JP2019136042A - Fgfr3病の予防および治療剤ならびにそのスクリーニング方法 - Google Patents
Fgfr3病の予防および治療剤ならびにそのスクリーニング方法 Download PDFInfo
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Abstract
Description
[1] 有効成分としてHMG-CoA還元酵素阻害薬を含む、FGFR3病の治療および/または予防用医薬。
[2] 前記HMG-CoA還元酵素阻害薬が、メバスタチン、アトルバスタチン、プラバスタチン、ロスバスタチン、フルバスタチンおよびロバスタチンから成る群より選択される薬剤である、[1]に記載の医薬。
[3] 前記FGFR3病が、致死性骨異形成症 (TD) および/または軟骨無形成症 (ACH)である、[1]または[2]に記載の医薬。
[4] FGFR3病を治療および/または予防するための方法であって、HMG-CoA還元酵素阻害薬を投与することを含む、方法。
[5] 前記HMG-CoA還元酵素阻害薬が、メバスタチン、アトルバスタチン、プラバスタチン、ロスバスタチン、フルバスタチンおよびロバスタチンから成る群より選択される薬剤である、[4]に記載の方法。
[6] 前記FGFR3病が、致死性骨異形成症 (TD) および/または軟骨無形成症 (ACH)である、[4]または[5]に記載の方法。
[7] FGFR3病の治療および/または予防用医薬の製造におけるHMG-CoA還元酵素阻害薬の使用。
[8] 前記HMG-CoA還元酵素阻害薬が、メバスタチン、アトルバスタチン、プラバスタチン、ロスバスタチン、フルバスタチンおよびロバスタチンから成る群より選択される薬剤である、[7]に記載の使用。
[9] 前記FGFR3病が、致死性骨異形成症 (TD) および/または軟骨無形成症 (ACH)である、[7]または[8]に記載の使用。
[10] 下記の工程を含む、FGFR3病の治療薬および/または予防薬をスクリーニングする方法;
(a)FGFR3に変異を有する人工多能性幹(iPS)細胞を、被験物質と接触させるおよび接触させない条件下で軟骨細胞へと分化させる工程、
(b)工程(a)で得られた培養物中における軟骨細胞外マトリックスの量、軟骨細胞マーカー遺伝子および線維芽細胞マーカー遺伝子の発現量から成る群より選択される一つ以上の指標を測定する工程、および
(c)被験物質と接触させる条件下において、接触させない条件下より軟骨細胞外マトリックス量または軟骨細胞マーカー遺伝子が増加した場合、もしくは線維芽細胞マーカー遺伝子の発現量が減少した場合、当該被験物質をFGFR3病の治療薬または予防薬として選出する工程。
[11] 前記工程(a)における軟骨細胞への分化が下記の工程を含む、[10]に記載の方法:
(i)多能性幹細胞を接着培養することにより中胚葉細胞を誘導する工程、
(ii)前記工程(i)で得られた細胞をbFGF、アスコルビン酸、BMP2、TGFβ、GDF5および被験物質を含む培養液中で接着培養する工程、および
(iii)前記工程(ii)で得られた細胞をアスコルビン酸、BMP2、TGFβ、GDF5および被験物質を含む培養液中で浮遊培養する工程。
[12] 前記軟骨細胞マーカー遺伝子が、SOX9、AGGRECANおよびCOL2からなる群から選択される少なくとも一つの遺伝子である、[10]または[11]に記載の方法。
[13] 前記線維芽細胞マーカー遺伝子が、COL1A1および/またはCOL1A2である、[10]または[11]に記載の方法。
[14] 前記FGFR3の変異が、FGFR3中のArg248CysまたはGly380Arg変異である、[10]から[13]のいずれか1項に記載の方法。
[15] 前記FGFR3病が、致死性骨異形成症 (TD)および/または軟骨無形成症 (ACH)である、[10]から[14]のいずれか1項に記載の方法。
[16] FGFR3病を治療および/または予防するために使用されるHMG-CoA還元酵素阻害薬。
[17] 前記HMG-CoA還元酵素阻害薬がメバスタチン、アトルバスタチン、プラバスタチン、ロスバスタチン、フルバスタチンロバスタチンから成る群より選択される薬剤である、[16]に記載のHMG-CoA還元酵素阻害薬。
[18] 前記FGFR3病が、致死性骨異形成症 (TD) および/または軟骨無形成症 (ACH)である、[17]または[18]に記載のHMG-CoA還元酵素阻害薬。
本発明において、iPS細胞は、ある特定の核初期化物質を、DNAまたはタンパク質の形態で体細胞に導入すること、または薬剤によって当該核初期化物質の内在性のmRNAおよびタンパク質の発現を上昇させることによって作製することができる、ES細胞とほぼ同等の特性、例えば分化多能性と自己複製による増殖能を有する体細胞由来の人工の幹細胞である(K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676、K. Takahashi et al. (2007) Cell, 131: 861-872、J. Yu et al. (2007) Science, 318: 1917-1920、M. Nakagawa et al. (2008) Nat. Biotechnol., 26: 101-106、国際公開WO 2007/069666および国際公開WO 2010/068955)。核初期化物質は、ES細胞に特異的に発現している遺伝子またはES細胞の未分化維持に重要な役割を果たす遺伝子もしくはその遺伝子産物であればよく、特に限定されないが、例えば、Oct3/4, Klf4, Klf1, Klf2, Klf5, Sox2, Sox1, Sox3, Sox15, Sox17, Sox18, c-Myc, L-Myc, N-Myc, TERT, SV40 Large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28, Lin28b, Nanog, Esrrb, Esrrg, Glis1などが例示される。これらの初期化物質は、iPS細胞樹立の際には、組み合わされて使用されてもよい。例えば、上記初期化物質を、少なくとも1つ、2つもしくは3つ含む組み合わせであり、好ましくは4つもしくは5つを含む組み合わせである。
遺伝子名 マウス ヒト
L-Myc NM_008506 NM_001033081
Lin28 NM_145833 NM_024674
Lin28b NM_001031772 NM_001004317
Esrrb NM_011934 NM_004452
Esrrg NM_011935 NM_001438
Glis1 NM_147221 NM_147193
iPS細胞から軟骨細胞を分化誘導する方法としては、当分野において用いられる任意の方法を採用することができ、本願出願時において当業者に知られていた方法のみならず、本願出願後において開発された分化誘導方法を採用することも可能である。軟骨細胞を分化誘導する方法として、例えば、Koyama, N. et al. Stem cells and development 22, 102-113 (2013)、Hwang, N.S., et al. PLoS ONE 3, e2498 (2008)、Oldershaw, R.A. et al. Nat. Biotechnol. 28, 1187-1194 (2010)、Bai, H.Y., et al. Journal of biomedical materials research. Part A 94, 539-546 (2010)、Yamashita, A. et al. Scientific Reports 3 (2013)などに記載の方法が挙げられるが、これらに限定されない。
本発明において、中胚葉細胞とは、動物の発生期の原腸胚期において、内胚葉と外肺葉の間に発生する細胞を意味し、好ましくは、BRACHYURYが陽性である細胞を意味する。本発明において、BRACHYURYには、NCBIのアクセッション番号として、ヒトの場合、NM_001270484またはNM_003181、マウスの場合、NM_009309に記載されたヌクレオチド配列を有する遺伝子並びに当該遺伝子にコードされるタンパク質、ならびにこれらの機能を有する天然に存在する変異体が包含される。
本工程(ii)では、前記工程(i)で得られた細胞培養物の培養液を除去し、bFGF、アスコルビン酸、BMP2、TGFβおよびGDF5を含有する培養液を添加して行い得る。従って、前記工程(i)において細胞培養物は、培養皿へ接着していることから、本工程(ii)は接着培養によって行い得る。
本工程(iii)では、前記工程(ii)で得られた細胞培養物を培養容器より剥離させ、浮遊培養することで行い得る。本工程(iii)において、細胞培養物を剥離させる方法は、力学的分離方法(ピペッティング等)により行うことが好ましく、プロテアーゼ活性および/またはコラゲナーゼ活性を有する分離溶液(例えば、トリプシンとコラゲナーゼの含有溶液であるAccutase(TM)およびAccumax(TM)(Innovative Cell Technologies, Inc)が挙げられる)を用いない方法が好ましい。
前記工程(iii)により、軟骨細胞を製造することが可能であるが、より成熟した軟骨細胞を得るために、前記工程(iii)で得られた細胞培養物をさらに、浮遊培養してもよい。
本発明は、前述のように得られたiPS細胞由と被験物質とを接触させ、各指標を用いて、FGFR3病の治療薬および/または予防薬の被験物質をスクリーニングする方法を提供する。
(a)FGFR3病を患う対象由来の体細胞から作製された人工多能性幹(iPS)細胞を、被験物質と接触させるおよび接触させない条件下で軟骨細胞へと分化させる工程、
(b)工程(a)で得られた培養物中における軟骨細胞外マトリックスの量、軟骨細胞マーカー遺伝子および線維芽細胞マーカー遺伝子の発現量から成る群より選択される一つ以上の指標を測定する工程、および
(c)被験物質と接触させる条件下において、接触させない条件下より軟骨細胞外マトリックス量または軟骨細胞マーカー遺伝子が増加した場合、もしくは線維芽細胞マーカー遺伝子の発現量が減少した場合、当該被験物質をFGFR3病の治療薬または予防薬として選出する工程。
本発明のスクリーニング方法によって得られる物質は、種々の軟骨疾患の治療剤および/または予防剤の有効成分として有用である。本発明の処置対象となる軟骨疾患は、通常よりも減少した軟骨の量を何らかの手段で増加させることにより治療および/または予防が可能である疾患であれば何でもよい。本発明の処置対象となる軟骨疾患として、例えば、変形性関節症、軟骨損傷、軟骨形成異常症などが挙げられるがこれらに限定されない。本発明の処置対象となる軟骨疾患は、軟骨の成長過程における軟骨形成不全による疾患が適応症として最も好ましく、このような病態であれば、いずれの疾患でもあっても特に限定されないが、例えば、FGFR(FGFR1、FGFR2およびFGFR3)における変異を有する病態が挙げられる。このような病態として、Warman et al., Am J Med Genet 155A(5):943-68 (2011)に記載の疾患が例示される。本発明において見出された治療剤を使用するにあたり、FGFR3病が好ましい適応症例である。
iPS細胞の作製
以下のすべての実験は、治験審査委員会、動物実験委員会および施設内生物安全委員会、ならびに京都大学の承認を得て行われた。
3人のTD患者由来のHDF (TD-714, TD10749およびTD-315H) を、コリエル医学研究所および埼玉県立小児医療センターから入手した。これらのHDFから抽出したゲノムcDNAを配列解析したところ、すべてのTD患者においてFGFR3遺伝子のヘテロ接合突然変異(Arg248Cys)が確認された。続いて各患者由来のHDFから以下に記載する方法でiPS細胞を作製し(以下、TD-iPSCと言う)、各患者から1種のiPS細胞株(TD-714-3、TD10749-2およびTD315H-2)を解析に用いた。また、2人の異なる新生児由来の対照HDFs(Strain #01491および#01439)をKURABOから購入し、同様の方法で初期化し、対照iPS細胞を作製した(KF4009-1およびHDF-11)。このほかにも、K. OkitaおよびS. Yamanaka(京都大学iPS細胞研究所)から入手した健常な個体由来のiPS細胞株(409B2)(Okita, K., et al. Nature methods 8, 409-412 (2011))も対照iPS細胞として用いた(以下、KF4009-1、HDF-11および409B2を、WT-iPSCと言う)。
mRNAは、RNeasy Mini Kit (Qiagen)を用いて、各細胞から単離した。得られた全RNAのうち500 ngを鋳型として、ReverTra Ace (TOYOBO)を用いて逆転写によりcDNAを合成した。定量PCR(リアルタイムPCR)のための標準曲線を作成して解析を行った。リアルタイムPCR解析は、KAPA SYBR FAST qPCR kit Master Mix ABI prism (KAPA BIOSYSTEMS)を用いて、Step One system (ABI)により測定した。使用したプライマーの配列およびAssayIDを表2に示す。
細胞溶解液は、SDS-PAGE法を用いて分離し、anti-FGFR3 antibody(Cell SIgnaling社 #4574)、anti-phosphorylated MAPK antibody(Cell Signaling #9109)またはanti β-actin antibody(Cell Signaling社 #49776)を用いて免疫染色した。
軟骨誘導
以前に報告された方法(Oldershaw, R.A., et al., Nat Biotechnol 28, 1187-1194 (2010))を修正した以下に記載した方法に従って、各iPS細胞から軟骨細胞に分化誘導した。
軟骨形成的に分化させた TD-iPSCの妨害された軟骨形成からの回復
TD-iPSCがFGFR3の機能獲得型突然変異が、軟骨細胞分化不全に起因しているかを確認するために、FGFR3をノックダウンする実験を行った。
TD-iPSCの軟骨細胞分化不全を正常化させるための既知薬剤の評価
FGFR3病の治療に有効な薬剤を見出すために、TD-iPSCからの軟骨細胞分化を正常化させるための物質についてスクリーニングを行った。
TD-iPSCの軟骨細胞分化不全を正常化させるための薬剤の探索
スタチンは、広く脂質降下剤として特徴づけられる薬剤クラスを形成する物質であり、メバロン酸合成を阻害し、その結果として、全コレステロール量および低密度リポタンパク質(LDL)レベルを減少させることが知られている。すべてのスタチン類は、心血管系疾患、神経系、免疫系、骨格系および腫瘍成長に対して有利な影響を与えることから、スタチン類の多面的な効果について関心が集まってきている。さらに、ロバスタチンは、髄核から単離した細胞を単層培養することで起きる脱分化によるII型コラーゲンの脱落を抑制することが報告されている(Hu et al., Artif Organs. 35, 411-416 2011)。
ACHにおけるロバスタチンの効果
ロバスタチンがACHに対しても有効であるか否かを調べるため、ACH患者由来のiPS細胞を用いて同様に評価を行った。
スタチンによるACHモデルマウスにおける減少した骨成長の回復
スタチンがin vivoでFGFR3病の表現型を正常化できるか否かを調べた。David Ornitz (Washington University School of Medicine)から入手したFGFR3Achマウス(Naski et al., Development 125, 4977-4988 (1998))を野生型マウスと交雑した(C57BL/6 background)。交雑マウスの出生後3日から14日まで、週に6回1.0 mg/kgロスバスタチン溶液を腹腔内に投与し、15日目に安楽死させ、躯体および骨形成をX線イメージ(Faxitron DX-50)によって評価した。このとき、ゲノムDNAをつま先から抽出し、遺伝子型を調べた。また同様に、交雑マウスの出生後3日から28日まで、週に6回0.4mg/kgロバスタチン溶液を腹腔内に投与し、29日目に安楽死させ、躯体および骨形成を評価した。
Claims (6)
- 下記の工程を含む、FGFR3病の治療薬および/または予防薬をスクリーニングする方法;
(a)FGFR3に変異を有する人工多能性幹(iPS)細胞を、被験物質と接触させるおよび接触させない条件下で軟骨細胞へと分化させる工程、
(b)工程(a)で得られた培養物中における軟骨細胞外マトリックスの量、軟骨細胞マーカー遺伝子および線維芽細胞マーカー遺伝子の発現量から成る群より選択される一つ以上の指標を測定する工程、および
(c)被験物質と接触させる条件下において、接触させない条件下より軟骨細胞外マトリックス量または軟骨細胞マーカー遺伝子が増加した場合、もしくは線維芽細胞マーカー遺伝子の発現量が減少した場合、当該被験物質をFGFR3病の治療薬または予防薬として選出する工程。 - 前記工程(a)における軟骨細胞への分化が下記の工程を含む、請求項1に記載の方法:
(i)多能性幹細胞を接着培養することにより中胚葉細胞を誘導する工程、
(ii)前記工程(i)で得られた細胞をbFGF、アスコルビン酸、BMP2、TGFβ、GDF5および被験物質を含む培養液中で接着培養する工程、および
(iii)前記工程(ii)で得られた細胞をアスコルビン酸、BMP2、TGFβ、GDF5および被験物質を含む培養液中で浮遊培養する工程。 - 前記軟骨細胞マーカー遺伝子が、SOX9、AGGRECANおよびCOL2からなる群から選択される少なくとも一つの遺伝子である、請求項1または2に記載の方法。
- 前記線維芽細胞マーカー遺伝子が、COL1A1および/またはCOL1A2である、請求項1または2に記載の方法。
- 前記FGFR3の変異が、FGFR3中のArg248CysまたはGly380Arg変異である、請求項1から4のいずれか1項に記載の方法。
- 前記FGFR3病が、致死性骨異形成症(TD)および/または軟骨無形成症(ACH)である、請求項1から5のいずれか1項に記載の方法。
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