドデンドリマーの添加の際に、このプレートに対してMTTアッセイを実施した。試験デンドリマーの添加前に、全てのプレートを24時間にわたり培養した。MTT溶液を調製するために、100mgのMTT(3−(4,5−ジメチルチアゾリル−2)−2,5−ジフェニルテトラゾリウムブロミド)(Cat#.M−128,Sigma,St Louis MO.)を、pH7.4の20mLのPBSと混合する。結果として生じる溶液を濾過滅菌(0.2μM シリンジフィルター)し、光から保護された状態で、使用するまで4℃にて保管する。MTT基質が増殖細胞中で開裂し、水に不溶性の塩を生じる。塩結晶の可溶化後、着色生成物が生成され、これを測定することにより培養細胞の増殖活性を定量し得る。
HT29細胞を、0.5%トリプシンEDTA(Invitrogen)を用いて採取し、5000細胞を200μL中に再懸濁させ、きれいなNUNC組織培養処理済み96ウェルプレート(NUNC)の各ウェルに加えた。細胞を10%(v/v)ウシ胎仔血清(FCS,Invitrogen)、1%L−グルタミン(Invitrogen)および2%1M HEPES緩衝液(Invitrogen)を補充したDMEM培地(Invitrogen)に播種した。次いで、細胞を一晩中37℃にてインキュベートした。翌日、この増殖培地を1%L−グルタミンおよび2%1M HEPES緩衝液を補充した100μLの血清非含有のDMEMと交換し、さらに24時間にわたり37℃にてインキュベートした。デンドリマーの最終濃度が0.1〜20μLAの範囲となるように、デンドリマーを試験ウェルの血清非含有培地中に加えた。各ウェルの総容量は200μLであり、細胞を5分〜4時間の範囲の時間にわたり37℃にてインキュベートした。細胞を血清による刺激に供した試験において、22μL FCS(10%(v/v)最終濃度)を37℃での培養期間の最後の10分間、ウェルに加えた。血清非含有培地(SFM)のみ(22μL)を、血清により刺激されていない細胞に加えた。
βインテグリンサブユニットβ3、β5またはβ6へのERK2の結合のいずれかに対するポリペプチドインヒビターのモノマー単位に連結されたリジン枝分かれ単位を含む図3に図示されている種類のペプチドデンドリマーを、以下の実施例において用いた。
ポリペプチドRSKAKNPLYR(配列番号6)を4(Dend4)または8(本明細書においてデンドリマーIK248、Dend8またはDend8 10(4)と呼ばれる)モノマー単位含むペプチドデンドリマーが、実施例1.2に記載されたMTTアッセイによって評価される場合に、HT29結腸癌細胞の増殖を阻害することが見出された。特に、IK248は、上記ポリペプチドを4モノマー含むペプチドデンドリマーと比べて、細胞成長/増殖を阻害するのに実質的により効果的であることが見出された(24時間のインキュベーション期間)。
HT29癌細胞の細胞質ゾル中への血漿細胞膜を超えてのポリペプチドの進入を促進するための異なるペプチド促進因子部分と連結させたポリペプチドRSKAKNPLYR(配列番号6)を含む種々の薬剤と比較される、実施例2に記載されたペプチドデンドリマーIK248の、HT29癌細胞中のERKの成長因子媒介性(すなわち、FCSにより刺激された)活性化の阻害における有効性を評価した。5μM IK248(Dend8)またはポリペプチド−促進因子部分薬剤での1時間にわたる処理(いずれも、5μM、同じ継続時間)後の細胞におけるホスホ−ERKレベルを、基本的に実施例1.3に記載されるとおりに測定した。利用した促進因子部分は、シグナルペプチドフラグメントAAVALLPAVLLALLA(配列番号14)、TAT−GペプチドGRKKRRQRRRPPQG(配列番号15)、改変貫通配列Tr−Pen RRQKWKKG(配列番号16)、および貫通ペプチドRQIKIWFQNRRMKWKKCS−SC(配列番号17)(ここで、S−Sは、隣接するシステイン残基間のジスルフィド架橋を示している)であった。
4.1 細胞培養および処理条件
ペプチドRSKAKNPLYR(配列番号6)、すなわちペプチドRSKAKNPLYR(配列番号6)を8(IK248)または10(IK248B)モノマー単位提示するリジン枝分かれ単位を含む図3に図示されている種類のペプチドデンドリマーを本試験で利用した。ホスホ−ERKレベルを、実施例1.3に記載されるとおりActive Motif FACE ERK1/2 ELISAキット(Australian Biosearch,WA,Australia)を利用して、ELISAにより評価した。
4.2.1 成長因子の不在下におけるホスホ−ERKレベル(HT29細胞をHI FCSで刺激せず)
用量応答試験において、より低い濃度のIK248デンドリマーで処理(1μM〜10μM、4時間のインキュベーション)したHT29細胞におけるホスホ−ERKのレベルはコントロール細胞と比較して増大することが見出され、それによりデンドリマーによる基礎の刺激されていないERKの活性化が示された。さらに、5μM IK248デンドリマーと共にインキュベートしたHT29細胞は、評価された全ての時点(5分、10分、30分および1時間)でコントロール細胞と比較してホスホ−ERKレベルの著しい上昇を示した。
HI FCSでの刺激に続き、HT29コントロール細胞におけるホスホ−ERKレベルは約3倍上昇すると認められた。20μM IK248で処理(1時間のインキュベーション)した細胞において、ホスホ−ERKレベルは、このデンドリマーで処理したFCS非刺激細胞におけるよりも低かったが、デンドリマーでの処理もFCSでの刺激も行わなかったコントロール細胞におけるホスホ−ERK基礎レベルよりも高いままであった(図5参照)。全ERKレベルは、IK248デンドリマーで処理したHT29細胞において本質的に影響されないままであった。
HT29細胞を、選択されたデンドリマーの存在下で48時間にわたって培養し、細胞の増殖を、基本的に実施例1.2に記載されるとおりにMTTアッセイにより評価した。結果を、コントロール細胞(デンドリマーで処理されない)に対するパーセント増殖として計算した。
HT29細胞をペプチドデンドリマーIK248Bで処理した。その結果を図4に示す。図から分かるように、デンドリマーにより細胞の増殖が阻害された。
ペプチドRSKAKNPLYR(配列番号6)を9(Dend9(10)4)または12(Dend12 10(4))モノマー単位提示する上記スキーム3に記載される種類のデンドリマーを、HT29細胞の増殖を阻害する能力について評価した。図5に示されるように、Dend12は、細胞増殖の阻害においてDend9より効果的であった。デンドリマーIK248B(ペプチドRSKAKNPLYR(配列番号6)を10モノマー単位提示する)と比較される場合、Dend12 10(4)は、IC50値に僅かな改善(1.8μMに対し1μM)を示したが、全滅に必要とされるデンドリマー濃度(すなわち、Dend12(10)4およびIK248Bの両方について10μM)の増加は全く得られなかった。デンドリマーIK248Bは、同様に、デンドリマーIK248(RSKAKNPLYRペプチド(配列番号6)を8モノマー単位提示する)よりも効果的であった(それぞれ、1.8μMおよび5μMのIC50)。
RSKAKNPLYRペプチド(配列番号6)のモノマー単位が完全にD−アミノ酸から成り、それらのカルボキシ末端にて2つのポリエチレングリコール(PEG)単位でペグ化されたペプチドデンドリマーIK248B(改変(Mod.)IK248BまたはPeg2 Dend10 D−10(4)と同定される)の、ヒトHT29細胞の増殖の阻害における有効性を、シスプラチン、イリノネクチン(irinonectin)(CPT−11)および5−フルオロウラシル(5FU)と比較した。細胞の増殖をMTTアッセイにより評価した。その結果を図6に示す。図から分かるように、改変IK248Bデンドリマーは、シスプラチン、CPT−11および5FUのみと比べてかなり大きな細胞増殖阻害をもたらした。
HT29結腸癌細胞を、β3ベースの10マーペプチド(RARAKNPLYK(配列番号7))(Dend8 β3)またはβ5ベースのペプチド(RSRARNPLYR(配列番号8)(Dend8 β5と同定される)を8モノマー単位提示する図3に図示されている種類のペプチドデンドリマーで処理した。試験細胞を、48時間にわたってデンドリマーに暴露し、細胞の増殖を、基本的に上記実施例1.2に記載されるとおりにMTTアッセイを用いて評価した。吸光度を、マイクロタイタープレートリーダーを用いて550nmで読み取り、試験細胞の増殖のパーセント阻害を、非処理コントロール細胞と比較して計算した。結果を図7に示す。図から分かるように、デンドリマーDend8 β3およびDend8 β5の両方が、HT29癌細胞の増殖を阻害したが、Dend8 β5はより効果的であった。
7.1 材料
HT−29ヒト結腸直腸腺癌細胞の培養のための試薬を以下の供給業者から入手した:RPMI 1640細胞培養培地、FBSおよびHBSSをInvitrogen Australia(Mt Waverley,VIC,Australia)から入手;ペニシリン−ストレプトマイシン、リン酸緩衝化生理食塩水(PBS)およびトリパンブルーをSigma−Aldrich(Castle Hill,NSW,Australia)から入手。
HT29ヒト結腸直腸腺癌細胞を、10%(v/v)FCSおよび50IU/mLペニシリン−ストレプトマイシンを補充したRPMI 1640細胞培養培地で培養した。細胞をトリプシン処理により採取し、HBSSで2回洗浄し、計数した。次いで、最終濃度が2×107細胞/mLとなるように細胞をHBSSに再懸濁した。
種:マウス(Mus musculus)
株:BALB/c nu/nu
供給者:University of Adelaide(Waite Campus,Urrbrae,SA,Australia)
試験における動物の総数:雌20匹
試験群数:2群(1試験群、1コントロール群)
1群当たりのマウス数:10匹
体重範囲:処置開始時20.61〜25.27g(平均22.27g)
週齢範囲:処置開始時10〜12週
接種前に、注射部位(背右脇)の皮膚にアルコールを塗った。動物の右肩直下の皮下空間に皮膚を通して針を挿入し、100μLの細胞(2×106細胞)を吐出させた。マウスの処置は、HT29細胞接種の9日後に開始し、平均腫瘍体積は68mm3(平均変動性6.1%)であった。
体重および腫瘍寸法(長さおよび直径)を、全ての動物について処置の初日(0日目)に測定し、次いで週に3回(試験の終了日(24日目)を含む)測定した。
試験の0日目に、マウスを体重に基づき無作為抽出し、2つの10匹のマウスから成る群に分けた。ペプチドRSKAKNPLYR(配列番号6)を10モノマー単位提示するペプチドデンドリマーIK248B(実施例8参照)を、本試験に用いた。ビヒクルコントロール(リン酸緩衝化生理食塩水(PBS))およびIK248Bデンドリマー(20mg/kg)の各々を、0日目に開始し、1日1回、5日間連続して腫瘍内注射により投与した。
死後に全てのマウスから腫瘍を摘出し、計量した。腫瘍体積を以下の方程式を用いて計算した。
V(mm3)=長さ×直径2×p/6
腫瘍の変動性を以下の方程式を用いて計算した。
変動性(%)=(標準誤差腫瘍体積/腫瘍体積平均)×100
ΔT/ΔC(%)を以下の方程式を用いて計算した。
ΔT/ΔC%=(Δ体積処置/Δ体積コントロール)×100
式中、Δ=0日目から最終測定日または対象の指定日までの体積変化
全ての統計的計算を、SigmaStat 3.0(SPSS Australasia,North Sydney,NSW,Australia)を用いて実施した。二標本t検定を用いて、0日目と4日目の間および0日目と試験終了日の間の処置群内の体重変化の有意性を決定した。一元配置分散分析(ANOVA)を、本試験の最後に全てのマウスにおいて測定された腫瘍体積に対して実施した。データに有意差が見出された場合は、全対多重比較法(ホルム−サイダック(Holm−Sidak)法)を実施した。二標本t検定を用いて、ビヒクルコントロールおよびDend10 10(4)処置群についての腫瘍重量データ間の有意差を証明した。0.05未満のp値は有意と見なされた。
8.1 材料
96ウェル細胞培養プレート(Becton−Dickinson,North Ryde,NSW,Australia)。CellTiter−Blue(登録商標)(Alamar Blue(商標))細胞生死判別アッセイ(Promega,Madison,WI,USA)。RPMI 1640細胞培養培地、FBSおよびHBSS(Invitrogen Australia,Mt Waverley,VIC,Australia)。ペニシリン−ストレプトマイシンおよびトリパンブルー(Sigma−Aldrich,Castle Hill,NSW,Australia)。生理的食塩水(Baxter Australia,NSW,Australia)。Spectramax Gemini XPS蛍光測定器(Adelab Scientific,Adelaide,SA,Australia)。マウス血清(MS)については、眼窩採血を行い、続いて遠心分離機にかけることにより、BALB/cマウスから採取した。
種:マウス(Mus musculus)
株:BALB/c
供給者:University of Adelaide(Waite Campus,Urrbrae,SA,Australia)
試験における動物の総数:9(インビボ試験用)
体重範囲:処置開始時17.8〜20.7g(平均19.12g)
開始時の週齢範囲:処置開始時8〜10週
ヒト結腸直腸腺癌細胞株HT−29を、American Type Culture Collection(ATCC)(Rockville,MD,USA)から調達した。HT−29細胞を、10%(v/v)FCSおよび50IU/mLペニシリン−ストレプトマイシンを補充したRPMI 1640細胞培養培地で培養した。全ての細胞を、95%空気/5%CO2が供給される加湿細胞培養インキュベータで37℃にて増殖させた。本試験で用いられた細胞は、2継代後に用いられた。
様態:白色粉末
分子量:14,843ダルトン
ロットNo.:T40800
使用期限:該当なし
保存条件:4℃
取扱い上の注意事項:標準的な実験注意事項
本試験において用いられる9匹のマウスを、3つの3匹から成る群に割り振った。群1のマウスを処置せずに放置し、採血を行った。群2のマウスの採血をPeg2 Dend10 D−10(4)処置の5分後および30分後に行い、群3の動物の採血をPeg2 Dend10 D−10(4)処置の15分後および60分後に行った。全ての場合において、眼窩採血により採血を行った。全血液試料を、ヘパリン処理したキャピラリ−チューブ中に収集し、新しいチューブに移して氷上に置いた。試料を、2分〜3分にわたり4℃にて遠心分離機(1000×g)にかけた。血漿成分を新しいチューブに回収し、4℃にてインビトロ試験に移した。全てのマウスを、イソフルオランで誘発した知覚麻痺下において、心穿刺による致命的な出血により安楽死させた。
HT−29細胞を2×96ウェルプレートに入れ、播種の24時間後、一方のプレートを、CellTiter−Blue(登録商標)試薬を用いてアッセイした(6ウェル)。他方のプレートについては、20%マウス血清中のPeg2 Dend10 D−10(4)デンドリマーの存在下でさらに72時間にわたってインキュベートし、次いで、CellTiter−Blue(登録商標)アッセイに供した。24時間の増殖後に得られた平均値を、96時間のインキュベーション(デンドリマーの存在下では72時間)の間に得られた値から引いた。パーセント阻害を、以下の式を用いて計算した。
阻害(%)=[1−(所与の用量における蛍光/非処置細胞の蛍光)]×100
細胞のインキュベーション後、20μLのCellTiter−Blue(登録商標)を各ウェルに加え、細胞培養インキュベータにおいて37℃にてインキュベートした。蛍光を、Spectramax Gemini XPS蛍光測定器を用いて測定した。
結果を図9に示す。インビトロ試験において、HT29癌細胞増殖の40%より大きな阻害が0.5μMの濃度のデンドリマーについて観察され、約58%阻害が1μMのデンドリマーの濃度で観察された。
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Claims (23)
- ERK MAPキナーゼに、βインテグリンサブユニットの細胞質結合を提供する少なくとも1種のポリペプチド、または前記MAPキナーゼが結合するその変異形態もしくは改変形態を8単位より多く提示するデンドリマー。
- 前記ポリペプチドが、前記βインテグリンサブユニットの結合ドメインを含む、または前記βインテグリンサブユニットの結合ドメインから成る、請求項1に記載のデンドリマー。
- 前記ポリペプチドが、前記結合ドメインの変異形態または改変形態を含む、請求項1に記載のデンドリマー。
- 前記βインテグリンサブユニットの前記結合ドメインは、前記MAPキナーゼに結合する両末端領域を有し、前記両末端領域は、前記MAPキナーゼとの結合に必須ではない介在アミノ酸リンカー配列によって互いに連結されている、請求項3に記載のデンドリマー。
- 前記βインテグリンサブユニットの前記結合ドメインと比較して、前記介在アミノ酸配列の1つ以上のアミノ酸が前記ポリペプチドにおいて欠失している、請求項4に記載のデンドリマー。
- 前記結合ドメインと比較して、前記介在アミノ酸配列中のアミノ酸の全てが前記ポリペプチドにおいて欠失している、請求項5に記載のデンドリマー。
- 前記結合ドメインの前記両末端領域が、前記ポリペプチドにおいて変化せずに残っている、請求項3〜6のいずれか1項に記載のデンドリマー。
- 前記ポリペプチドが、R/KxR/K*R/K−xx*x*NPLY/FR/Kによって表されるアミノ酸配列を含み、またはR/KxR/K*R/K−xx*x*NPLY/FR/Kによって表されるアミノ酸配列からなり、ここで、各R/Kは、独立して、アルギニンまたはリジンのいずれかであり、Y/Fは、チロシンまたはフェニルアラニンのいずれかであり、xは任意のアミノ酸であり、*は疎水性アミノ酸であり、−はアミノ酸であり、配列−xx*x*の1つ以上のアミノ酸が必要に応じて欠失している、請求項3に記載のデンドリマー。
- −により規定される前記アミノ酸がセリンである、請求項8に記載のデンドリマー。
- −により規定される前記アミノ酸が欠失している、またはスレオニン、チロシン、アスパラギンおよびグルタミンからなる群より選択されるアミノ酸である、請求項8に記載のデンドリマー。
- 前記ポリペプチドが、R/KxR/K*R/KNPLY/FR/Kによって規定されるアミノ酸配列を有する、請求項8〜10のいずれか1項に記載のデンドリマー。
- 前記βインテグリンサブユニットの前記結合ドメインの前記変異形態または改変形態が、少なくとも2つの正に荷電したアミノ酸残基を含む、請求項3または4に記載のデンドリマー。
- 前記ポリペプチドが、RSKAKWQTGTNPLYR(配列番号2)、RARAKWDTANNPLYK(配列番号3)、RSRARYEMASNPLYR(配列番号4)、KEKLKSQWNNDNPLFK(配列番号5)、RSKAKNPLYR(配列番号6)、RARAKNPLYK(配列番号7)、RSRARNPLYR(配列番号8)、およびKEKLKNPLFK(配列番号9)から成る群より選択されるアミノ酸配列を含む、またはその群より選択されるアミノ酸配列から成る、請求項3または4に記載のデンドリマー。
- 前記ポリペプチドが、10アミノ酸長以上である、請求項1〜13のいずれか1項に記載のデンドリマー。
- 前記デンドリマーが、前記ポリペプチドを10単位提示する、請求項1〜14のいずれか1項に記載のデンドリマー。
- 前記ポリペプチドが、1つ以上のD−アミノ酸を含み、かつ/またはタンパク質分解からN末端もしくはC末端が保護されている、請求項1〜15のいずれか1項に記載のデンドリマー。
- 前記βインテグリンサブユニットが、β2、β3、β5、およびβ6から成る群より選択される、請求項1〜16のいずれか1項に記載のデンドリマー。
- 前記MAPキナーゼがERK2である、請求項1〜17のいずれか1項に記載のデンドリマー。
- 請求項1〜18のいずれか1項に記載のデンドリマーを、薬学的に許容可能なキャリアと一緒に含む、薬学的組成物。
- 請求項1〜18のいずれか1項に記載のデンドリマーの有効量で癌細胞を処置する工程を包含する、癌細胞の成長を阻害するための方法。
- 哺乳動物における癌の予防または処置のための方法であり、前記デンドリマーを前記哺乳動物に投与する工程を包含する、請求項20に記載の方法。
- 前記βインテグリンサブユニットが前記癌の癌細胞によって発現される、請求項20に記載の方法。
- 前記癌が、乳癌、結腸癌、胃癌および前立腺癌から選択される、請求項21に記載の方法。
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Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20140819 |