JP4255382B2 - 骨形成タンパク質(bmp)、bmpレセプターおよびbmp結合タンパク質ならびに緑内障の診断および処置におけるそれらの使用 - Google Patents
骨形成タンパク質(bmp)、bmpレセプターおよびbmp結合タンパク質ならびに緑内障の診断および処置におけるそれらの使用 Download PDFInfo
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Description
本発明は、緑内障および関連障害の診断および処置のための方法および試薬を開示する。
「緑内障」は、消耗性の眼の疾患の一群であり、米国および他の先進国において回復不能の失明の主要原因である。緑内障のもっとも一般的な形態である原発性開放隅角緑内障(「POAG」)は、虹彩と角膜との間の空間(例えば、「角」)の閉鎖無しに、房水が眼を離れる正常な能力の閉塞を起こす、小柱網の変性によって特徴づけられる(Vaughan,D.ら、(1992))。この疾患におけるこのような閉塞の特徴は、眼内圧力(「IOP」)上昇であり、もし適切にかつ適時な様式で処置されなければ、進行性の視力喪失および失明を起こす。この疾患は、40歳を越える全ての成人のうち0.4%と3.3%との間の人々に影響を及ぼすことが推測される(Leske,M.C.ら、(1986);Bengtsson,B.(1989);Strong,N.P.(1992))。さらに、この疾患の有病率は、年齢とともに増加して、75歳以上では6%を超える(Strong,N.P.、(1992))。
本発明は、先行技術のこれらおよび他の欠点を、緑内障の初期診断のための方法およびキット、緑内障の処置のための方法およびキット、ならびに緑内障の処置において有用な化合物の同定のための方法およびキットを提供することによって克服する。
a)緑内障を有すると疑われる患者から組織サンプルまたは体液サンプルを得る工程;
b)このサンプルからDNAを抽出する工程;
c)複数のPCRプライマーを得る工程であって、これらプライマーの各々は、配列番号1、配列番号3、配列番号5、配列番号7、配列番号37、配列番号39、配列番号41、配列番号43、配列番号45、配列番号47または配列番号53由来の18〜1547連続するヌクレオチドからなる配列を含む、工程;
d)これらのプライマーを使用して、抽出されたDNAの領域を増幅してPCR産物を得る工程;
e)このPCR産物を分離する工程;ならびに
f)このPCR産物の配列と正常な遺伝子配列との間の違いを同定する工程、を包含し、
ここで、この増幅配列と正常な遺伝子配列との間の違いは、緑内障の診断特徴である。
a)BMP−2A、BMP4、BMP−5またはBMP7を発現する組換え細胞の集団を含む第一の組成物を得ること;
b)候補物質を得ること;
c)この組成物およびこの候補物質をインキュベートすること;
BMP誘導性Smadシグナル伝達経路および/またはBMP調節性の遺伝子発現を開始する能力について、この化合物を試験すること;ならびにBMPのこれらの下流の効果を阻害、または刺激する候補物質を同定すること。
小柱網は、房水の正常な流れにおいて、重要な役割を担うことが提案されており、そして緑内障の眼における流出抵抗の主な部位であると推測されている。ヒト小柱網(HTM)細胞は、房水が眼から出る流出チャネルを裏打ちする特殊化した細胞である。細胞の変化した合成機能は、POAG、ステロイド緑内障、および他のタイプの緑内障の病因に関与し得る。
(a)局所的な眼用処方 重量%
眼のBMP−4の発現を上昇させる薬剤 0.01〜2
HPMC 0.5
塩化ナトリウム 0.8
BAC 0.01
EDTA 0.01
NaOH/HCl qs pH7.4
精製水 qs 100mL
(b)局所的な眼用処方 重量%
グレムリンアンタゴニスト 0.01〜2
HPMC 0.5
塩化ナトリウム 0.8
BAC 0.01
EDTA 0.01
NaOH/HCl qs pH7.4
精製水 qs 100mL
(c)局所的な眼用処方 重量%
Smad1/5アゴニスト 0.01〜2
HPMC 0.5
塩化ナトリウム 0.8
BAC 0.01
EDTA 0.01
NaOH/HCl qs pH7.2
精製水 qs 100mL
本発明の化合物は、眼内挿入デバイス中で処方されることがさらに企図される。
本発明はまた、BMPシグナル伝達経路に関与する新しい抗緑内障治療剤の発見のために有用である(図5を参照のこと)。選択的BMPリガンドは、BMP I型およびBMP II型セリン/スレオニンキナーゼレセプター(BMP−RIおよびBMP−RII)に結合し、そしてSmadタンパク質を介してシグナルを伝達する。BMPシグナルは、タンパク質−タンパク質相互作用およびタンパク質−DNA相互作用を介して、Smadによって伝搬される(AttisanoおよびTuen Lee−Hoeflich 2001)。調節的Smad1およびSmad5は、BMPレセプターへのリガンドの結合によって(リン酸化を介して)活性化される(von BubnoffおよびCho 2001)。これらの調節的Smadは、その後、Smad4と相互作用して、核に転移するヘテロマー複合体を形成する。この複合体は、どの核補助因子が存在するかに依存して、この転写複合体を認識する選択的遺伝子の転写を活性化することまたは抑制することが可能である。
本発明は、緑内障の初期検出のための方法、化合物およびキットを提供する。このキットは、BMPポリペプチドまたはBMPタンパク質をコードする核酸セグメントを含み得る。このキットはさらに、サンプルと本発明の核酸またはペプチドとの間の相互作用を検出するための試薬を含み得る。提供された試薬は、放射性標識化、蛍光標識化、または酵素的標識化され得る。このキットは、本発明の核酸またはペプチドもしくはタンパク質と結合または相互作用し得る、公知の放射性標識された薬剤を含み得る。
細胞培養:ヒトTM細胞およびONH細胞を、ドナーの眼から記載されるように生成した(Steelyら、1992;Steelyら、2000;Wilsonら、1993;Clarkら、1994;Clarkら、1995b;Clarkら、1995c;Clarkら、1996;Clarkら、2001a;Clarkら、2001b;Dickersonら、1998;Wordingerら、1998;Wordingerら、1999;Wordingerら、2000;Wordingerら、2002;Lambertら、2001;Agarwalら、1999;Liuら、2001)。TM細胞を、年齢6日齢から90歳の範囲のドナーのTM外移植体から増殖させた。ヒト視神経乳頭星状細胞および篩板(LC)細胞を、慎重に解剖した視神経乳頭(2日齢〜90歳までの年齢のドナー)から生成し、そして以前の報告に従って特徴付けた(Lambertら、2001;Clarkら、1995a)。細胞を、以下の培地:TM細胞については10%ウシ胎仔血清(HyClone,Logan,UT)および抗生物質(Gibco BRL−Life Technologies,Grand Island,NY)を含むHam’s F10培地(JRH Biosciences、Lenexa、KS);LC細胞については10%FBSを含むダルベッコ改変イーグル培地(DMEM,HyClone);そしてONH星状細胞については5%FBSを含む星状細胞増殖培地(AGM,Clonetics,San Diego,CA)内で、コンフルエントまで増殖させた。
以下の参考文献は、例示的な手順、または本明細書中に記載される手順に対して補助的な他の詳細を提供する程度にまで、本明細書中で参考として具体的に援用される。
(書籍)
Claims (6)
- 骨形態形成ファミリーメンバー遺伝子の発現の変化を検出することによる、緑内障の診断を補助するための方法であって、該方法は、以下の工程:
g)緑内障を有すると疑われる患者からの組織または液体のサンプルから得られたサンプルからDNAを抽出する工程;
h)複数のPCRプライマーを得る工程であって、該プライマーの各々は、配列番号1、配列番号3、配列番号5、配列番号7、配列番号37、配列番号39、配列番号41、または、配列番号45由来の、18〜1547連続するヌクレオチドからなる配列を含む、工程;
i)該プライマーを使用して、該抽出されたDNAの領域を増幅してPCR産物を得る工程;
j)該PCR産物を分離する工程;ならびに
k)該増幅した抽出されたDNA配列と該プライマーの配列との間の発現レベルの違いを同定する工程、を包含し、
ここで該増幅配列とプライマーとの間の発現レベルの違いが緑内障の診断特徴として使用されるである、方法。 - 請求項1に記載の方法であって、前記組織サンプルまたは前記液体サンプルは、血液または頬の細胞である、方法。
- 請求項1に記載の方法であって、前記プライマーは、配列番号1、配列番号3、配列番号5、配列番号7、配列番号37、配列番号39、配列番号41、または、配列番号45の、20〜100連続するヌクレオチドからなる配列を含む、方法。
- 請求項1に記載の方法であって、前記プライマーは、配列番号3の20〜50連続するヌクレオチドからなる配列を含む、方法。
- 請求項1に記載の方法であって、前記PCR産物は、SSCP、DGGE,ASOまたはRFLPによって分離される、方法。
- 請求項1に記載の方法であって、前記プライマーは、配列番号45の18〜1547連続するヌクレオチドからなる配列を含む、方法。
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PCT/US2002/035251 WO2003055443A2 (en) | 2001-10-31 | 2002-10-31 | Bone morphogenic proteins (bmp), bmp receptors and bmp binding proteins and their use in the diagnosis and treatment of glaucoma |
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