JP6088982B2 - Ampk活性化を測定するための全血アッセイ - Google Patents
Ampk活性化を測定するための全血アッセイ Download PDFInfo
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- JP6088982B2 JP6088982B2 JP2013548429A JP2013548429A JP6088982B2 JP 6088982 B2 JP6088982 B2 JP 6088982B2 JP 2013548429 A JP2013548429 A JP 2013548429A JP 2013548429 A JP2013548429 A JP 2013548429A JP 6088982 B2 JP6088982 B2 JP 6088982B2
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Description
35U.S.C.§119(e)に基づき、本願は、2011年1月6日に出願された米国仮特許出願第61/430,472号の出願日の優先権を主張するものであり、上記出願の開示は参照により本明細書に組み込まれる。
用語「生物学的試料」は本明細書で使用されるとき、生きた材料を含有するまたは生きた材料から作られる任意の試料を指す。生物学的試料は、多細胞生物から得られたインタクトな細胞を含有することができる。生物学的試料は、個体(例えば軟組織もしくは体液)から、またはインビトロで増殖される細胞培養物から単離することができる。生物学的試料は、脳、副腎、皮膚、肺、脾臓、腎臓、肝臓、脾臓、リンパ節、骨髄、膀胱、胃、小腸、大腸また筋肉等などの軟組織から作ることができる。体液には、血液、血漿、唾液、粘液、痰、脳脊髄液(cerebral spinal fluid)、胸膜液、涙液、乳糜管液(lactal duct fluid)、リンパ液、喀痰、脳脊髄液(cerebrospinal fluid)、滑液、尿、羊水、および精液等が含まれる。生物学的試料には、インビトロでの培養下で増殖された細胞も含まれる。
下記の方法は、血液の試料を使用する。しかし、血液は、該方法において使用することができる多くの生物学的試料の1つにすぎない。他の実施形態において、他の組織(例えば、肝臓または脾臓等などの他の軟組織)由来のインタクトな細胞、または組織培養で増殖された細胞が使用されてもよい。フローサイトメトリーに適した細胞懸濁液を提供するための、このような組織の処理方法が知られている。ひとたび作製されると、細胞懸濁液は、下記の方法と類似した方法で使用することができる。
上記の方法は、例えば、AMPK活性化を調節する化合物の同定、投与された化合物が所望の効果を有しているかどうかの判定、または化合物の最適用量がAMPK活性化を調節することが知られていることの判定に使用することができる。これらの実施形態において、上述の方法を用いて得られた測定は、血液の参照試料から得られた結果と比較することができる。上述の通り、血液の試験試料は、試験薬剤とエクスビボまたはインビボで接触させることができる。血液の参照試料は、例えば、試験薬剤と接触させていなくてもよく、または異なる量の試験薬剤と接触させていてもよい。1つの実施形態において、試験試料および参照試料はいずれも、同じ量の試験化合物と、ただし、異なる回数または異なる期間接触させていることができる。試験試料および参照試料は、同じ被験者、または異なる被験者から得ることができる。被験者は、少なくとも8〜12時間絶食していてもよく、または、ある場合には、方法は運動前、運動中または運動後に行うことができる。方法は、被験者の健康の評価を提供するために、コレステロール試験(すなわち、脂質パネル)または血糖試験などの他の医学的試験と併用することができる。試験薬剤の標的は、AMPKの上流であってもよく、AMPK自体またはAMPKの下流であってもよい。幾つかの場合において、試験薬剤の作用機序は未知である場合がある。
MEFまたはC2C12細胞をDMSOまたは化合物2で処理し、Cell Signaling抗pAMPK抗体を用いたウェスタンブロッティングにより調べた。抗体はpAMPKのみを認識し、他のバンドは観察されない。同じ抗体をこの後の実験におけるFACSに使用した。
ヒト全血を、丸底プレートに1ウェル当たり100μlで等分し、1μlの1uM化合物1/DMSO溶液またはDMSO単独を添加し、混合し、37℃で1時間インキュベートし、次いで900μlのlyse−fix溶液(BD)を添加した。手順の残りは、1:100希釈のpAMPKウサギ抗体(Cell Signaling)を一次としてpACCの代わりに使用をした以外は、上述のHepG2細胞に関する記載の通りに行った。リンパ球および顆粒球を示されているようにゲートした。
ヒト全血およびマウス全血を、丸底プレートに1ウェル当たり100μlで等分し、1μlの1uM化合物1DMSO溶液またはDMSO単独を添加し、混合し、37℃で1時間インキュベートし、次いで900μlのlyse−fix溶液(BD)を添加した。手順の残りは、1:100希釈のpAMPKウサギ抗体(Cell Signaling)を一次としてpACCの代わりに使用をした以外は、上述のHepG2細胞に関する記載の通りに行った。結果を図4に示す。抗体は、ヒトおよびげっ歯類血液を用いて両方とも機能する。
pAMPKについて染色した、ゲートされたヒトまたはマウスリンパ球および顆粒球に関するFL−1チャネルヒストグラムを比較した。両方の種においてリンパ球は、顆粒球より良好なウィンドウを一貫して示した。IL−2の存在下で増殖された精製T細胞は、刺激の極めて狭いウィンドウのため、全血の代わりとしては不足であることを証明した。この結果、全ての追跡実験はWBリンパ球を用いてEC50を生成した。
インビトロで増殖されたヒトT細胞ならびにヒトおよびマウス血液細胞における、化合物2によるAMPKリン酸化の刺激に関するEC50判定。結果を図5に示す。精製T細胞のウィンドウは、恐らく細胞ストレスにより誘導されたAMPKリン酸化の高いバックグラウンドレベルのため極めて狭い。マウス血液のEC50は、ヒトのEC50より著しく(平均で約10倍)高い。ヒトおよびマウスEC50は両方とも、HepG2の対応するEC50より著しく高い(最大数百倍)。
マウス血液におけるAMPKリン酸化に対する飢餓の影響。C57B1/6Jオスマウスは、採血の前に14時間絶食させるか、または普通に給餌した。化合物2および化合物1によるAMPK刺激は、50μlで1時間、エクスビボで行った。染色手順は、一次および二次抗体の両方に1:500希釈を使用した以外は、HepG2に関してと同じであった。絶食させた動物におけるAMPKリン酸化の最大レベルは、給餌した動物と比べた場合、一貫してほぼ2倍高かったが、バックグラウンドレベルは元の状態のままであった。
マウス血液におけるAMPKによるACCリン酸化に対する飢餓の影響。AMPKリン酸化に関する全ての観察は、同様にpACC染色に関連する。結果を図6に示す。両方のタンパク質のEC50は、同じ化合物を使用する場合、予想通り同一である。
抗pAMPKウサギモノクローナルおよびポリクローナル抗体を用いた染色は、高度に特異的であり、ドナー依存性である。2名のヒトドナー由来の全血試料を用いて、AMPKリン酸化を誘導する化合物2 10μMの非存在下(青いプロフィール)および存在下(赤いプロフィール)でpAMPKを染色した。抗体は両方とも、化合物2処理をした際のAMPKリン酸化の類似の増加をロバストに検出し、染色選択性を裏付けた。
リンパ球の染色に2つの異なる抗体を用いた場合、2名の異なるドナー由来のヒト全血における化合物2のEC50は同一であった。結果を図7に示す。モノクローナルウサギ抗体を用いた場合、ウィンドウは著しく良好であった。
全血中の化合物をエクスビボで滴定した後、pAMPに関して血液細胞を染色して得られた曲線は、顆粒球ゲートよりリンパ球ゲートを用いた場合に再現性がより高いが、両方の方法により得られたEC50は、抗体、細胞型またはドナーにかかわりなく本質的に同じである。
pAMPKアッセイの一貫性および安定性。同じドナーの血液を用いて異なる週に得られた化合物1および化合物2の滴定曲線およびEC50。最終EC50は互いに密接に一致する。図8A〜8Cは、2つのAMPK活性化因子を用いたpAMPKおよびpACC刺激には、ドナーによって低い変動性があることを示している。
WBアッセイでの得られたEC50に対する、化合物とのインキュベーション時間の影響。単一のドナー由来のヒト全血を、37℃で一定期間、さまざまな量の化合物1により処理した。血液を標準的方法により処理して、FACSによりAMPKリン酸化を検出した。EC50はmatlabにより判定した。結果を図9に示す。リンパ球に対する影響はほとんど観察されなかった(右の2つのパネル)。EC50の著しい変化をもたらさないにもかかわらず(左の2つのパネル)、顆粒球は、検出されたpAMPKのベースラインレベルにおける著しいおよび着実な上昇のため、インキュベーション時間に極めて感受性である。故に、インキュベーション時間が長いほど、アッセイのウィンドウおよび全般的ロバスト性を予想外に減少させる。両方の細胞型において、化合物2は、AMPK活性化の極めて速い動態を示す−5分間隔で、化合物の飽和濃度で該キナーゼの完全な刺激を見ることができる。最大活性化レベルは同じままであり、非刺激レベルはわずかに上昇する。
図10は、HepG2細胞においてpACC in−cell−western(ICW)により得られたEC50(X軸)と、ヒト全血においてpAMPK FACSにより得られたEC50(Y軸)との間の強い線形相関を示す。両方の軸に対数目盛を用いた。恐らく試験化合物の、高いタンパク質結合および赤血球への高レベルの分布(高VSS)の組み合わせのため、化合物の大多数が、HepG2細胞と比べて血液における有効性の著しい喪失(最大100倍)を示す。
図11は、試験化合物に関するヒト全血におけるpACC EC50とpAMPK FACS EC50(Y軸)との間の極めて良好な線形相関を示している。ACCは、AMPKの下流の標的であり、AMPKは、ACC上の阻害性リン酸化部位を標的にする唯一のキナーゼである。
インビボでの血液バイオマーカーとしてのpAMPK。結果を図14に示す。上、5および20mg/kgで化合物2を単回投与により、または示されているようにビヒクル対照により処理したマウスの血液由来リンパ球におけるpAMPKシグナルの幾何平均。処理前に採取した血液試料からのpAMPKレベルの測定結果を青で示し、処理1時間後に採取した対応する測定結果を赤で示す。下、同じ動物群に関する、処理前1時間でのシグナル幾何平均対処理前0.5時間のシグナル幾何平均の比。シグナルの明らかな用量依存的増加が、化合物2で処理した動物由来の血液試料に関して観察される。
図15は、FACSベースのpAMPKアッセイが、血液以外の組織に対する化合物の影響を検出するのに使用できることを示している。化合物2(上)またはR043(下)で処理した動物由来の脾臓を、単細胞懸濁液にホモジナイズし、得られた試料を血液と同じ方法で処理し、pAMPKについて染色した。上、ビヒクル処理(右)または化合物2処理(左)した動物由来の試料の幾何平均(ビヒクル対照と比較した倍)。非刺激、いずれの追加処理もせずに処理した試料。刺激、懸濁液中の細胞に関する最大AMPK刺激レベルを判定するための処理の前に、37℃で5分間、3.2uM化合物1で処理した試料。
図16Aおよび16Bは、pAMPKの最大刺激レベルは、給餌し肥満したマウスと比べて絶食させ痩せたマウスで著しく高いが、非刺激レベルは変わらないままであることを示している。図15Aは、いずれの試験化合物のEC50も、満腹度に依存しなかったことを示している。AMPKおよびACCリン酸化の両方のベースラインレベルは両群で変わらないままであったが、両方についての最大刺激レベルは絶食群で著しく高かった。図15Bの上のパネルは、化合物1によるエクスビボ刺激の前後の肥満したマウスおよび痩せたマウスの血液におけるpAMPKレベルを示している。図15Bの下のパネルは、R283によるエクスビボ刺激の前後の肥満したマウスおよび痩せたマウスの脾臓細胞におけるpAMPKレベルを示している。
Claims (17)
- 被験者のエネルギー状態を評価するための方法であって、
a)ホスホ−AMPKまたはホスホ−アセチルCoAカルボキシラーゼに特異的に結合する抗体を用いて被験者由来の血液試料の細胞を標識し、標識試料を作製するステップと、
b)フローサイトメトリーを用いて前記標識試料の血液細胞の集団の個々の細胞による抗体結合を測定し、これにより細胞の前記集団におけるAMPK活性化の評価を得るステップであって、前記細胞の集団における評価されたAMPK活性化が被験者のエネルギー状態と相関しているステップと
を含む、方法。 - 前記評価が血液細胞の前記集団の幾何平均蛍光である、請求項1に記載の方法。
- 血液細胞の前記集団が白血球またはその亜集団である、請求項1または2に記載の方法。
- 前記亜集団がリンパ球または顆粒球から構成される、請求項3に記載の方法。
- 前記抗体がホスホ−AMPKに特異的に結合する、請求項1から4のいずれか一項に記載の方法。
- 前記抗体がホスホ−ACCに特異的に結合する、請求項1から4のいずれか一項に記載の方法。
- 前記方法が、
前記標識ステップa)の前に、血液をエクスビボで試験薬剤と接触させるステップと、
前記評価を血液細胞の参照試料から得られた結果と比較し、これにより血液細胞の前記集団におけるAMPK活性化に対する前記化合物の影響を検出するステップ
をさらに含む、請求項1から6のいずれか一項に記載の方法。 - 前記影響が、血液細胞の前記集団の幾何平均蛍光および血液細胞の前記参照試料の幾何平均蛍光の差を計算して測定される、請求項7に記載の方法。
- 前記接触させるステップが、被験者から採取された血液と前記試験薬剤を一定期間接触させるステップを含む、請求項7または8に記載の方法。
- 前記試験薬剤がAMPK活性化に影響を与えることが知られている、請求項7から9のいずれか一項に記載の方法。
- AMPK活性化に対する前記試験薬剤の影響が知られていない、請求項7から9のいずれか一項に記載の方法。
- 前記参照試料が、前記血液試料と同じ個体から得られた血液細胞を含む、請求項7から11のいずれか一項に記載の方法。
- 前記参照試料は前記試験薬剤と接触させていない、請求項7から12のいずれか一項に記載の方法。
- 前記血液試料を前記試験薬剤の第1の量と接触させ、参照試料を前記試験薬剤の第2の量と接触させる、請求項7から12のいずれか一項に記載の方法。
- 前記方法が、
前記標識ステップa)の前に、哺乳動物に生活様式の変化を経験させるステップと、
前記評価を前記哺乳動物から得られた血液細胞の参照試料から得られた結果と比較し、これにより前記哺乳動物のAMPK活性化に対する前記生活様式の変化の影響を検出するステップ
をさらに含む、請求項1から14のいずれか一項に記載の方法。 - 前記生活様式の変化が食事である、請求項15に記載の方法。
- 前記生活様式の変化が運動の増加である、請求項15に記載の方法。
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US9005909B2 (en) | 2015-04-14 |
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CA2823582C (en) | 2021-05-04 |
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US20120178098A1 (en) | 2012-07-12 |
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