JP2016179973A - 化合物またはその塩および医薬組成物 - Google Patents
化合物またはその塩および医薬組成物 Download PDFInfo
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Abstract
Description
式
式
上記本発明の第1の観点に係る化合物の塩である。
上記本発明の第1の観点に係る化合物または上記本発明の第2の観点に係る塩を有効成分として含む。
GPR56発現抑制剤であってもよい。
白血病の治療または予防に使用するためのものであってもよい。
本実施の形態に係る化合物GPR56−1は、次の式で表される。
本実施の形態に係る医薬組成物は、GPR56−1もしくはGPR56−2またはその塩を有効成分として含む。上記実施の形態1に記載のように、GPR56−1およびGPR56−2は、EVI1を介したGPR56の発現を抑制する。このため、当該医薬組成物は、GPR56発現抑制剤として使用できる。
GPR56のプロモータ領域におけるEVI1が結合する部位(D2B)を含む領域の塩基配列ACGGAAGATを認識してDNAに結合するPIポリアミドとして、GPR56−1(Dp−β−ImPy−β−Im−ImPy−γ−ImPy−Py−β−Py−Py−Ac、化学式C76H95N30O15+、分子量1667.76)を以下のように合成した。なお、ここでは、Acはアセチルを、Pyはピロールを、Imはイミダゾールを、βはβ−アラニンを、γはγ−酪酸を、DpはN,N−ジメチル−1,3−プロパンジアミンを、意味する。
EVI1を発現するプラスミドは、pCMV26ベクター(シグマアルドリッチ社製)に、EVI1遺伝子の全長をサブクローニングすることで作製した。GPR56遺伝子の転写調節を評価するために、GPR56遺伝子のプロモータ領域としてGPR56遺伝子の転写開始点から上流約3000塩基対の領域を、pGL4.10 Luc2ベクター(プロメガ社製)に組み込んだ。組み換え実験で用いたコンピテントセルはDH5α株である。組み換えDH5α株の培養に使用したLB培地の組成は、1%バクトトリプトン(ベクトン・ディッキンソン アンド カンパニー(BD)社製)、0.5%酵母エキス(BD社製)、pH7.4の0.5%塩化ナトリウム(ナカライテスク社製)である。また、LBプレートは、アガロース(BD社製)を使用して作成した。
図1は、ルシフェラーゼの相対的な活性を示す。ルシフェラーゼの活性は、組み込んだプロモータ領域の転写活性に相当する。GPR56−1を添加しないD2B/WTで見られた転写活性は、GPR56−1を添加することで、ほとんど消失した。
GPR56−1によるGPR56遺伝子のプロモータ領域へのEVI1の結合抑制能を評価するために、クロマチン免疫沈降(Chromatin immunoprecipitation;ChIP)アッセイを実施した。293T細胞に、EVI1を発現するプラスミド(EVI1 WT)または8〜10番目のzinc finger部位を欠くEVI1を組み込んだプラスミド(EVI1Δ8−10)、および上記GPR56遺伝子のプロモータ領域を組み込んだプラスミドを導入した。導入後、AP1−2あるいはGPR56−1を、0.1、1、10μMの濃度で添加した。添加から48時間経過後、細胞を回収してPBS(−)で洗浄した。ChIP Lysisバッファーで細胞を溶解し、FLAG M2抗体(シグマアルドリッチ社製)とDynaBeads(ベリタス社製)用いて免疫沈降を行った。
GPR56 ChIP Fw:3’−GCAAGACAGGTGAGACAGCA−5’(配列番号1)
GPR56 ChIP Rv:3’−CTGAGAAGCCTTCCATGACC−5’(配列番号2)
図2に示すように、EVI1 WTでは、GPR56遺伝子のプロモータ領域へのEVI1の結合が確認された。一方、EVI1Δ8−10では、EVI1がzinc finger部位を欠くため、GPR56遺伝子のプロモータ領域に結合しないことが示された。AP1−2を添加した場合、GPR56遺伝子のプロモータ領域へのEVI1の結合が維持されているが(図2(a)参照)、GPR56−1では、濃度依存的にバンドが消失した(図2(b)参照)。
GPR56の発現量が高い難治性白血病細胞株であるUCSD/AML1細胞(カリフォルニア大学サンディエゴ校より提供)を用いて、GPR56−1がGPR56の発現を抑制することを確認した。
hGPR56 RT Fw:5’−TTGATCTTCTTCTCCTTTGCTTC−3’(配列番号3)
hGPR56 RT Rv:5’−CGCATGGACCAGTACCAGAT−3’(配列番号4)
β−アクチンに対するプライマーの塩基配列を次に示す。
hβ−actin RT Fw:5’−GACAGGATGCAGAAGGAGATCACT−3’(配列番号5)
hβ−actin RT Rv:5’−TGATCCACATCTGCTGGAAGGT−3’(配列番号6)
図3は、フローサイトメトリーの結果を示す。AP1−2を添加しても、処理なしと同等のGPR56の発現が確認された(図3(a)参照)。一方、GPR56−1を添加することで、濃度依存的にGPR56の発現が抑制された(図3(b)参照)。
GPR56−1の薬効を評価するために、USCD/AML1細胞の増殖への影響を評価した。AP1−2またはGPR56−1を0.1、0.3、1、3、10μM添加した培地でUSCD/AML1細胞を5日間培養した。培養中、細胞を1日1回回収し、トリパンブルーで染色後、生細胞を光学顕微鏡下で計数した。
図5は、生細胞数の経時変化を示す。AP1−2は、USCD/AML1細胞の増殖を抑制してないのに対し(図5(a)参照)、GPR56−1は、USCD/AML1細胞の増殖を濃度依存的に抑制した(図5(b)参照)。
GPR56−1によるUSCD/AML1細胞におけるアポトーシスの誘導を、フローサイトメトリーを用いて評価した。USCD/AML1細胞をプレートに撒いて、AP1−2またはGPR56−1を1μMで添加した。添加から3日経過後、7−AAD抗体(559925、BD社製)およびFITCで標識したアネキシンV抗体(640906、BioLegend社製)を加え、FACS Calibur(BD社製)で解析した。
図6は、フローサイトメトリーで得られた結果を示す。AP1−2に対して、GPR56−1では、7−AADおよびアネキシンVの発現が増加していた。このため、GPR56−1は、USCD/AML1細胞のアポトーシスを誘導することが示された。
さらに、各種マーカー遺伝子の発現を、Western blot法で評価した。マーカー遺伝子は、アポトーシス関連遺伝子であるp53、MDM2、p21、p27、CDK4およびCDK6である。USCD/AML1細胞にAP1−2およびGPR56−1をそれぞれ1μMまたは3μMで添加した。添加から3日経過後、USCD/AML1細胞を、RIPA Bufferで溶解し、BCA法でタンパク質濃度を測定した。アクリルアミドゲルは常法に従って作製し、10%アクリルアミドゲルを用いて泳動した。各レーンには10μgのタンパク質を泳動し、100V、1時間で転写を行った。その後、5%スキムミルク/TBS−Tを用いてブロッキングし、TBS−Tにて3回洗浄し、1/1000に希釈した抗体液で、4℃で一晩処理した。抗体はCan Get Signal(商標)液(東洋紡社製)にて希釈した。処理後、TBS−Tで3回洗浄し、1/5000に希釈した二次抗体液(抗体はGE社製、希釈液はCan Get Signal(商標)液)で、常温で一時間処理した。その後、TBS−Tで3回洗浄の後、Lumi light plus(ロシュ社製)で検出した。検出に用いた装置は、LAS3000(フジフィルム社製)である。
図7は、各種マーカー遺伝子の発現を示す。GPR56−1によって、MDM2の発現が抑制され、p53の蓄積が見られた。この結果から、GPR56−1が、USCD/AML1細胞のp53を介したアポトーシスを誘導することが示された。
GPR56−1およびGPR56−2の薬効を評価するために、EVI1高発現AML細胞株の増殖への影響を評価した。GPR56−1もしくはGPR56−2、または対照群としてジメチルスルホキシド(DMSO)を1μM添加した培地で、USCD/AML1細胞またはMOLM1細胞(株式会社林原生物化学研究所製)を5日間培養した。培養中、細胞を1日1回回収し、トリパンブルーで染色後、生細胞を光学顕微鏡下で計数した。
図8(a)および(b)にそれぞれUSCD/AML1細胞およびMOLM1細胞の生細胞数の経時変化を示す。DMSO添加群と比較して、GPR56−1添加群及びGPR56−2添加群では、USCD/AML1細胞およびMOLM1細胞ともに細胞増殖が有意に抑制された。GPR56−1添加群とGPR56−2添加群には差は見られなかった。この結果から、GPR56−1およびGPR56−2は、同等にEVI1高発現白血病細胞の増殖を抑制することが示された。
公益財団法人実験動物中央研究所より提供されている6週齢の高度免疫不全マウス(NOD/Shi−scid−IL2Rγnullマウス、以下単に「NOGマウス」とする)に、1個体あたり5×106個のUCSD/AML1細胞を移植した。詳細には、UCSD/AML1細胞を移植当日にサブコンフルエントになるように培養し、UCSD/AML1細胞を100μLの滅菌PBS(−)に懸濁した。UCSD/AML1細胞の懸濁液と等量のマトリゲル(BD社製)とを混合し、29Gのシリンジを用いてNOGマウスの皮下へ注入して移植した。移植後、腫瘍が5mm四方のサイズになったことを確認し(通常約1ヶ月間)、薬剤(AP1−2またはGPR56−1)の投与を開始した。薬剤は、週に1回、1mg/kgずつ投与し、腫瘍の大きさを測定した。なお、当該投薬スケジュールおよび薬剤濃度は、予備実験において1ヶ月間投与しても外観に目立った影響がないことを確認して設定した。
図9に示すように、AP1−2投与群と比較して、GPR56−1投与群では、有意な腫瘍サイズの抑制が確認された。図10は、腫瘍の重量を示す。GPR56−1投与群では、腫瘍重量はほとんど増加しなかった。
薬剤を投与した実施例9に係るマウスの血液を採取し、全自動血球計数器(MK−6450 セルタックα、日本光電工業社製)で血液を検査した。また、骨髄画分を次のようにフローサイトメトリーで評価した。
表1は、血液検査の項目ごとの測定値の平均を示す。いずれの項目においても、AP1−2投与群とGPR56−1投与群との間に有意差はなかった。
全身白血病モデルを作製するために、熊本大学CARDで樹立された免疫不全マウスBalb/c−RJマウス(Rag2/Jak3 dKO)に、UCSD/AML1細胞を注射した。マウス1個体あたり5×106個のUCSD/AML1細胞を、尾静脈に注射した。薬剤(AP1−2またはGPR56−1)は、週に1回、1mg/kgで投与した。薬剤の投与は、観察期間(90日)の間継続した。
図13は、90日間のマウスの生存率を示す。GPR56−1投与群の約半数は長期に渡って生存した。GPR56−1の投与によって、全身白血病モデルのマウスの有意な延命が確認された。このため、白血病の病態により近い全身白血病モデルに対してもGPR56−1は、非常に有効である。
薬剤を投与した実施例11に係るマウスより骨髄、脾臓、肝臓および末梢血を分取し、セルストレイナー(352340、BD社製)を用いて細胞を含む試料を取得した。試料に溶血試薬(555899、BD社製)を加えて溶血させて、試料中の細胞数をカウントした。1x106個の細胞を100μLのMACS Bufferに懸濁し、PEで標識されたヒトCD45抗体(304008、BioLegend社製)で染色した。染色は、氷上で30分後静置し、MACS Bufferで3回洗浄した。染色した試料を、FACS Calibur(BD社製)で解析した。
図14は、骨髄(a)、脾臓(b)、肝臓(c)および末梢血(d)それぞれにおけるCD45陽性、すなわち白血病細胞の割合を示す。骨髄、脾臓、肝臓および末梢血のいずれにおいても、GPR56−1投与群は、PBS投与群およびAP1−2投与群と比較して、白血病細胞の割合が有意に低かった。したがって、GPR56−1は、全身白血病モデルにおける各臓器への白血病細胞の浸潤を抑制することが示された。
Claims (4)
- 式
式
- 請求項1に記載の化合物またはその塩を有効成分として含む医薬組成物。
- GPR56発現抑制剤である請求項2に記載の医薬組成物。
- 白血病の治療または予防に使用するための請求項2または3に記載の医薬組成物。
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