JP2004510743A - Method of modulating apoptosis by administration of relaxin agonist or antagonist - Google Patents

Abstract

The present invention relates to the discovery that relaxin is involved in the development or maturation of body tissues. Knockout of the gene encoding relaxin results in various abnormalities in the development of various body tissues. The present invention provides a method of modulating apoptosis by administering a relaxin agonist or antagonist to a subject. In one aspect, the invention provides a method of modulating apoptosis, the method comprising: providing a subject in need thereof with a method for reducing apoptosis in cells that express the relaxin receptor. Administering an effective amount of a relaxin agonist for a sufficient time.

Description

^１精巣および^３前立腺の各々のサイズを、組織の長さを幅で乗じて、その面積によって測定した。精巣上体および精嚢のサイズ（面積）は、正確には測定できなかったので、各組織の緊密な近似を、以下のように計算した：精巣上体（組織の長さに、精巣上体頭部の幅を乗じて測定した^２）；精嚢（各器官の長さに、組織の平均幅を乗じて測定した^４）。^＊は、ｐ＜０．０５を示す。
【０１９２】
（雄性生殖管の組織学に対する、レラキシン遺伝子ノックアウトの影響）
Ｈ＆Ｅ＆Ｍａｓｓｏｎトリクロム染色：ＲＬＸ＋／＋マウスおよびｒｌｘ−／−マウス由来の雄性生殖管組織切片を、精子成熟、管サイズ／コンパクトさ（精巣、精巣上体）およびコラーゲンにおける差異について最初に観察した。
【０１９３】
精巣：１週齢で、レラキシン野生型動物の精細管（生殖細胞／精母細胞、セルトリ細胞を含む区画であり、精子成熟が生じる）は、より小さく、より円筒形の形状であり（表２）、そして各器官の白膜（卵型の本体を被覆する膜）を取り巻くコラーゲンの薄層によって主に支持される。対照的に、ｒｌｘ−／−マウスの精巣に由来する細管は、より大きくかつ拡大していたが、精巣内のコラーゲンによって完全に取り囲まれ、支持されていた。このことは、誕生直後にさえ、レラキシンの非存在下で、精巣の内部組織における差異を直ぐに示唆した。しかし、１週齢では、組織切片のいずれの群においても、未成熟な精子は、検出されなかった。１月齢までに、ＲＬＸ＋／＋マウスに由来する精巣細管は、（１週齢の細管サイズと比較して）サイズがより大きくなり、わずかにコンパクトでなく、そして主に未成熟精子を含んだ。対照的に、１月齢のＲＬＸホモ接合体マウスに由来する細管は、１週齢のノックアウトマウスから測定された細管サイズと異ならず、そしてＲＬＸ＋／＋マウス由来のサンプルと比較して、あまり未成熟でない精子を含んだ。このことは、レラキシンヌル変異体マウスが、３月齢の組織切片と比較した場合にさらに確認される、遅延した精子成熟のプロセスを受け；ＲＬＸ＋／＋マウス組織由来の切片が、主に成熟した精子を有する（１月齢の細管サイズと比較して）より大きい細管を含むことをさらに示唆した。逆に、ｒｌｘ−／−マウス由来の切片は、（３月齢のＲＬＸ＋／＋組織切片と比較して）あまり成熟していない精子およびなおいくらかの未成熟の精子を有する、僅かにより小さい細管（これは、１週齢／１月齢のｒｌｘ−／−マウス由来の細管サイズと異ならなかった）を含んだ。ＲＬＸ＋／＋マウス由来の精巣における精子成熟レベルは、ｒｌｘ−／−マウスの精巣において観察された精子成熟レベルよりも有意に（ｐ＜０．０５）高いことが示された（使用された等級付けの尺度については、表２を参照のこと）。成体ＲＬＸノックアウトマウスの精巣における精子成熟レベルが、未成熟の（１月齢）ＲＬＸ野生型マウス組織において観察されるレベルに類似したこともまた注目された。１〜３月齢で、正常マウスの精巣は、白膜を覆うコラーゲンの薄膜によって取り囲まれたままであったが；いくつかの組織において、コラーゲンの散在する薄い裏打ちがまた、精細管を支持することが観察された。しかし、コラーゲンの内部層は、年齢と共に減少することが見出された。ｒｌｘ−／−マウスにおいて、細管構造の中間で検出されるコラーゲンは、精巣内のより大きい細管サイズの構造に起因して消失したが、野生型動物由来の組織サンプルにおいて観察されたものと比較して、なおより密であり、かつ堅実であった。非常に若い齢でのＲＬＸヌル変異体マウスの生殖管において観察された増加したコラーゲンが、組織をより硬く堅固にし得ることが予想され、これは、上で考察された細管の組成および精子成熟における変化に関連し得る。興味深いことに、正常な成体マウスにおけるレラキシンの存在はまた、正常な未成熟（１月齢）マウスから観察された構造と比較して、精細管の細管構造の弛緩（増大した間質間隔）を誘導した。対照的に、１月齢および３月齢のｒｌｘ−／−マウスに由来する精巣由来の細管の組織化（サイズ／コンパクトさ）における差異は、存在しなかった。さらに、１月齢、および３月齢まで、ｒｌｘ−／−マウス由来の精巣細管は、ＲＬＸ野生型動物に由来する細管細胞と比較して、増加した数の死細胞を含むようであった。未成熟組織において見られた増加した数の死細胞は、動物間で異なったが、マウスが年齢と共に成熟するにつれ一貫して検出された。
【０１９４】
精巣上体：１ヶ月齢および３ヶ月齢のＲＬＸ＋／＋およびｒｌｘ−／−の生殖管の精巣上体において、細管のサイズに有意な差異は注目されなかった。しかし、成体において、細管のコンパクトさは、ＲＬＸ野生型動物由来の組織と比較して、ＲＬＸホモ接合体マウス由来の組織においてより明らかであった。ｒｌｘ−／−マウスの精巣上体細管もまた、結合組織によってより大きな程度まで支持された。３ヶ月齢のＲＬＸ＋／＋マウス組織由来の細管構造は、形が緩み、コラーゲンによって部分的に維持されるのみであった。逆に、ＲＬＸノックアウト動物から得た組織切片は、コラーゲンの薄い層によって非常にきれいに囲まれたよりコンパクトな細管を有することが示された。精巣の場合、ｒｌｘ−／−マウスから得た組織は、ＲＬＸ＋／＋マウスに由来する組織サンプルと比較して、観察した全ての年齢において増加した濃度のコラーゲンによって支持された。表２に示されるように、成熟した精子（精巣上体細管中）の密度は、ＲＬＸ野生型マウスから得た組織切片と比較して、１ヶ月齢および３ヶ月齢の各それぞれの年齢群において、ＲＬＸヌル（ｎｕｌｌ）変異マウスから得た組織切片においてより低かったことも見出された。しかし、この差異は、統計学的に有意ではなかった。レラキシンを欠くマウスの精巣上体における成熟精子のこの減少は、おそらくこれらの動物の精巣で生じる遅延した精子成熟プロセスに原因がある（表２）。
【０１９５】
【表２】

表２：組織切片を、Ｈ＆Ｅで染色し、以下のように等級に分けた：
^ａ細管サイズ−１＝全ての細管が小さく、環状の形態である；２＝ほとんどの細管が１と比較してわずかに伸張しているが、いくつかのより小さい環状細管が観察される；３＝細管は１と比較して著しく伸張しているが、いくつかのより小さい環状細管および中サイズの細管が観察される；４＝全ての細管が１および２と比較して著しく伸張している。
^ｂ細管のコンパクトさ−１＝全ての細管の形が緩んでいる；２＝形が緩んでいる細管の割合のほうが、コンパクトな細管の割合よりも多い；３＝コンパクトな細管の割合のほうが、形が緩んでいる細管の割合よりも多い；４＝全ての細管がコンパクト。
^ｃ精子の成熟度−０＝精子は検出されず；１＝未成熟な精子のみが検出された；２＝未成熟な精子の割合のほうが成熟な精子の割合よりも多い；３＝成熟な精子の割合のほうが未成熟な精子の割合よりも多い；４＝成熟な精子のみが検出された。
示した数値は、測定した各パラメーターについて使用した等級分けスケールの平均±ＳＥ（ｎ）である。^＊はｐ＜０．０５を示す。
【０１９６】
【表３】

表３：組織切片を、Ｍａｓｓｏｎトリクロム染色によって染色し、以下のように等級に分けた：
^ａコラーゲン−１＝構造の外部層を取り囲むコラーゲンの薄い内層のみ；２＝組織の内部構成成分を取り囲むコラーゲンのさらなる内層；３＝組織の外部層および内部構成成分を取り囲むより厚いコラーゲンの内層；４＝３＋コラーゲンで充填された細管／構成成分との間の空間。
【０１９７】
示した数値は、等級分けスケールの平均±ＳＥ（ｎ）である。^＊はｐ＜０．０５を示す。
【０１９８】
前立腺：１ヶ月齢において、ＲＬＸ＋／＋由来組織とｒｌｘ−／−由来組織との間に前立腺構造に有意な差異は注目されなかった。しかし、正常動物由来の前立腺の管の間の空間は、微量のコラーゲンのみと会合していた。対照的に、ｒｌｘ−／−マウス由来の前立腺の管は、形の緩んだ結合組織の層が介在していた。成体（３ヶ月）では、ｒｌｘ−／−マウスは、より小さい腺および管を有するより小さい前立腺を含んだ。ＲＬＸノックアウトマウス由来の組織の管は、よりコンパクトであり、そして結合組織によって完全に支持されたが、ＲＬＸ野生型マウスから得た管は、より広がり（形が緩んでいる）、そしてなおコラーゲンによってほとんど支持されなかった。さらに、ｒｌｘ−／−マウスから得た成体前立腺の管は、より小さい上皮を有するようであり（含まれる細胞が少ない）、一方、ＲＬＸ＋／＋前立腺サンプルの管は、より大きい上皮層／腺組織を含んだ。これらの結果が精巣および精巣上体に関する本発明者らの最初の知見に加えられて、これらのことは、雄性生殖組織の遅延した成熟プロセスが、機能的に活性なレラキシン遺伝子を欠くマウスで生じ、そしてこれらの組織内のコラーゲンの蓄積に関連するということを示す。
【０１９９】
（抗体染色による細胞アポトーシスの検出）
Ｈ＆Ｅ染色からなされた観察（これによるとｒｌｘ−／−マウス由来の組織が低下した精子成熟および増大した細胞死（精巣）を引き起こし、そして減少した上皮（細胞）層（前立腺）を含む）に基づいて、観察された増大した細胞死がアポトーシス経路の結果であるか否かを調査することを決定した。
【０２００】
Ｂａｘ抗体染色：Ｂａｘの過剰発現は、サイトカイン遮断によって誘導されるアポトーシス死を加速させ、そしてまた、Ｂｃｌ−２の死リプレッサー活性を相殺する（Ｋｒａｊｅｗｓｋｉら、Ａｍ．Ｊ．Ｐａｔｈｏｌ．１４５：１３２３−３６（１９９４））。Ｂａｘモノクローナル抗体を用いて、Ｂａｘタンパク質のインビボ分布を、雄性生殖管中で評価した。精巣：未成熟（１ヶ月）野生型動物由来の精細管は、Ｂａｘに対して弱い免疫染色を示し（濃い褐色の細胞染色）、これは、基底膜付近の胚芽細胞において主に観察された。これらの知見は、以前の報告と一致する（Ｂｅｎ−Ｈｕｒら、Ｃａｌｃｉｆ．Ｔｉｓｓ．Ｉｎｔ．５３：９１−９６（１９９６））。比較において、増大した数の細胞が、ｒｌｘ−／−マウス由来の精巣において、Ｂａｘについてポジティブに染色された。Ｂａｘポジティブ細胞の数を、血球計を用いて計数し、そしてＲＬＸ＋／＋マウス由来の組織から観察されたアポトーシス細胞数（１３．１±３．５細胞／精巣（ｎ＝６））と比較して、ｒｌｘ−／−マウスの精巣において有意により多い（３２．７±４．８細胞／精巣（ｎ＝６））が示された。増大したＢａｘ染色はｒｌｘ−／−組織において一貫して観察されたが、Ｂａｘに関してポジティブに染色された細管の数は、組織サンプル間で変化し、そしていくつかの細管がまたＢａｘを含まないことも観察された。成熟した雄性の生殖管の場合、成体ＲＬＸ野生型マウスの精巣は、Ｂａｘについて減少した染色を示し（３．４±１．４細胞／精巣（ｎ＝６））、これは、これらの動物が経験した精子成熟および組織成熟のレベルの増大と相関する。逆に、ｒｌｘ−／−マウス生殖管中で観察されたよりゆっくりとした組織成熟の速度は、３ヶ月齢のＲＬＸノックアウトマウスにおけるＢａｘ染色のさらなる増加（４４．４±８．１細胞／精巣（ｎ＝６））と相関する。観察した細胞の多くは、アポトーシスの最終段階のようであり、ことによるとアポトーシス体を示した。しかし、Ｂａｘ染色は、成熟精子細胞と関連しなかった。精巣上体：より強力なＢａｘ染色が、１ヶ月のＲＬＸ＋／＋マウス組織から得た精巣上体細管の上皮細胞の細胞内膜に存在した。１ヶ月のｒｌｘ−／−マウス組織から得た精巣上体細管の細管は、相対的により強いレベルのＢａｘ染色を含み、これはさらに、３ヶ月の組織切片で増大しない場合であっても、維持された。対照的に、成体野生型マウスの精巣上体は、この組織が年齢と共に成熟するにつれ、１ヶ月の正常マウス由来の組織と比較して、より弱いＢａｘ発現の染色を含んだ。前立腺：正常な１ヶ月および３ヶ月の動物の前立腺の上皮細胞は、Ｂａｘについて弱いポジティブ染色を示した。研究した他の組織に関して、特にｒｌｘ−／−動物の（前立腺管の）上皮層内の細胞は、未成熟（１ヶ月）組織および成体（３ヶ月）組織の両方において、Ｂａｘについて増大した染色を示した。これらの知見は、レラキシンが雄性生殖管内の細胞アポトーシスの調節に新たな役割を果たし得ることを示唆するが、細胞アポトーシスを検出する別の抗体を用いたさらなる研究を、レラキシンの作用を確認するために実施した。
【０２０１】
カスパーゼ−９（中心的な死のプロテアーゼ）は、他の記載されたプロテアーゼファミリーと配列、構造および基質特異性が異なる、システインプロテアーゼの特有なファミリーに属する。このカスパーゼファミリーのメンバー（これらは、通常、タンパク質分解性切断事象のカスケードに関与する）は、細胞活性に重要である特定の標的タンパク質を破壊するように作用することによって、アポトーシス機構の重要な構成成分として機能する。精巣：中程度のカスパーゼ−９染色が、１ヶ月ＲＬＸ＋／＋精巣において観察され（２８．１±５．６細胞／精巣（ｎ＝７））、これは、精原細胞（ｓｐｅｒｍａｔａｇｏｎｉａ）または精母細胞よりもむしろ、精細管内の胚細胞と主に関連するようである。Ｂａｘに関して、カスパーゼ−９染色の有意に（ｐ＜０．０５）増大したレベルは、１ヶ月のｒｌｘ−／−マウスの精巣（７６．６±１０．２細胞／精巣（ｎ＝６））で検出されたが、重度のカスパーゼ−９染色は、ほとんど細管と関連せず、一方で多くの細管は、ほとんどまたは全くカスパーゼ−９タンパク質を含まないことが観察された。年齢に関して、正常な未成熟組織中で検出されたカスパーゼ−９のレベルは、３ヶ月齢のより高齢の組織において一貫して見出された（２６．９±４細胞／精巣（ｎ＝６））。しかし、精巣の大きさが増大するにつれ、検出されるアポトーシス細胞の数は、少ない画分の高齢組織を示した。ｒｌｘ−／−マウス由来の精巣細管は、１ヶ月の組織におけるカスパーゼ９染色細胞の密度と比較して、３ヶ月においてわずかに少ないアポトーシス細胞（６１．６±９．８細胞／精巣（ｎ＝５））を含んだ。ＲＬＸノックアウトマウスの組織由来のポジティブに染色されたアポトーシス細胞の数にもかかわらず、残りは、研究した全年齢の野生型対応物よりも有意に（ｐ＜０．０５）大きかった。精巣上体：カスパーゼ−９の染色は、１ヶ月のＲＬＸ＋／＋精巣上体細管から検出されず、一方、微量のポジティブ細胞がｒｌｘ−／−組織切片において観察された。しかし、ポジティブに染色された細胞は、まばらに散在し、そしてその細管の上皮層において検出された。年齢に関して、カスパーゼ−９の明確な染色は、３ヶ月齢のＲＬＸ＋／＋マウス組織およびｒｌｘ−／−マウス組織において検出されなかった。前立腺：カスパーゼ−９の染色は、調査した全ての年齢（１〜３ヶ月齢）において、ＲＬＸ＋／＋組織およびｒｌｘ−／−組織の前立腺中で同定されなかった。それにもかかわらず、得られた結果は、最初に、このレラキシンが細胞アポトーシスの調節に関連していることを示唆する。しかし、レラキシンが細胞増殖経路に関与するか否かを確立するためのさらなる研究が必要とされた。
【０２０２】
（抗体染色による細胞増殖の検出）
ＰＣＮＡ染色：増殖細胞核抗原（ＰＣＮＡ）抗体を使用して、ＤＮＡ合成が生じる核領域と関連する能力に基づいて、雄性生殖管における細胞増殖を検出した。精巣：ＰＣＮＡを未成熟な精細管および成熟な精細管において検出したが、ＰＣＮＡ染色における有意な差異は、１ヶ月齢および３ヶ月齢のＲＬＸ＋／＋マウスおよびｒｌｘ−／−マウス由来の精巣管において検出されなかった。未成熟組織において、ＰＣＮＡ染色は、通常、有糸分裂活性な精原細胞で高度に発現され、時に、いくつかのセルトーリ細胞で高度に発現され、これらの染色は、増殖活性に対応する。この研究において、これらの細胞は、マウス成体においても良好に、より均一かつ絶え間なくＰＣＮＡについて標識され、これはおそらく、これらの増殖細胞の不断の活性化を介して、再生を開始し得る両方の群の能力を反映する。精巣上体：ＰＣＮＡについての染色は、１ヶ月齢および３ヶ月齢のＲＬＸ＋／＋マウスまたはｒｌｘ−／−マウスのいずれか由来の精巣上体においても検出されなかった。これらの知見は、成熟精子のレザバーとして作用する精巣上体の役割と一致する。前立腺：ＰＣＮＡについての弱い染色が１ヶ月のＲＬＸ＋／＋マウスおよびｒｌｘ−／−マウス由来の前立腺管の上皮細胞と関連していた。しかし、３ヶ月齢の群からはいずれも、ＰＣＮＡについての染色は検出されなかった。白血球増殖研究は、ある程度の生殖組織に限定され、この蓄積された知見によって、おそらく、レラキシンはおそらく、細胞増殖に対してよりも細胞アポトーシスの調節において、影響のある役割を果たすということが、確認された。
【０２０３】
（他の身体細胞の組織学に対するレラキシン遺伝子ノックアウトの影響）
ＲＬＸ野生型（ＲＬＸ＋／＋）の雄および雌ならびにＲＬＸ遺伝子ノックアウト（ｒｌｘ−／−）の雄および雌を、上記に記載されるように、ＲＬＸヘテロ接合体（ＲＬＸ＋／−）の親から発生させた。マウスを、組織を回収するために、１週間、１ヶ月および／または３ヶ月で重量し、屠殺した。以下の組織（脳、心臓、肝臓、腎臓、胸腺、脾臓および雄性生殖管を含む）を回収し、重量を量り、詳細な組織学的分析のために１０％ホルマリン中においた。これらの組織重量のまとめを表４〜８に示す。
【０２０４】
１週齢において、雄性ｒｌｘ−／−マウスは、ＲＬＸ＋／＋動物と比較して、有意に（ｐ＜０．０５）小さい肝臓、腎臓および脾臓を含んだ。しかし、これらの重量の差異は、１ヶ月齢においてもはや明らかではなかった。その代わり、１ヶ月齢の雄性ｒｌｘ−／−マウスの胸腺は、そのＲＬＸ＋／＋対応物よりも小さかった（ｐ＜０．０５）。この重量の差異は、３ヶ月齢においてもはや明らかではなかった。雄性生殖管の場合、ＲＬＸ＋／＋群とｒｌｘ−／−群との間で１週齢または１ヶ月齢において有意な差異は注目されなかった。しかし、３ヶ月齢までに、ｒｌｘ−／−マウスの生殖管は、ＲＬＸ＋／＋動物から得たものよりも有意に（ｐ＜０．０５）小さかった。
【０２０５】
雌性の１週齢のＲＬＳ＋／＋マウスとｒｌｘ−／−マウスとの間で注目に値する差異は観察されなかった。しかし、１ヶ月齢までに、ｒｌｘ−／−雌性マウスは、そのＲＬＸ＋／＋対応物よりも、有意に小さい肝臓を有した。３ヶ月齢までに、この重量の差異は、もはや観察されず、そして他の器官の他の有意な差異ももはや注目に値しなかった。
【０２０６】
ＲＬＸ−／−マウス（ＲＬＸ＋／−の親由来）における器官および組織の差異が、ｒｌｘ−／−子孫をＲＬＸ−／−の親から得た場合にもさらに強調されるか否かを決定するために、ＲＬＸ＋／＋マウスおよびｒｌｘ−／−マウスを、対応するの親のセットから得て、そして１週齢、１ヶ月齢および３ヶ月齢において屠殺した。脳、心臓、肝臓、腎臓、胸腺、脾臓、雄性生殖管、腸、および肺を回収し、そして重量を量った。これらの器官の重量を図４〜８に示す。
【０２０７】
器官重量または組織重量がマウスが齢を重ねるにつれ標準化されるようであるが、通常の重量単独では、通常の器官とも通常の組織とも等しくないことに、さらに注目する。このデータは、成長サイクルの任意の時期における器官（組織）重量　対　体重の比率が、潜在する器官発達もしくは組織発達または細胞構成における変化を示すことを示す。１つの例において、線維症によって浸潤された（変更された）小さい肝臓が、より大きい正常な肝臓または脂肪で充填された肝臓と同じ重量またはより重い重量である。従って、雌性および雄性のＲＬＸ−／−マウスの、脳、心臓、肝臓、腎臓、胸腺、脾臓、腸および肺（レラキシンの非存在下で発達する）は、増大したアポトーシスおよび細胞外マトリクス（コラーゲン）蓄積の証拠を示す。
【０２０８】
（表４：平均±ＳＥ（ｎ）で示した、それぞれ、ＲＬＸ＋／＋の親およびｒｌｘ−／−の親由来の、１週齢マウスの組織比較）
【０２０９】
【表４】

（表５：平均±ＳＥ（ｎ）で示した、ＲＬＸ＋／−の親由来の、１ヶ月齢マウスの組織比較）
【０２１０】
【表５】

（表６：平均±ＳＥ（ｎ）で示した、それぞれ、ＲＬＸ＋／＋の親およびｒｌｘ−／−の親由来の、３ヶ月齢雄性マウスの組織比較）
【０２１１】
【表６】

（表７：平均±ＳＥ（ｎ）で示した、それぞれ、ＲＬＸ＋／＋の親およびＲＬＸ−／−の親由来の、１ヶ月齢マウスの組織比較）
【０２１２】
【表７】

（表８：平均±ＳＥ（ｎ）で示した、それぞれ、ＲＬＸ＋／＋の親およびｒｌｘ−／−の親由来の、３ヶ月齢マウスの組織比較）
【０２１３】
【表８】

（皮膚の組織学に対するレラキシン遺伝子ノックアウトの影響）
組織リモデリングの調節におけるレラキシンの役割を、ｒｌｘ−ヌルマウス（ｒｌｘ−／−）における皮膚組織学を研究することによって調べた。これらのマウスは、ｒｌｘ−／−のオス親およびメス親の子孫であった。少なくとも５匹のオスマウスおよび５匹のメスマウスの、耳および背中からの連続した皮膚サンプルを、Ｈ＆Ｅ染色およびＭａｓｓｏｎトリクロム染色によって染色し、各時点で調べた。同じ時点の類似のサンプルを、同じ系統のＲｌｘ＋／＋マウスから得た。ｒｌｘヌルマウスの組織学的試験の際に、真皮が、時間の進行につれ厚くなること、および真皮一帯の増大した線維症を有することを見出した。ｒｌｘヌルマウスの皮膚サンプルは、１週齢において正常であった；しかし、１ヶ月までに初期の皮膚線維症が明らかとなった。３ヶ月齢までに、皮膚線維症の顕著な増大が存在し、これは、６ヶ月齢までに密度が増大した。これらの皮膚に関する知見は、雄性および雌性のｒｌｘヌルマウスにおいて類似していた。
【０２１４】
これらのマウスにおいて、表皮は正常であり、１ヶ月齢までにＲｌｘ＋／＋マウスからは識別不可能になるｒｌｘヌルマウスにおける初期のより明るい外被色以外は、毛も変化しなかった。ｒｌｘマウスからの、血清化学、血液学的パラメーターおよび尿の試験は、注目に値しなかった。
【０２１５】
これらの知見は、レラキシンが、重要なマトリクス分子、マトリクス分解酵素および増殖因子を変更することによって、インビトロおよびインビボにおいてマトリクスターンオーバーに影響を与えるという、以前の観察を支持する。間質性コラーゲンを産生する多くの間質細胞は、雄性供給源由来または雌性供給源由来にかかわらず、レラキシンレセプターを有する。抗体に基づくアッセイによって、雌性の月経周期の黄体期の間に、低いピコグラムレベルでの血清中レラキシンが検出され得、そしてこれらのレベルは、妊娠中（このとき、組織リモデリングが最も明らかである）に上昇する。対照的に、雄性における血清レラキシンは検出不可能であることが報告されている。
【０２１６】
Ｒｌｘヌルマウスは、正常な皮膚におけるコラーゲンターンオーバーの普遍的な変更に対してレラキシンを関連付ける最初の直接的な証拠を提供する。これらの結果は、レラキシンが、雄性および雌性における検出流動レベルより低い生物学的に関連する濃度で、産生され得るかまたは循環し得ることを示す。レラキシンは、他のマトリクス調節分子と協力して、マトリクスターンオーバーの規則正しい維持に関与する。レラキシンは、組織リモデリング、血管形成の促進および血管拡張の刺激に影響を与え得る。これらの結果によってまた、レラキシン合成経路、およびレラキシンレセプターが、線維症状態（例えば、強皮症）と関連することが示される。
【０２１７】
これまでの例は、例示するために提供され、特許請求される本発明の範囲を限定するために提供されない。本発明の他の改変は、当業者に容易に明らかであり、添付の特許請求の範囲によって含まれる。全ての刊行物、特許、特許出願、およびその中に引用される他の参考文献は、本明細書によって参考として援用される。[0001]
(Cross-reference of related applications)
No. 60 / 238,232 (filed on Oct. 4, 2000), US Provisional Patent Application No. 60 / 241,991 (filed on Oct. 20, 2000) and US Provisional Application No. 60 / 238,232. No. 60 / 242,037 (filed Oct. 20, 2000), the disclosures of which are incorporated herein by reference.
[0002]
(Background of the Invention)
Normal tissue growth and development is achieved by programmed cell growth, differentiation and cell death. Cell proliferation and differentiation is required for the formation of new cells and tissues. Conversely, programmed cell death (also called apoptosis) is required to remove existing cells, including immature or damaged cells. Apoptosis occurs naturally in virtually all tissues of the body. Apoptosis plays an important role in tissue homeostasis, that is, ensuring that the number of new cells produced is offset by a corresponding number of dying cells. For example, the cells in the intestinal lining divide rapidly, so the body must eliminate cells after only three days to prevent overgrowth of the intestinal lining.
[0003]
Disruption of the genetic program, either by abnormally increasing or decreasing the rate of cell proliferation and / or apoptosis, can result in abnormal tissue development. For example, a decrease in cell proliferation below normal levels can lead to immature tissue and other tissue abnormalities. Increased cell proliferation above normal levels is considered to be a major event in the development of neoplasia and cancer, as well as other cell proliferative disorders. An abnormal increase in apoptosis can also lead to precancerous lesions. Premalignant lesions include breast lesions (which can develop into breast cancer), prostate lesions (which can develop into prostate cancer) or skin lesions (which can develop into malignant melanoma or basal cell carcinoma), Colon adenomatous polyps, which can develop into colon cancer, and other such neoplasias. Such lesions show a strong tendency to develop into a malignant tumor or cancer.
[0004]
Precancerous lesions can result from the accumulation of damage to existing cells in various tissues of the body. Such injuries may include exposure to sunlight, radiation, mutagens, and carcinogens, chemicals (eg, pesticides, herbicides, preservatives) and the like that are commonly found in the diet. These injuries can result in the accumulation of mutations in the cells, which can lead to hyperplastic conditions (ie, an abnormal increase in cell number) (eg, overgrowth of liver, kidney, spleen, thymus, intestine, lung or prostate tissue). (Like formation). Down-regulation of apoptosis may also lead to the accumulation of cells in these hyperplastic states.
[0005]
An abnormal increase in apoptosis can interfere with the normal development and / or differentiation of a tissue. For example, apoptosis is required during pregnancy and maturation of male genital tract tissue. An abnormal increase in apoptosis may also interfere with the formation of new cells and tissues, thereby preventing normal tissue maturation or development.
[0006]
Therefore, a need exists for a method of modulating apoptosis by administering an agonist or antagonist of apoptosis. In particular, there is a need for methods of treating conditions associated (directly or indirectly) with abnormally fast or slow apoptosis. The present invention satisfies this need by providing a method for the administration of a relaxin agonist or antagonist to treat abnormalities in such tissues by modulating apoptosis in the relaxin-related tissues.
[0007]
(Summary of the Invention)
The present invention relates to the discovery that relaxin is involved in the development of body tissue. Knockout of the gene encoding relaxin results in various abnormalities in the development of various body tissues, such as reduced size of such tissues, increased collagen deposition in such tissues, and immature such tissues . Conversely, the accumulation of relaxin-responsive cells leads to abnormalities in body tissues. The present invention provides a method of modulating apoptosis in a tissue by administering a relaxin agonist or a relaxin antagonist.
[0008]
In one aspect, the present invention provides a method of modulating apoptosis in a subject, the method comprising: providing a subject in need thereof with an apoptosis sufficient to reduce apoptosis in cells expressing the relaxin receptor. Time, by administering an effective amount of a relaxin agonist. Tissues typically affected by administration of a relaxin agonist useful in the methods according to the invention include, for example, liver, kidney, spleen, thymus, brain, heart, intestine, skin, lung, male and female reproductive tracts Is mentioned. The male reproductive tract tissue includes, for example, prostate, epididymis, seminiferous tissue, or testicular tissue. Female genital tract tissue includes, for example, connective tissue in the uterus, cervix, pubis, or lower extremity girdle.
[0009]
In certain embodiments, administration of a relaxin agonist typically reduces the number of apoptotic cells. In another embodiment, the relaxin agonist stimulates tissue maturation. For example, a relaxin agonist can stimulate the maturation of male genital tract tissue (eg, prostate tissue, epididymal tissue, ring sperm tissue, testicular tissue or semen). In one embodiment, the maturation results in an increase in the number of cells in the underdeveloped testis, such as an increase in the number of mature testis cells. In another embodiment, maturation of male genital tract tissue results in an increase in the number of live sperm cells as compared to a tissue not contacted with a relaxin agonist. In yet another embodiment, fibrosis can be reduced by a relaxin agonist and / or excess collagen deposition can be reduced.
[0010]
A subject in need of a relaxin agonist administration can have a relaxin deficient condition, such as, for example, immature tissue, excessive collagen deposition and / or low sperm count. For example, the immature tissue can be an immature male genital tract (eg, a testis with poor development). Such immature tissue may be present in other mature or immature animals.
[0011]
The relaxin agonist can be a relaxin, a relaxin analog, a small molecule relaxin effector or a relaxin nucleic acid. Relaxin is typically vertebrate relaxin, and more typically human relaxin.
[0012]
Compositions comprising relaxin agonists useful in the methods according to the invention include, for example, administration by injection, injection, oral delivery, nasal delivery, and pulmonary, rectal, transdermal, intratissue or subcutaneous delivery. May be prescribed for: Such compositions may also be formulated for delayed release of the relaxin agonist into the tissues and circulation of the subject. Certain embodiments include compositions formulated for infusion or injection, or pulmonary, subcutaneous, or transdermal delivery. In certain embodiments, a nucleic acid encoding a relaxin agonist may be formulated for administration to a subject with a vector encoding a relaxin agonist. For example, the vector can be an expression vector that expresses a relaxin agonist in the cell. Alternatively, a relaxin or relaxin analog nucleic acid can be formulated for delivery to a subject. The subject can be pre-pubertal or post-pubertal.
[0013]
In another aspect, the invention provides a method for modulating apoptosis in a subject in need thereof, wherein the method is effective for a sufficient time to increase apoptosis in a cell population expressing a relaxin receptor. By administering an amount of a relaxin antagonist. In one embodiment, the relaxin antagonist inhibits the binding of relaxin to the relaxin receptor. In another embodiment, the relaxin antagonist reduces relaxin-related tissue remodeling.
[0014]
Tissues typically affected by administration of a relaxin antagonist useful in the methods according to the invention include, for example, liver, kidney, spleen, thymus, brain, heart, intestine, skin, lung, male genital tract, female genital tract And the like. The male reproductive tract tissue includes, for example, prostate, epididymis, seminiferous tissue, or testicular tissue. In certain embodiments, the male genital tract tissue can be prostate tissue and can be mature or non-mature. Suitable target female genital tract tissues include, for example, the uterus, cervix, pubis, or connective tissue in the lower extremity girdle. Alternatively, the cell population can include cells that express the relaxin receptor, such as fibroblasts, osteoblasts, monocytes, epithelial cells or endothelial cells.
[0015]
A relaxin antagonist can be, for example, a relaxin binding agent, a relaxin receptor binding agent, a relaxin antisense nucleic acid, and the like. The relaxin binding factor can be, for example, an anti-relaxin antibody, a soluble relaxin receptor, a small molecule relaxin antagonist, and the like. The relaxin receptor binding factor can be, for example, an anti-relaxin receptor antibody, a relaxin analog, a small molecule relaxin receptor antagonist, and the like. Examples of relaxin or an antibody that binds to relaxin include polyclonal antibodies, monoclonal antibodies, Fab, Fab ′, F (ab ′) ₂ , Fv, single heavy chains, chimeric antibodies and the like.
[0016]
Compositions comprising relaxin antagonists useful in the methods of the invention include, for example, administration by infusion, injection, oral delivery, nasal delivery, and pulmonary, rectal, transdermal, tissue or subcutaneous delivery. May be prescribed for: The compositions can also be formulated for delayed release of the relaxin antagonist into the tissues and circulation of the subject. Certain embodiments include compositions formulated for infusion or injection, or pulmonary, subcutaneous, or transdermal delivery.
[0017]
In certain embodiments, a nucleic acid encoding a relaxin antagonist can be administered to a subject in a vector encoding a relaxin antagonist. In one embodiment, the vector is an expression vector that expresses a relaxin antagonist in a cell population. Alternatively, relaxin or a relaxin receptor antisense nucleic acid can be delivered directly to a subject according to any of the methods described above. In an exemplary embodiment, administration of a relaxin antagonist increases apoptosis and reduces unwanted cell accumulation. For example, these unwanted cells can be hyperplasia, hypertrophy, cancer or neoplasia.
[0018]
(Definition)
Before describing the present invention in more detail, it may be helpful to provide a definition of certain terms as used herein below to better understand the present invention.
[0019]
The term "nucleic acid" refers to a polymer composed of a plurality of nucleotide units (ribonucleotides or deoxyribonucleotides or related structural variants) linked via phosphodiester bonds. Nucleic acids can be of virtually any length, typically from about 6 nucleotides to about 10 nucleotides. ⁹ Nucleotides or more. Unless otherwise indicated, the conventional notation used herein for nucleic acids is as follows: the left hand end of a single stranded nucleic acid is the 5 'end; the left hand direction of a double stranded nucleic acid is 5 Called 'direction'. The direction of 5 'to 3' addition of the initial RNA transcript is referred to as the direction of transcription. The sequence region of the DNA strand that has the same sequence as the RNA and is 5 'to the 5' end of the RNA transcript is referred to as the "upstream sequence"; The sequence region of the DNA strand 3 'to the 3' end of the transcript is referred to as the "downstream sequence."
[0020]
As will be readily understood by those skilled in the art, nucleic acids include RNA, mRNA, cDNA, genomic DNA, synthetic forms, and mixed polymers (both sense and antisense strands), and nucleic acids also include Can be modified chemically or biochemically, or can include unnatural or derivatized nucleotide bases. Such modifications include, for example, labeling, methylation, replacement of one or more naturally occurring nucleotides by analogs, internucleotide modifications (eg, uncharged linkages (eg, methylphosphonates, phosphotriesters, phosphoramidates). , And carbamates), charged linkages (eg, phosphorothioates and phosphorodithioates), pendant moieties (eg, polypeptides), intercalators (eg, acridine and psoralen), chelators, alkylators, and modified linkages ( For example, α-anomeric nucleic acid)). Also included are synthetic molecules that mimic nucleic acids in their ability to bind to the indicated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art, and include, for example, peptides in which the peptide bonds are substituted for phosphate bonds in the backbone of the molecule.
[0021]
As used herein, the term “amino acid” or “amino acid residue” refers to an L amino acid or a D amino acid as further described below. Single letter and three letter abbreviations of commonly used amino acids are used herein (eg, Alberts et al., Molecular Biology of the Cell, Garland Publishing, Inc., New York, 3rd ed., 1994)).
[0022]
When describing a polypeptide, the term "conservative substitution" refers to a change in the amino acid composition of a polypeptide that does not substantially alter the activity of the polypeptide. Thus, "conservative substitutions" of a particular amino acid sequence refer to those amino acid substitutions or similar properties that are not important for polypeptide activity (eg, acidic, basic, positive or negative charge, polar or non-polar, etc.). Refers to the replacement of an amino acid by another amino acid that does not substantially alter the activity, even by substitution of the important amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each include amino acids that are conservatively substituted for each other: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E). 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), threonine (Y), tryptophan (W) (see also Creighton, Proteins, WH Freeman and Company (1984), which is incorporated herein by reference). In addition, individual substitutions, deletions, or additions that change, add, or delete a single amino acid or a few percent of amino acids in the encoded sequence are also "conservative substitutions."
[0023]
The term "relaxin analog" refers to a modified relaxin polypeptide that increases or decreases the functional activity of the molecule or its interaction with the relaxin receptor.
[0024]
The term “soluble” in the context of a polypeptide refers to the ability of the polypeptide to dissolve (ie, be molecularly or ionically dispersed) in an aqueous solution (eg, water, blood, or plasma).
[0025]
The term "small molecule effector" in the context of relaxin or relaxin receptor refers to an agent that binds to relaxin or relaxin receptor and stimulates relaxin or relaxin receptor activity.
[0026]
The term "small molecule antagonist" in the context of relaxin or relaxin receptor refers to an agent that binds to relaxin or relaxin receptor and reduces or inhibits relaxin or relaxin receptor activity.
[0027]
The term “effective amount” refers to a dosage sufficient to provide relief of the symptoms or treatment of the abnormal condition (eg, disease, disorder, or condition) to be treated. The dosage will vary depending on the subject, the condition being treated, and the treatment being effected.
[0028]
The terms “subnormal,” “subnormal,” and “underdeveloped” refer to the normal age of a subject's tissues and / or cells of the same age and species in the context of body tissue and / or cells. A smaller size, reduced cell number, and / or immature developmental state as compared to size, cell number, and / or developmental state.
[0029]
The term "tissue" generally refers to a cell or a population of cells consisting of cells of the same type that perform the same or similar function. A tissue can be part of an organ or bone, or it can be a loose connection of cells (eg, cells of the immune system).
[0030]
The term "maturation" in the context of bodily tissue refers to the evolutionary progression and / or differentiation of a tissue to a fully developed state.
[0031]
The term "mature", in the context of bodily tissue, refers to achieving the full development, differentiation, and / or growth of a tissue.
[0032]
The term "immature" refers to a tissue that has not fully developed and / or differentiated in the context of body tissue.
[0033]
The term "prepubertal male" refers to a male that has not completely completed the pubertal process.
[0034]
The term "post-pubertal male" refers to a male who has completed the process of puberty.
[0035]
The term "puberty" refers to a series of phenomena in which a child becomes a young adult, characterized by gametogenesis, secretion of gonadal hormones, development of secondary sexual characteristics, and onset of reproductive function.
[0036]
The term "accumulation", in the context of body tissue, refers to an increase in the number of normal (ie, non-mutated, non-malignant) cells in that tissue.
[0037]
The terms “biologically active” and “functionally active” refer to the ability of a molecule (eg, a relaxin agonist or a relaxin antagonist) to bind to relaxin or a relaxin receptor, and apoptosis, cell accumulation, And / or the ability to stimulate or inhibit tissue maturation.
[0038]
The term “relaxin deficiency condition” refers to a disease, disorder, or condition in a subject in which relaxin levels or relaxin receptor levels are lower than normal in an associated tissue, cell, or subject.
[0039]
The term “relaxin responsive” refers to an increase in the state of cell number or maturity (ie, tissue development and / or differentiation) in response to relaxin binding to a relaxin receptor.
[0040]
The term "relaxin-related" refers to a cell or tissue that is directly or jointly affected by an increase or decrease in functionally active relaxin levels or a functionally active relaxin receptor. A property, state, or response.
[0041]
The term “hyperplasia” or “hyperplastic tissue” refers to an increase in the number of cells in a tissue.
[0042]
The term “hypertrophy” or “hypertrophic” refers to an increase in tissue size.
[0043]
The terms "cancer" and "malignant disease" generally refer to various types of malignant neoplasms, most of which invade surrounding tissues.
[0044]
The terms "cancerous" and "malignant" relate to cells or tissues that have the properties of a cancer or malignancy.
[0045]
The term "tissue remodeling" refers to the formation of new cells or tissues and the destruction of existing cells through the apoptotic pathway in response to relaxin or relaxin receptor mediated signals.
[0046]
The term "apoptosis" induces a selected form of cell suicide, and readily observable morphological and biochemical events such as deoxyribonucleic acid (DNA) fragmentation, chromatin condensation (endonuclease activity Whether or not related), chromosomal migration, marginal propensity in the nucleus of cells, formation of apoptotic bodies, mitochondrial swelling, expansion of mitochondrial crystals, opening of the mitochondrial transduction pore, and / or (Dissipation of the mitochondrial proton gradient).
[0047]
(Description of a specific embodiment)
The present invention provides a method of administering a relaxin agonist or antagonist for modulating apoptosis. Relaxin agonists and antagonists can be used to reduce, manage, treat, or prevent relaxin-related abnormalities. A relaxin-related disorder includes a disease, disorder, or condition in a subject that is associated with increased or decreased levels of apoptosis as compared to the level of apoptosis in a normal subject. Relaxin-related abnormalities include, for example, relaxin deficiency states and relaxin responsive states.
[0048]
In one aspect, a relaxin agonist is used to treat a relaxin-related disorder that has increased apoptosis as compared to a corresponding cell from a normal subject (ie, a cell that does not have a relaxin-related disorder). Administered to the subject. Such relaxin-related abnormalities include, for example, immature body tissue, increased collagen deposition, fibrosis (ie, the presence of abnormal amounts of fibrous tissue as compared to normal tissue), low tissue weight, Includes increased apoptosis of cells in the cell population, and the like. Relaxin-related abnormalities are typically associated with reduced levels of relaxin and / or relaxin receptor as compared to normal cells or tissues.
[0049]
In certain embodiments, the relaxin-related abnormality is a relaxin deficiency condition (eg, immature tissue, presence of excess collagen, low sperm count, etc.). The immature tissue can be, for example, immature male reproductive tract tissue (eg, immature prostate, epididymis, insemination, or testicular tissue or sperm). Immature tissue can be characterized, for example, by increased collagen deposition, lower weight, and / or reduced cell number as compared to a corresponding sample of normal tissue. Such tissue may also be characterized by lack of organization, incomplete development, and / or lack of or incomplete differentiation as compared to a corresponding sample of normal tissue. In certain other embodiments, fibrosis (eg, fibrosis of the skin of these or other organs or tissues, fibrosis of the lung, fibrosis of the kidney, or fibrosis associated with aging) may be reduced.
[0050]
The relaxin agonist is administered in an amount effective to reduce, manage, treat, or prevent a relaxin-related disorder. For example, administration of a relaxin agonist can reduce apoptosis of target cells, stimulate maturation, development, and / or differentiation of immature tissue, increase tissue weight, increase the number of cells in a tissue, And reduce collagen deposition.
[0051]
In another aspect according to the present invention, a relaxin antagonist is administered to a subject to reduce, manage, treat, or prevent a relaxin-related disorder by increasing apoptosis in target cells. Such relaxin-related abnormalities can be, for example, accumulation of relaxin-related cells, reduced apoptosis to normal cells or tissues, hyperplasia, hypertrophy, cancer, neoplasm, and the like. In certain embodiments, a relaxin antagonist can reduce remodeling of relaxin-related tissues, reduce unwanted cell accumulation, reduce or prevent hyperplasia, hypertrophy, cancer, neoplasms, and the like.
[0052]
As a body tissue having a relaxin-related or relaxin-responsive tissue abnormality, for example, brain, heart, liver, kidney, spleen, thymus, intestine, skin, lung, male reproductive tract (eg, prostate, epididymis, seminal vesicles) Or testis), or the tissues of the female reproductive tract (eg, the uterus, cervix, interpubic ligament, or connective tissue of the lower limb girdle).
[0053]
(Relaxin agonist)
In one aspect according to the present invention, a relaxin agonist is administered to a subject to treat a relaxin-related disorder. Relaxin agonists can be, for example, relaxin, relaxin analogs, small molecule relaxin effectors, relaxin nucleic acids, and the like.
[0054]
(Relaxin and relaxin analog)
In one aspect of the invention, the relaxin agonist is a relaxin polypeptide or a fragment or analog of a relaxin polypeptide. The term "relaxin" refers to a vertebrate relaxin polypeptide, including a full-length relaxin polypeptide or a portion of a relaxin polypeptide that retains biological activity.
[0055]
Relaxin is well defined in its natural human, animal and synthetic forms. In particular, relaxin is widely described in U.S. Patent Nos. 5,166,191 and 4,835,251, both of which are hereby incorporated by reference. In this application, “relaxin” generally refers to the terms “relaxin,” “human relaxin,” “native relaxin,” and “synthetic relaxin,” as described in US Pat. No. 5,166,191. Refers to the terms "human relaxin" and "human relaxin analog" as described in U.S. Patent No. 4,835,251. In an exemplary embodiment, relaxin is described, for example, in U.S. Patent Nos. 5,179,195; 5,023,321; and 4,758,516 (the disclosures of which are incorporated herein by reference). And human relaxin as described in US Pat. In this application, "relaxin" also refers to relaxin isolated in pigs, rats, horses, or other mammals or vertebrates, and cDNA clones of rat, pig, or other mammals or vertebrates relaxin. Refers to relaxin produced by recombinant technology.
[0056]
Methods for producing relaxin and its analogs are known in the art. Additionally, methods for isolating and purifying relaxin are known in the art. Some sources of these methods are described in US Pat. No. 5,166,191 (the following references: US Pat. No. 4,835,251, Barany et al., The Peptides 2: 1 (1980); Treager et al.). EP 0251 615; EP 0107 782; EP 0107 045; and WO 90/13659 (all of which are incorporated herein by reference), including Biology of Relaxin and it's Role in the Human, pp. 42-55). ).
[0057]
Additional methods of producing relaxin are described in U.S. Patent No. 5,464,756 and PCT / US94 / 06997, the disclosures of which are incorporated herein by reference. Relaxin is also described in EP 0 251 615 (published Jan. 7, 1998, the disclosure of which is incorporated herein by reference) and the synthesis of A and B chains and their purification. And can be prepared by construction. For in vitro construction of relaxin, a 4: 1 molar ratio of A chain: B chain is commonly used. The resulting product is then purified by any means known to those of skill in the art, including, for example, reverse phase HPLC, ion exchange chromatography, gel filtration, dialysis, or any combination of such procedures. You. Untreated or partially treated forms of relaxin, such as preprorelaxin or prorelaxin, can also be used.
[0058]
In certain embodiments, the relaxin polypeptide includes H1 and H2 forms of human relaxin. The predominant human relaxin is the H2 relaxin form with a truncated B chain in which the four C-terminal amino acids of the B chain have been deleted such that the B chain ends at serine at position 29 (ie, relaxin H2 (B29 A24 )). Either of this form (referred to as "short relaxin" or "long relaxin", comprising a 33 amino acid B chain) can be used.
[0059]
Relaxin agonists further include analogs (eg, variants of naturally occurring amino acid sequences of relaxin). Relaxin analogs also include analogs that have been altered by substitution, addition, or deletion of one or more amino acid residues to provide a functionally active relaxin polypeptide. Such relaxin analogs include the amino acid sequence of a relaxin polypeptide (where one or more functionally equivalent amino acid residues are substituted for residues within this sequence and silent functional changes (eg, conservative changes). ), Including but not limited to, all or a portion of an altered sequence that results in a primary amino acid sequence.
[0060]
In another aspect, the relaxin agonist is a polypeptide consisting of or comprising a relaxin polypeptide having at least 10 contiguous amino acids of the relaxin polypeptide. Alternatively, the fragment comprises at least 20-25 contiguous amino acids of the relaxin polypeptide. In another embodiment, the fragment is less than 20 or 30 amino acids.
[0061]
A relaxin analog is substantially similar to a relaxin polypeptide or a fragment thereof (eg, in various embodiments, at least 60%, 70%, 75%, 80%, 90% or 95% identical or similar) or, when compared, substantially similar to an aligned sequence that is aligned by a computer sequence comparison / alignment program known in the art, or if the encoding nucleic acid is , A polypeptide comprising a region that can hybridize to relaxin nucleic acid under conditions of high, moderate, or low stringency (eg, Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Unsch, J. Mol. Biol. 48: 443 (1970); Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988); Gap, BESTFIT, FASTA, and TEASTA in WI; Ausubel et al. (Eds.), Current Protocols in Molecular Biology, 4th Edition, John Wiley and Sons, New York (1999), the disclosure of which is hereby incorporated by reference. Incorporated by reference in the book))). Relaxin agonists further include functionally active relaxin polypeptides, analogs or fragments that bind to a relaxin receptor.
[0062]
Relaxin agonists (eg, relaxin polypeptides, analogs, and fragments) can be produced by various methods known in the art. The operations that produce them can be performed at the genetic or polypeptide level. For example, a cloned relaxin nucleic acid can be modified by any of a number of strategies known in the art (eg, creating conservative substitutions, deletions, insertions, etc.) (eg, Sambrook et al., Molecular Cloning: A). Laboratories Manual, 3rd edition, Cold Spring Harbor Laboratory Press, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, 4th edition, John Y. New York. Which is incorporated herein by reference)). This sequence can be cleaved at the appropriate site by restriction endonuclease and then subjected, if desired, to further enzymatic modification, isolation and ligation in vitro. In generating relaxin nucleic acids encoding analogs or fragments, the modified nucleic acid is typically retained in a suitable translation reading frame, such that the reading frame is responsible for the translation stop signal or the synthesis of the relaxin analog or fragment. Blocked by other blocking signals. Relaxin nucleic acids can also be mutated in vitro or in vivo to create and / or destroy translation initiation and / or termination sequences. Relaxin nucleic acids may also be used to create variations in the coding region and / or create new restriction endonuclease sites or destroy existing sites, and to facilitate further in vitro modifications. Can be mutated. Any technique of mutagenesis known in the art (chemical mutagenesis, in vitro site-directed mutagenesis (see, for example, Hutchison et al., J. Biol. Chem. 253: 6551-60 (1978)), TAB (See, eg, Sambrook et al., Supra; Ausubel et al., Supra), including, but not limited to, the use of a ® Linker (Pharmacia).
[0063]
In certain embodiments, a relaxin analog is prepared from a relaxin-encoding nucleic acid that has been modified to introduce a codon aspartate at a particular site within at least a portion of the relaxin-encoding region (eg, US Pat. No. 945,402, the disclosure of which is incorporated herein by reference. The resulting analog can be treated with a dilute acid to release the desired analog, thereby allowing the protein to be more easily isolated and purified. Other relaxin analogs are described in U.S. Patent Nos. 4,656,249; 5,179,195; 5,945,402; 5,811,395; and 5,795. , 807, the disclosures of which are incorporated herein by reference.
[0064]
Manipulation of the relaxin polypeptide sequence can also be done at the polypeptide level. Relaxin polypeptides, analogs, or fragments that are differentially modified during and after synthesis (eg, in vivo or in vitro translation) are within the scope of the invention. Such modifications include conservative substitutions, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, antibody molecules, other polypeptides or other cellular ligands And the like. Any of a number of chemical modifications can be performed by known techniques (specific chemical cleavage (eg, cleavage with cyanogen bromide)), enzymatic cleavage (eg, cleavage with trypsin, chymotrypsin, papain, V8 protease, etc.); eg, NaBH ₄ Acetylation, formylation, oxidation and reduction, including, but not limited to, modifications by metabolic synthesis in the presence of tunicamycin).
[0065]
Relaxin polypeptides, analogs, and fragments can be purified from natural sources by standard methods (eg, immunoaffinity purification) as described herein. Relaxin polypeptides, analogs, and fragments can also be obtained by standard methods (including chromatography (eg, ion exchange, affinity, sizing column chromatography, high pressure liquid chromatography), centrifugation, differential lysis). Alternatively, it can be isolated and purified by any other standard technique for purifying polypeptides. Relaxin polypeptides can be synthesized by standard chemical methods known in the art (eg, Hunkapiller et al., Nature 310: 105-11 (1984); Stewart and Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Co., Rockford, IL (1984), the disclosure of which is incorporated herein by reference.
[0066]
In addition, analogs of the relaxin polypeptide can be chemically synthesized. For example, peptides corresponding to fragments of a relaxin polypeptide that contain a desired domain or mediate a desired activity in vivo can be synthesized, for example, by using chemical synthesis methods using an automated peptide synthesizer. In addition, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition to the relaxin polypeptide sequence. Non-classical amino acids include D-isomers of common amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, ε-aminohexanoic acid, 6-aminohexanoic acid, 2-aminoisobutyric acid, and 3-amino Propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sacrosin, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, selenocysteine, fluoro-amino acid, designer amino acid (eg, , Β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and common amino acid analogs, and the amino acids are D (dextrorotary) or L (levorotary). obtain.
[0067]
In another embodiment, the relaxin agonist is a relaxin polypeptide or a fragment thereof (typically consisting of at least one domain or motif of the relaxin polypeptide, or at least 10 contiguous amino acids of the relaxin polypeptide). Or a fusion protein, which is linked to the amino acid sequence of a different protein via a peptide bond at its amino or carboxy terminus. In one embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the chimeric polypeptide. Chimeric products are made by linking the appropriate nucleic acid sequences (encoding the desired amino acid sequence) to one another in an appropriate reading frame and expressing the chimeric product by methods known in the art. Can be done. Alternatively, the chimeric product can be made by protein synthesis techniques (eg, by using an automated peptide synthesizer).
[0068]
In certain embodiments, the fusion protein is a relaxin-ubiquitin fusion protein. For example, US Pat. No. 5,108,919, the disclosure of which is incorporated herein by reference, discloses a method for preparing a fusion protein of a relaxin chain and ubiquitin.
[0069]
In a preferred embodiment, the relaxin analog or fragment is functionally active (ie, may exhibit one or more functional activities associated with a full-length wild-type relaxin polypeptide). In one example, an analog or fragment that retains a desired relaxin property of interest (eg, binding to a relaxin binding partner (eg, a relaxin receptor) and / or modulating (eg, inhibiting) apoptosis) It can be used as an inducer of such properties and their physiological relevance. Characteristic embodiments relate to relaxin analogs or fragments that bind to the relaxin receptor and induce a relaxin-related decrease in apoptosis. An analog or fragment of relaxin can be tested for the desired activity by procedures known in the art, including but not limited to the functional assays described herein.
[0070]
(Relaxin nucleic acid)
The present invention provides relaxin nucleic acid sequences for expression of relaxin polypeptides, fragments and analogs in vivo or in vitro. The relaxin nucleic acid can be vertebrate or mammalian relaxin, including, for example, human, mouse, rat, pig, bovine, dog or monkey relaxin. Relaxin nucleic acids can include genomic nucleic acids, cDNA, relaxin coding regions or fragments thereof. Relaxin nucleic acids further include mRNA corresponding to the relaxin locus. Relaxin nucleic acids can also include analogs (eg, nucleotide sequence variants) that encode the same amino acid, such as a naturally occurring allelic variant, or other codon choices possible for conservative amino acid substitutions thereof. )including. Due to the degeneracy of the nucleotide coding sequence, other nucleic acid sequences encoding substantially the same amino acid sequence as the relaxin cDNA or open reading frame can be used in the practice of the present invention. These nucleic acid sequences include relaxin altered by substitution of different codons (thus resulting in silent changes) that encode identical or functionally equivalent amino acid residues (eg, conservative substitutions) within the sequence. Nucleic acid sequences that include all or part of a gene include, but are not limited to.
[0071]
The invention further provides a relaxin nucleic acid fragment (eg, a hybridizable portion) of at least 6 contiguous nucleotides. In other embodiments, the nucleic acid is at least 8 contiguous nucleotides, at least 25 contiguous nucleotides, at least 50 contiguous nucleotides, at least 100 nucleotides, or at least 150 nucleotides of the relaxin sequence. Including more. In another embodiment, the nucleic acid is less than 150 nucleotides in length. A relaxin nucleic acid can be single-stranded or double-stranded. As will be readily understood, as used herein, "a nucleic acid encoding a fragment of a relaxin polypeptide" refers to a contiguous sequence as opposed to other contiguous portions of the relaxin polypeptide, It is to be understood that the nucleic acid encodes only the listed fragments or portions of the relaxin polypeptide. Fragments of relaxin nucleic acids encoding one or more relaxin domains are also provided.
[0072]
The relaxin nucleic acid or a functionally active analog or fragment thereof can be inserted into a suitable vector, such as an expression vector (ie, a vector containing the essential elements for the transcription and translation of the inserted polypeptide coding sequence). Essential transcriptional and translational signals can also be provided by the native relaxin gene and / or its flanking regions. Various host-vector systems can be utilized to express the relaxin nucleic acid sequence. These include, but are not limited to: mammalian cell lines infected with a virus (eg, vaccinia virus, adenovirus, adeno-associated virus, etc.), insect cells infected with a virus (eg, baculovirus) Systems, microorganisms (eg, yeast containing yeast vectors or bacteria transformed with bacteriophage), DNA, plasmid DNA or cosmid DNA. Vector expression factors vary in their strength and specificities. Any of a number of suitable transcription and translation factors may be used, depending on the host-vector system utilized. In certain embodiments, the nucleic acid sequence encoding a human relaxin nucleic acid or a functionally active portion of human relaxin is expressed in yeast or bacteria. In yet another embodiment, a fragment of relaxin comprising a domain of a relaxin polypeptide is expressed.
[0073]
For insertion of a nucleic acid into a vector, any method known in the art can be used to construct an expression vector containing a chimeric gene consisting of appropriate transcription / translation control signals and a relaxin nucleic acid sequence. These methods include recombinant DNA and synthetic techniques in vitro and recombination in vivo (genetical recombination). Expression of a nucleic acid sequence encoding a relaxin polypeptide, analog or fragment can be regulated by a second nucleic acid sequence, such that the relaxin polypeptide, analog or fragment is transformed into a host transformed with the recombinant DNA molecule. Is expressed in For example, expression of a relaxin polypeptide can be controlled by any promoter / enhancer element known in the art. Promoters that can be used to control gene expression include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, Nature 290: 304-10 (1981)), Rous sarcoma virus 3 'Promoters containing long terminal repeats (Yamamoto et al., Cell 22: 787-97 (1980)), herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78: 1441-45 (1981)), metallothionein (Metallothionen) gene regulatory sequences (Brinster et al., Nature 296: 39-42 (1982)), prokaryotic expression vectors (e.g., beta-lactamase promoter ( illa-Komaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-31 (1978) or the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 (1983)), cauliflower. Plant expression vector containing the mosaic virus 35S RNA promoter (Gardner et al., Nucl. Acids Res. 9: 2871-88 (1981)), the promoter of the photosynthetic enzyme ribulose diphosphate carboxylase (Herrera-Estrellala et al., Nature 310: 115-20). (1984)), promoter elements from yeast or other fungi (eg, Gal7 and Gal4 promoters), ADH (alcohol dehydrogenase) Promoter, PGK (phosphodiester glycerol kinase) promoter, alkaline phosphatase promoter.
[0074]
Animal transcription regulatory regions that exhibit the following tissue specificity have been utilized in transgenic animals: Elastase I gene regulatory regions that are active in pancreatic acinar cells (eg, Swift et al., Cell 38: 639-46 (1984)). Ornitz et al., Cold Spring Harbor Symp.Quant.Biol.50: 399-409 (1986); MacDonald, Hepatology 7 (1 Suppl.): 42S-51S (1987)); Insulin gene regulation active in pancreatic β cells; Regions (eg, Hanahan, Nature 315: 115-22 (1985)), immunoglobulin gene regulatory regions active in lymphoid cells (eg, Grosschedl et al., Cell 38: 647-5). 8 (1984); Adams et al., Nature 318: 533-38 (1985); Alexander et al., Mol. Cell. Biol. 7: 146-44 (1987)), in testis cells, breast cells, lymphoid cells and mast cells. A mouse mammary adenocarcinoma virus regulatory region that is active (eg, Leder et al., Cell 45: 485-95 (1986)), an albumin gene regulatory region that is active in the liver (eg, Pinkert et al., Genes Dev. 1: 268-76 ( 1987)), α-fetoprotein gene regulatory regions that are active in the liver (eg, Krumlauf et al., Mol. Cell. Biol. 5: 1639-48 (1985); Hammer et al., Science 235: 53-58 (1987)); In the liver A1-antitrypsin gene regulatory region that is sexual (eg, Kelsey et al., Genes and Level. 1: 161-71 (1987)); β-globin gene regulatory region that is active in myeloid cells (eg, Magram et al., Nature) 315: 338-40 (1985); Kollias et al., Cell 46: 89-94 (1986)); a myelin basic protein gene regulatory region active in oligodendrocytes of the brain (eg, Readhead et al., Cell 48: 703). -12 (1987)); a myosin light chain-2 gene regulatory region that is active in skeletal muscle (eg, Shani, Nature 314: 283-86 (1985)); and a gonadotropin-releasing hormone gene regulatory region that is active in the hypothalamus. (For example, M son et al., Science 234: 1372-78 (1986)). In a preferred embodiment, the tissue-specific promoter is a prostate-specific antigen promoter (see, eg, US Patent No. 6,100,444, the disclosure of which is incorporated herein by reference). ).
[0075]
In another embodiment, a vector is used that comprises a promoter operably linked to the relaxin nucleic acid, one or more origins of replication and, optionally, one or more selectable markers (eg, antibiotic or drug resistance markers). Is done. For example, an expression construct can be made by subcloning a relaxin nucleic acid into a restriction site of a pRECT expression vector. Such a construct allows expression of a relaxin polypeptide, analog or fragment under the control of a T7 promoter having a histidine amino-terminal flag sequence for affinity purification of the expressed polypeptide. In another specific embodiment, comprising a prostate-specific antigen promoter operably linked to a relaxin nucleic acid, one or more origins of replication and, optionally, one or more selectable markers (eg, drug resistance markers). Vectors are used.
[0076]
Expression vectors containing relaxin nucleic acids can be identified by general approaches well known to those of skill in the art, including: (a) nucleic acid hybridization; (b) the presence or absence of "marker" gene function; (C) expression of the inserted sequence, (d) polymerase chain reaction (PCR) and the like. In a first approach, the presence of the relaxin nucleic acid inserted into the expression vector can be detected by nucleic acid hybridization using a probe containing a sequence homologous to the inserted relaxin nucleic acid. In a second approach, the recombinant vector / host system is used to generate specific "marker" gene functions (eg, thymidine kinase activity, antibiotic resistance, transformation phenotype) caused by inserting relaxin nucleic acid into the vector. , Baculovirus, closed body formation, changes in colorimetry, etc.) can be identified and selected based on the presence or absence. For example, if the relaxin nucleic acid is inserted within a marker gene sequence of a vector, recombinants containing the relaxin nucleic acid can be identified by the absence of marker gene function.
[0077]
In a third approach, a recombinant expression vector can be identified by assaying a recombinantly expressed relaxin polypeptide, analog or fragment. Such assays include, for example, the physical or functional properties of a relaxin polypeptide, analog or fragment (eg, binding to an anti-relaxin antibody, binding to a relaxin receptor, etc.) in an in vitro assay system. Can be based on In a fourth approach, a recombinant expression vector can be identified by the polymerase chain reaction. (See, for example, U.S. Patent Nos. 4,683,202, 4,683,195 and 4,889,818; Gyllensten et al., Proc. Natl. Acad. Sci. USA 85: 7652-56 (1988). Ochman et al., Genetics 120: 621-23 (1988); Loh et al., Science 243: 217-20 (1989)). Once a particular recombinant vector has been identified and isolated, several methods known in the art can be used to amplify it.
[0078]
Once a suitable host system and growth conditions are established, recombinant vectors can be amplified and prepared in large quantities. As explained above, vectors that may be used include, but are not limited to, the following vectors or analogs thereof, to name a few: human or animal viruses (eg, vaccinia virus or Adenovirus); insect viruses (eg, baculovirus); yeast vectors; bacteriophage vectors (eg, λ); and plasmid and cosmid DNA vectors.
[0079]
Furthermore, in the particular manner desired, a host cell strain may be chosen to modulate the expression of the inserted nucleic acid, or to modify or process relaxin, a relaxin analog or fragment. Expression from a particular promoter may be increased in the presence of a particular inducer: the expression of a relaxin polypeptide, analog or fragment may be controlled. In addition, different host cells with characteristic and specific mechanisms for translation and post-translational processing and modification (eg, glycosylation and / or phosphorylation) can be used. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the expressed polypeptide, analog or fragment. For example, expression in a bacterial system can be used to produce an unprocessed core protein product. Expression in mammalian cells can be used to ensure "native" processing of a relaxin polypeptide or relaxin analog or fragment of a mammalian cell. Furthermore, different vector / host expression systems can affect the processing reaction to different degrees.
[0080]
(Relaxin antagonist)
In another aspect of the invention, a relaxin antagonist is provided for modulating apoptosis. For example, relaxin antagonists can include relaxin binding agents, relaxin receptor binding agents, antisense nucleic acids, and the like.
[0081]
(Relaxin antibody)
Relaxin antagonists include antibodies that immunospecifically recognize relaxin or a relaxin receptor polypeptide and antibodies that stimulate apoptosis and / or reduce or inhibit the accumulation of relaxin-bound cells in a cell population or tissue. May be included. Anti-relaxin and anti-relaxin receptor antibodies include, but are not limited to: polyclonal, monoclonal, chimeric (eg, fully humanized or human chimeric), single chain, antibody fragments ( For example, Fab, F (ab '), F (ab') ₂ , Fv or hypervariable regions), single heavy chains, and Fab expression libraries. In certain embodiments, full length polyclonal and / or monoclonal antibodies, vertebrate or mammalian relaxin or relaxin receptor polypeptide are generated and selected for an antibody that selectively binds to relaxin or relaxin receptor polypeptide. , Thereby functionally inactivating such polypeptides. In another embodiment, antibodies are raised to a domain of a vertebrate relaxin polypeptide or a relaxin receptor polypeptide. In yet another embodiment, a vertebrate relaxin polypeptide or a fragment of a relaxin receptor polypeptide, which is identified as hydrophilic, is used as an immunogen for antibody production, and Selected for immunospecific binding to the peptide and inhibition of its biological activity.
[0082]
Various procedures known in the art can be used for the production of polyclonal antibodies against relaxin or a relaxin receptor polypeptide or a fragment or analog thereof. For the production of such antibodies, a variety of host animals, including but not limited to rabbits, mice, rats, sheep, goats, etc., may be prepared by native relaxin or relaxin receptor polypeptides or fragments thereof. Or it can be immunized by injecting an analog. Alternatively, a transgenic animal having a human immune system can be immunized by injecting native relaxin or a relaxin receptor polypeptide. (See, for example, U.S. Patent Nos. 6,114,598 and 6,111,166, which are incorporated herein by reference). Various adjuvants can be used to enhance the immune response (depending on the host species), including but not limited to: Freund's adjuvant (complete or incomplete), minerals Gels (eg, aluminum hydroxide), surfactants (eg, lysolecithin), pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol and potentially useful Human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum.
[0083]
For the preparation of monoclonal antibodies to relaxin or relaxin receptor polypeptides, fragments or analogs thereof, any technique provided for the production of antibody molecules by cell lines continuing in culture may also be used. Such techniques include, for example: the hybridoma technology first developed by Kohler and Milstein (Nature 256: 495-97 (1975)) and the trioma technology, the human B cell hybridoma technology (eg, Kozbor et al.). , Immunology Today 4:72 (1983)) and EBV-hybridoma technology for producing human monoclonal antibodies (see, eg, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Lis, Inc., 77-96). P. (1985)). Mammalian antibodies can be used and human B cells with EBV virus in vitro, using hybridomas (see, eg, Cote et al., Proc. Natl. Acad. Sci. USA 80: 2026-30 (1983)). It can be obtained by using transformation (see, eg, Cole et al., (1985), supra). Selection of hybridomas that produce antibodies with appropriate biological functions is well known in the art or described herein below. Human monoclonal antibodies can also be prepared by preparing hybridomas from animals having a human immune system immunized by injecting native relaxin or a relaxin receptor polypeptide (see, eg, US Pat. No. 6,114,598). No. 6,111,166, which are incorporated herein by reference).
[0084]
Further, the present invention provides that “chimeric” or “humanized” antibodies can be prepared (eg, Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-55 (1984); Neuberger et al., Nature 312: 604-08 (1984); Takeda et al., Nature 314: 452-54 (1985)). Such chimeric antibodies are prepared by splicing a non-human gene for an antibody molecule specific for relaxin or a receptor polypeptide, typically with a gene from a human antibody molecule of appropriate activity. Antigen binding region of non-human antibody (eg, F (ab ') ₂ , F (ab '), Fv or hypervariable region) may be transferred into the framework of a human antibody by recombinant DNA technology, essentially producing human molecules. In a preferred embodiment, the antibodies are fully humanized.
[0085]
Methods for producing such “chimeric” molecules are generally well known and are described, for example, in: US Pat. Nos. 4,816,567; 4,816,397; Nos. 693,762; 5,712,120; 5,821,337; 6,054,297; International Patent Publications WO 87/02671 and WO 90/00616; and European Patent Publications EP 0 239 400 (the disclosures of which are incorporated herein by reference). Alternatively, a human monoclonal antibody or a portion thereof encodes an antibody that specifically binds to relaxin or a relaxin receptor polypeptide, according to the general method described by Huse et al. (Science 246: 1275-81 (1989)). A human B cell cDNA library for the DNA molecule of interest can be identified by first screening. The DNA molecule can then be cloned and amplified to obtain a sequence encoding the antibody (or binding domain) of the desired specificity. Phage display technology provides another technique for selecting antibodies that bind to relaxin or a relaxin receptor polypeptide, fragment or analog thereof. (See, for example, International Patent Publications WO 91/17271 and WO 92/01047; and Huse et al., Supra).
[0086]
According to another aspect of the present invention, techniques for the production of single-chain antibodies (see, for example, US Pat. Nos. 4,946,778 and 5,969,108) comprise relaxin or the relaxin receptor. Can be adapted to produce specific single chain antibodies. (See also Riechmann and Muyldermans, J. Immunol. Methods 231: 25-38 (1999); Muyldermans and Lauwereys, J. Mol. Recognit. 12: 131-40 (1999)).
[0087]
A further aspect of the invention utilizes the techniques described for the construction of Fab expression libraries (see, eg, Huse et al. (1989), supra) to obtain the desired relaxin polypeptides, fragments or analogs thereof. The identification of monoclonal Fab fragments having specificity and biological activity for is rapid and easy.
[0088]
Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include, but are not limited to: F (ab '), which can be generated by pepsin digestion of an antibody molecule ₂ Fragment, F (ab ') ₂ Fab ′ fragments that can be generated by reducing the disulfide bonds of the fragments, Fab fragments that can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments. Recombinant Fv fragments can also be produced in eukaryotic cells, for example, using the methods described in US Pat. No. 5,965,405.
[0089]
In generating antibodies, screening for the desired antibody can be accomplished by techniques known in the art, such as ELISA (enzyme-linked immunosorbent assay) In one aspect, specificity of relaxin or a relaxin receptor polypeptide. Antibodies that recognize the domain can be used to assay hybridomas for products (eg, antibodies) that bind to relaxin or a fragment of the relaxin receptor, including the domain. To select an antibody that specifically binds to a polypeptide but does not specifically bind to a second, different relaxin polypeptide, the antibody is positively bound to the first polypeptide and bound to the second polypeptide. Selection based on loss of antibody binding to different polypeptides Get.
[0090]
(Soluble relaxin receptor)
In another aspect of the invention, the relaxin antagonist is a relaxin binding agent that comprises a soluble relaxin receptor that binds relaxin, or a fragment or analog thereof. The term "soluble relaxin receptor" refers to a relaxin receptor polypeptide that does not bind to cell membranes. The relaxin receptor is approximately 200 kilodaltons (see Palejwala et al., Endocrinology 139 (3): 1208-12 (1998), the disclosure of which is incorporated herein by reference). Soluble forms of the relaxin receptor retain the ability to bind vertebrate relaxin, but typically lack the transmembrane and / or cytoplasmic domains. A soluble relaxin receptor may include additional amino acid residues (eg, an affinity tag) that provide a means of purifying the polypeptide or attach the polypeptide to another polypeptide or immunoglobulin sequence. To provide a site for
[0091]
The soluble relaxin receptor can optionally include a transmembrane domain that cannot bind to cell membranes. By "transmembrane protein" is meant a domain of the relaxin receptor polypeptide that contains a sufficient number of hydrophobic amino acids, which allows the polypeptide to be inserted and anchored in the cell membrane. A “transmembrane domain that cannot bind to cell membranes” refers to a transmembrane domain that has been altered by a mutation or deletion, such that it is not sufficiently hydrophobic to allow insertion or other binding to the cell membrane. Such transmembrane domains, for example, prevent fusion of the relaxin receptor polypeptide or a fragment thereof (with a secretory signal sequence useful for polypeptide secretion from cells). Amino acid sequence substitutions or changes useful to achieve an inactive transmembrane domain include, but are not limited to, deletion or substitution of amino acids in the transmembrane domain. Methods for making soluble receptors are known in the art (see, eg, US Pat. Nos. 6,033,903; 6,037,450; and 5,925,549; These disclosures are incorporated herein by reference).
[0092]
Soluble relaxin receptors include naturally occurring amino acid sequence variants of the soluble relaxin receptor. Soluble relaxin receptors further include those receptors that are altered by substitution, addition or deletion of one or more amino acid residues that provide for a functionally activated relaxin receptor polypeptide. Such relaxin receptors include the amino acid sequence of the relaxin receptor polypeptide (where one or more functionally equivalent amino acid residues are substituted for residues within this sequence, resulting in silent functional changes (eg, conservative changes). ), Including, but not limited to, a sequence that results in a substitution).
[0093]
In another aspect, the soluble relaxin receptor is a polypeptide that consists of or comprises a fragment of the relaxin receptor polypeptide having at least 10 contiguous amino acids of the relaxin receptor polypeptide. More typically, the fragment comprises at least 20 or at least 50 contiguous amino acids of a relaxin receptor polypeptide. In other embodiments, the fragment is greater than 100 amino acids or even greater than 200 amino acids.
[0094]
A relaxin receptor polypeptide can comprise a region substantially similar to a relaxin receptor polypeptide or a fragment thereof (eg, in various embodiments, at least 60%, 70%, 75%, (E.g., 80%, 90%, or even 95% identity or similarity), or the sequences are aligned by computer sequence comparison / alignment programs known in the art or by visual inspection. And a polypeptide as compared to the aligned sequence. (For example, Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); Pearson and Lipman, Proc. Natl. Acad. Sci. USA. 85: 2444 (1988); GAP, BESTFIT, FASTA and TEASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI; see, et al .; Which is hereby incorporated by reference). Sequence identity or similarity can also be determined by identifying nucleic acids that can hybridize to a relaxin receptor nucleic acid under conditions of high, moderate, or low stringency. (See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, New York (2001); Ausubel et al., (1996), supra, the disclosures of which are incorporated herein by reference. Incorporated by reference in the book).
[0095]
Soluble relaxin receptor and fragments thereof can be produced by various methods known in the art. The operations leading to these productions can take place at the genetic or protein level. For example, a cloned relaxin receptor nucleic acid can be modified by any of a number of strategies known in the art (eg, making conservative substitutions, deletions, insertions, etc.) (eg, Sambrook et al., Supra). See Ausubel et al., Supra). This sequence can be cleaved at the appropriate site using a restriction endonuclease and then, if desired, further enzymatically modified, isolated and ligated in vitro. In the production of relaxin receptor nucleic acids, the modified nucleic acid typically maintains its proper translational reading frame, such that the reading frame is a translational stop signal, or the synthesis of a soluble relaxin receptor or fragment thereof. Not disturbed by other signals that interfere with A relaxin receptor nucleic acid can also be mutated in vitro or in vivo to create and / or disrupt translation initiation and / or translation termination sequences. Relaxin receptor nucleic acids can also be mutated to create variations in coding regions (eg, amino acid substitutions) and / or create new restriction endonuclease sites or destroy existing sites, and reduce in vitro modifications. It could be easier. Any technique for mutagenesis known in the art may be used, including chemical mutagenesis, in vitro site-directed mutagenesis (eg, Hutchison et al., J. Biol. Chem. 253: 6551-). 60 (1978)), the use of TAB® linkers (Pharmacia), and the like.
[0096]
Manipulation of the relaxin receptor polypeptide sequence can also be made at the polypeptide level. Included within the scope of the invention are relaxin receptor polypeptides that are differentially modified (eg, by in vivo or in vitro translation) during or after synthesis. Such modifications include conservative substitutions, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, antibody molecule, protein or other cellular ligand. Bonding and the like. Any of a number of chemical modifications can be performed by known techniques, including specific chemical cleavage (eg, with cyanogen bromide), enzymatic cleavage (eg, trypsin, chymotrypsin, papain, V8). Protease, etc.); for example, NaBH ₄ Acetylation, formylation, oxidation and reduction, modification by metabolic synthesis in the presence of tunicamycin, and the like.
[0097]
Relaxin receptor polypeptides and fragments thereof can be purified from natural sources by standard methods such as those described herein (eg, immunoaffinity purification). Relaxin receptor polypeptides and fragments can also be obtained by standard methods, including chromatography (eg, ion exchange chromatography, affinity chromatography, sizing column chromatography, high pressure liquid chromatography), centrifugation, differential solubility. Alternatively, it can be isolated and purified by any other standard technique for polypeptide purification. Relaxin receptor polypeptides and fragments thereof can be synthesized by standard chemical methods known in the art (eg, Hunkapiller et al., Nature 310: 105-11 (1984); Stewart and Young, Solid Phase Peptide Synthesis). , 2nd Edition, Pierce Chemical Co., Rockford, IL, (1984)). In addition, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the relaxin receptor polypeptide sequence. Non-classical amino acids include D-isomers of normal amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, ε-aminohexanoic acid, 6-aminohexanoic acid, 2-aminoisobutyric acid, -Aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, selenocysteine, fluoro-amino acid, designer ( designer amino acids (eg, β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids), and common amino acid analogs. Further, the amino acid can be D (dextrorotary) or L (levorotary).
[0098]
In another embodiment, the soluble relaxin receptor is a relaxin receptor polypeptide, or a fragment thereof (typically relaxin) that binds at its amino or carboxy terminus to the amino acid sequence of a different protein via a peptide bond. A chimeric protein or a fusion protein comprising at least a domain or motif of a receptor polypeptide or at least 10 contiguous amino acids of a relaxin receptor polypeptide. In one embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the chimeric polypeptide. Chimeric products can be made by linking the appropriate nucleic acid sequences encoding the desired amino acid sequences together in an appropriate reading frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, chimeric products can be made by protein synthesis techniques (eg, by using an automated peptide synthesizer). In certain embodiments, the fusion protein is a relaxin receptor-ubiquitin fusion protein.
[0099]
(Relaxin analog)
The relaxin antagonist can further be a relaxin analog (eg, a relaxin polypeptide that binds to a relaxin receptor but does not induce a response by that receptor). For example, a relaxin analog can be a competitive inhibitor of relaxin binding, or a conventional antagonist of the relaxin receptor. The term "relaxin" refers to a vertebrate relaxin polypeptide and includes a full-length relaxin polypeptide or a portion of a relaxin polypeptide that retains biological activity. Relaxin is well defined in its natural human, animal and synthetic forms. In particular, relaxin is disclosed in U.S. Patent Nos. 5,179,195; 5,166,191; 5,023,321; 4,835,251; and 4,758,516. (The disclosures of which are incorporated herein by reference). Methods for making relaxin and its analogs are known in the art (supra). A relaxin analog can be prepared by modifying a relaxin polypeptide such that the relaxin analog retains relaxin receptor binding activity but does not induce a response by the relaxin receptor. For example, a relaxin analog can be an amino acid sequence variant of relaxin that retains relaxin receptor binding activity but does not induce a response by the relaxin receptor. Relaxin analogs further include relaxin polypeptides that have been modified by the addition or deletion of one or more amino acid residues and that maintain the relaxin receptor binding function but do not induce a response by the relaxin receptor.
[0100]
In various aspects according to the invention, the relaxin analog is a fragment of the relaxin polypeptide that consists of or comprises at least 10 contiguous amino acids of the relaxin polypeptide. Alternatively, the fragment comprises at least 20 or 40 contiguous amino acids of the relaxin polypeptide. In other embodiments, these fragments are no longer than 35 amino acids.
[0101]
A relaxin analog can be a region substantially similar to a relaxin polypeptide (eg, in various embodiments, at least 60%, 70%, 75%, 80%, 90%, Or even 95% identity or similarity) or a polypeptide as aligned and compared to the aligned sequence by computer sequence comparison / alignment programs known in the art. Or the encoding nucleic acid can be a polypeptide capable of hybridizing to relaxin nucleic acid under conditions of high, moderate, or low stringency (see above). .
[0102]
Relaxin analogs can be produced by various methods known in the art. The operations leading to these productions can take place at the genetic or protein level. For example, a cloned relaxin nucleic acid can be modified by any of a number of strategies known in the art (eg, by making conservative or non-conservative substitutions, deletions, insertions, etc.) (eg, See Sambrook et al., Supra; Ausubel et al., Supra). The sequence can be cleaved at the appropriate site using a restriction endonuclease and then, if desired, further enzymatically modified, isolated and ligated in vitro. In the production of relaxin analog nucleic acids, the modified nucleic acid typically maintains its proper translational reading frame, such that this reading frame is a translation termination signal, or other signal that interferes with the synthesis of the relaxin analog. Is not disturbed by Relaxin nucleic acids can be mutated in vitro or in vivo to create and / or disrupt translation initiation and / or translation termination sequences. Relaxin nucleic acids can also be mutated to create variations in the coding region, and / or create new restriction endonuclease sites or destroy existing sites, and further facilitate in vitro modification. Any technique for mutagenesis known in the art may be used, including chemical mutagenesis, in vitro site-directed mutagenesis (eg, Hutchison et al., J. Biol. Chem. 253: 6551-). 60 (1978)), the use of TAB® linkers (Pharmacia), and the like.
[0103]
In certain embodiments, a relaxin analog is prepared from a relaxin-encoding nucleic acid that has been modified to introduce a codon aspartate at a particular position in at least a portion of the relaxin-encoding region (eg, US Pat. No. 5,945,402, the disclosure of which is incorporated herein by reference). The resulting analog can be treated with a dilute acid to release the desired analog, thereby making the protein more easily isolated and purified.
[0104]
Manipulation of the relaxin polypeptide sequence can also be done at the polypeptide level. Included within the scope of the invention are relaxin analogs that are differentially modified (eg, by in vivo or in vitro translation) during or after synthesis. Such modifications include amino acid substitutions (either conservative or non-conservative), glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, antibodies Binding to molecules, proteins or other cellular ligands, and the like. Any of a number of chemical modifications can be performed by known techniques, including specific chemical cleavage (eg, with cyanogen bromide), enzymatic cleavage (eg, trypsin, chymotrypsin, papain, V8). Protease, etc.); for example, NaBH ₄ Acetylation, formylation, oxidation and reduction, modification by metabolic synthesis in the presence of tunicamycin, and the like.
[0105]
Relaxin analogs can be purified from natural sources by standard methods such as those described herein (eg, immunoaffinity purification). Relaxin analogs can also be obtained by standard methods including chromatography (eg, ion exchange chromatography, affinity chromatography, sizing column chromatography, high pressure liquid chromatography), centrifugation, differential solubility, It can be isolated and purified by any other standard technique for purification. Relaxin analogs can be synthesized by standard chemical methods known in the art (eg, Hunkapiller et al., Nature 310: 105-11 (1984); Stewart and Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Co., Rockford, IL, (1984)). In addition, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the relaxin polypeptide sequence. Non-classical amino acids include D-isomers of normal amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, ε-aminohexanoic acid, 6-aminohexanoic acid, 2-aminoisobutyric acid, -Aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, selenocysteine, fluoro-amino acid, designer amino acid (Eg, β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids), and common amino acid analogs. Further, the amino acid can be D (dextrorotary) or L (levorotary).
[0106]
In another embodiment, the relaxin analog comprises a relaxin polypeptide (typically at least a domain of the relaxin polypeptide or a relaxin polypeptide that binds at its amino or carboxy terminus to the amino acid sequence of a different protein via a peptide bond. A motif, or consisting of at least 10 contiguous amino acids of a relaxin polypeptide). In one embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the chimeric polypeptide. Chimeric products can be made by linking the appropriate nucleic acid sequences encoding the desired amino acid sequences together in an appropriate reading frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, chimeric products can be made by protein synthesis techniques (eg, by using an automated peptide synthesizer).
[0107]
In certain embodiments, the fusion protein is a relaxin analog-ubiquitin fusion protein. For example, US Pat. No. 5,108,919, the disclosure of which is incorporated herein by reference, discloses a method for preparing a fusion protein of a relaxin chain and ubiquitin.
[0108]
(Nucleic acid)
The invention further provides, according to the invention, nucleic acids for use as antagonists or for expressing antagonists. Such nucleic acids include nucleic acids encoding soluble relaxin receptors or relaxin analogs for the synthesis of soluble relaxin receptors or relaxin analogs, respectively. Antisense nucleic acids for inhibition of relaxin or relaxin receptor expression are provided. Nucleic acids encoding relaxin or relaxin receptor polypeptides are also provided for the preparation of antibodies (see above).
[0109]
In one aspect, the invention provides a nucleic acid sequence encoding a relaxin receptor or relaxin analog for expression in vivo. A relaxin receptor or relaxin analog or antisense nucleic acid can be expressed in vivo for gene therapy. The relaxin receptor, relaxin analog or antisense nucleic acid can also be used in vivo to prepare a recombinant soluble relaxin receptor, recombinant relaxin analog or recombinant antisense nucleic acid for exogenous administration to a subject. Alternatively, it can be expressed in vitro.
[0110]
The nucleic acid can be a vertebrate nucleic acid, for example, a relaxin receptor, relaxin, or a relaxin analog derived from vertebrate relaxin of a human, mouse, rat, pig, cow, dog, or monkey. These nucleic acids can include genomic DNA, genomic cDNA, or the coding region for a relaxin receptor, relaxin or relaxin analog. These nucleic acids can further include the relaxin receptor locus or the mRNA corresponding to the relaxin locus. These nucleic acids may also include variants encoding other possible codon choices for the same amino acid, or variants encoding conservative amino acid substitutions thereof (eg, naturally occurring allelic variants). , Nucleotide sequence variants. Due to the degeneracy of the nucleotide coding sequence, other nucleic acid sequences encoding substantially the same amino acid sequence as the relaxin receptor, or relaxin coding sequences, can be used in the practice of the invention. These nucleic acid sequences include all of the relaxin genes that have been modified by substitution of different codons (eg, conservative substitutions) that encode the same or a functionally equivalent amino acid residue in the sequence, thus producing a silent change. Or a nucleotide sequence comprising a moiety.
[0111]
The invention further provides a nucleic acid fragment of at least 6 contiguous nucleotides (eg, a hybridizable portion); in other embodiments, the nucleic acid comprises at least 8 contiguous nucleotides of the relaxin sequence, at least 25 contiguous nucleotides. At least 50 contiguous nucleotides, at least 100 nucleotides, 150 nucleotides or more. In another embodiment, these nucleic acids are less than 100 or 150 nucleotides in length. These nucleic acids can be single-stranded or double-stranded. As is readily apparent, as used herein, a nucleic acid encoding a relaxin or a fragment of a relaxin receptor polypeptide encodes only the referenced fragment or portion of the relaxin polypeptide, and It is to be understood that nucleic acids that do not encode a continuous sequence of the relaxin receptor or other contiguous portions of the relaxin polypeptide as a continuous sequence. Fragments of the nucleic acids encoding relaxin or one or more domains of the relaxin receptor are also provided.
[0112]
The relaxin receptor, relaxin analog or antisense nucleic acid can be provided in a suitable vector, such as an expression vector containing the necessary elements for transcription or transcription and translation of the inserted polypeptide coding sequence, as desired. It can be inserted in either the sense or antisense direction. The necessary transcriptional and translational signals may also be provided by the native relaxin or relaxin receptor gene and / or its flanking regions. Various host vector systems may be utilized to express the polypeptide coding sequence. These include mammalian cell lines infected with viruses (eg, vaccinia virus, adenovirus, adeno-associated virus, etc.), insect cell lines infected with viruses (eg, baculovirus), microorganisms such as yeast containing yeast vectors Or a bacterium transformed with a bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors differ in their strength and specificities. Any of a number of suitable transcription and translation elements may be used, depending on the host vector system utilized. In certain embodiments, the nucleic acid can be expressed, or the nucleic acid sequence encoding a functionally active portion of a relaxin receptor or relaxin analog is expressed in a mammalian cell, yeast, or bacterium. In yet another embodiment, a relaxin receptor or relaxin analog fragment comprising the respective polypeptide domain is expressed.
[0113]
Using any of the methods known in the art for inserting a nucleic acid into a vector, the expression vector containing the chimeric gene, having the appropriate transcriptional, translational, and / or polypeptide coding sequences, can be prepared. Can build. These methods include in vitro recombinant DNA and synthetic techniques, and in vivo recombination (genetic recombination). Expression of a nucleic acid encoding a relaxin receptor or relaxin analog can be regulated by a second nucleic acid sequence such that the nucleic acid or polypeptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a relaxin receptor or relaxin analog nucleic acid can be controlled by any promoter / enhancer element known in the art. Promoters that can be used to control expression include, but are not limited to, the SV40 early promoter region (see, eg, Benoist and Chambon, Nature 290: 304-10 (1981)), The promoter contained in the 3 'long terminal repeat of the Rous sarcoma virus (see, for example, Yamamoto et al., Cell 22: 787-97 (1980)), the herpes thymidine kinase promoter (see, for example, Wagner et al., Proc. Natl. Acad. ScL USA 78: 1441-45 (1981)), the regulatory sequences of the metallothionein gene (see, eg, Brinster et al., Nature 296: 39-42 (1982)). Prokaryotic expression vectors, such as the β-lactamase promoter (see, eg, Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-31 (1978)) or the tac promoter (see See, e.g., deBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 (1983)), a plant expression vector containing the cauliflower mosaic virus 35S RNA promoter (e.g., Gardner et al., Nucl. Acids Res. 9: 2871-88 (1981)), and the promoter of the photosynthetic enzyme ribulose diphosphate carboxylase (e.g., Herrera-Estrella et al., Nature 310: 115-20 ( 984) see), GAL7 and Gal4 promoter, ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, such as the alkaline phosphatase promoter, such as yeast or other probe motor elements from fungi.
[0114]
The following animal transcription regulatory regions, which show tissue specificity, have been utilized in transgenic animals: Elastase I gene regulatory regions active in pancreatic acinar cells (eg, Swift et al., Cell 38: 639). -46 (1984); See Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50: 399-409 (1986); MacDonald, Hepatology 7 (Supplement 1): 42S-51S (1987); Insulin gene regulatory regions active in cells (see, eg, Hanahan, Nature 315: 115-22 (1985)); immunoglobulin gene regulatory regions active in lymphoid cells (eg, Grosschedl et al., Ce. l 38: 647-58 (1984); Adams et al., Nature 318: 533-38 (1985); Alexander et al., Mol. Cell. Biol. 7: 1436-44 (1987)), testis, breast, Mouse mammary adenocarcinoma virus regulatory regions active in lymphoid and mast cells (see, eg, Leder et al., Cell 45: 485-95 (1986)), albumin gene regulatory regions active in the liver (eg, See Pinkert et al., Genes Dev. 1: 268-76 (1987), a-fetoprotein gene regulatory region active in the liver (eg, Krumlauf et al., Mol. Cell. Biol 5: 1639-48 (1985)). Hammer et al., Science 2; 5: 53-58 (1987)); α1-antitrypsin gene regulatory region active in the liver (see, for example, Kelsey et al., Genes and Level. 1: 161-71 (1987)); Β-globin gene regulatory region active in myeloid cells (see, eg, Magram et al., Nature 315: 338-40 (1985); Kollias et al., Cell 46: 89-94 (1986)); rare in the brain Myelin basic protein gene regulatory region active in process glial cells (see, for example, Readhead et al., Cell 48: 703-12 (1987)); myosin light chain-2 gene regulatory region active in skeletal muscle (See, for example, Shani, Nature 314: 283-86 (19 5) See); and activity in the hypothalamus, gonadotropin-releasing hormone gene control region (e.g., Mason, et al., Science 234: 1372-78 See (1986)). In a preferred embodiment, the tissue-specific promoter is a prostate-specific antigen promoter (see, eg, US Patent No. 6,100,444, the disclosure of which is incorporated herein by reference). ).
[0115]
In another embodiment, a vector is used that comprises a promoter operably linked to the nucleic acid, one or more origins of replication, and, optionally, one or more selectable markers (eg, antibiotic or drug resistance markers). Is done. For example, an expression construct can be made by subcloning a relaxin receptor or a relaxin analog nucleic acid into a restriction site of a pRSEC expression vector. Such a construct allows expression of a relaxin analog or relaxin receptor polypeptide under the control of a T7 promoter, which has a histidine amino terminal flag sequence for affinity purification of the expressed polypeptide. In another specific embodiment, a prostate-specific antigen promoter operably linked to a relaxin analog nucleic acid, one or more origins of replication, and, optionally, one or more selectable markers (eg, drug resistance markers) A vector containing is used.
[0116]
Expression vectors containing such nucleic acids can be identified by general approaches well known to those of skill in the art, including: (a) nucleic acid hybridization or polymerase chain reaction, (b) "markers" Presence or absence of gene function, (c) expression of the inserted sequence, or polymerase chain reaction (PCR). In a first approach, the presence of a nucleic acid inserted into the expression vector can be detected by nucleic acid hybridization or polymerase chain reaction using a probe containing a sequence homologous to the inserted nucleic acid. In a second approach, the recombinant vector / host system is a system in which a particular “marker” gene function (eg, thymidine kinase activity, antibiotics) is triggered by insertion of a vector containing relaxin receptor, relaxin or relaxin analog nucleic acid. , Phenotype, transformation phenotype, formation of occlusion bodies in baculovirus, etc.). For example, if the nucleic acid is inserted within a marker gene sequence of a vector, a recombinant containing the nucleic acid can be identified by the absence of marker gene function.
[0117]
In a third approach, a recombinant expression vector can be identified by assaying the polypeptide expressed by the recombinant. Such assays include, for example, physical or functional properties of the polypeptide in an in vitro assay system (eg, binding to an anti-relaxin antibody, binding to relaxin or a relaxin analog, binding to a relaxin receptor, etc.). Based on Once a particular recombinant vector has been identified and isolated, several methods known in the art can be used to propagate this vector. Once the appropriate host system and growth conditions are established, the recombinant expression vector can be propagated and prepared in large quantities. As previously described, expression vectors that may be used include, but are not limited to, vectors or derivatives thereof, to name a few: human or animal viruses (eg, vaccinia virus or adenovirus) Insect viruses (eg, baculovirus); yeast vectors; bacteriophage vectors (eg, λ); and plasmid and cosmid DNA vectors. In a fourth approach, PCR is used to detect nucleic acids in a vector (see above).
[0118]
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies or processes the gene product in the specific fashion desired. Expression from a particular promoter may be increased in the presence of a particular inducer, and thus, expression of the polypeptide may be controlled. In addition, different host cells that have characteristic and specific mechanisms for translational processing, post-translational processing and modification of the polypeptide (eg, glycosylation, phosphorylation) can be used. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce an unprocessed core protein product. Expression in mammalian cells can be used to ensure "native" processing of a mammalian receptor polypeptide. In addition, different vector / host expression systems can affect processing reactions to different extents.
[0119]
(Functional assay for relaxin and relaxin receptor agonists and antagonists)
The activity of a relaxin agonist and a relaxin antagonist, and the activity of a relaxin receptor agonist and antagonist can be determined by standard assays for relaxin and / or relaxin receptor activity. In one aspect of the invention, the activity of a relaxin agonist is assayed. For example, the ability of a relaxin agonist to reduce apoptosis or stimulate tissue maturation is assayed.
[0120]
In another aspect of the invention, the ability of a relaxin antagonist to inhibit the functional activity of relaxin by binding to relaxin is assayed. Similarly, the ability of a relaxin antagonist to inhibit relaxin function or relaxin receptor function can be determined, for example, by adding the relaxin antagonist to a relaxin receptor assay and comparing the inhibition to a control without the relaxin antagonist. Can be assayed. A suitable measure of the activity of a relaxin antagonist is the IC which is% inhibition. ₅₀ And the like.
[0121]
Suitable assays for measuring the activity of relaxin, or a relaxin receptor agonist or antagonist, include, for example, the assays described in the following references, which are incorporated herein by reference. : MacLennan et al., Ripening of the Human Cervix and Induction of Labor with Intracervical Purified Porcine Relaxin, Obstetrics & Gynecology 68: 598-601 (1986); Poisner et al., Relaxin Stimulates the Synthesis and Release of Prorenin From Human Decidual Cells: Evi ence For Autocrine / Paracrine Regulation, J. Clinical Endocrinology and Metabolism 70: 765-67 (1990); O'Day-Bowman et al., Hormonal Control of the Cervix in Pregnant Gilts. III. Relaxin's Influence on Cervical Biochemical Properties in Ovaritomized Horne-Treated Pregnant Gilts, Endocrinology, 129, 1967-76, 1991. J. Obstetrics & Gynecology and Reproductive Biology 41: 197-201 (1991).
[0122]
Other assays include those disclosed by Bullesbach et al., The Receptor-Binding Sites of Human Relaxin II, J. Am. Biol. Chem. 267: 22957-60 (1992); Hall et al., Influence of Ovarian Steroids on Relaxin-Induced Uterine Growth in Ovariectomized Gilts, Endocrinology 130: 3159-66 (1992); Kibblewhite et al., The Effect of Relaxin on Tissue Expansion, Arch. Otolaryngol. Head Neck Surg. 118: 153-56 (1992); Lee et al., Monoclonal Antibodies Special for Rat Relaxin. VI. Passive Immunization with Monoclonal Antibodies Throughout the Second Half of Pregnancy Disrupts Histological Changes Associated with Cervical Softening at Parturition in Rats, Endocrinology 130: 2386-91 (1992); Bell et al., A Randomized, Double-Blind Placebo-Controlled Trial of the Safety of Vaginal Recombinant Human Relaxin for Medical Ripping, Obstrics & Gynecol gy 82: 328-33 (1993); Bryant-Greenwood et al, Sequential Appearance of Relaxin, Prolactin and IGFBP-1 During Growth and Differentiation of the Human Endometrium, Molecular and Cellular Endocrinology 95: 23-29 (1993); Chen et al, The Pharmacokinetics of Recombinant Human Relaxin in Nonpregnant Woman After Intravenous, Intravaginal, and Intravascular Administration, Pharmaceutical tical Research 10: 834-38 (1993); Huang et al., Stimulation of Collagen Secretion by Relaxin and Effect of Oestrogen on Relaxin Binding in Uterine Cervical Cells of Pigs, Journal of Reproduction and Fertility 98: 153-58 (1993).
[0123]
Further assays are disclosed below: Saxena et al., Isthe Relaxin System a Drug for Drug Development Cardiac Effects of Relaxin, TiPS 14: 231 (internal, lt. IV. Relaxin Promotes Changes in the Histological Characteristics of the Cervix that are Associated with Cervical Softening During Late Pregnancy in Gilts, Endocrinology 133: 121-28 (1993); Colon et al., Relaxin Secretion into Human Semen Independent of Gonadotropin Stimulation, Biology of Reproduction 50: 187-92 (1994); Golub et al., Effect of Short-Term Infusion of Recombinant Hum. n Relaxin on Blood Pressure in the Late-Pregnant Rhesus Macaque (Macaca Mulatta), Obstetrics & Gynecology 83: 85-88 (1994); Jauniaux et al., The Role of Relaxin in the Development of the Uteroplacental Circulation in Early Pregnancy, Obstetrics & Gynecology 84: 338-342 (1994); Johnson et al., The Regulation of Plasma Relaxin Levels During Human Pregnancy, J. Am. Endocrinology 142: 261-65 (1994); Lane et al., Decidualization of Human Endometrial Stromal Cells in Vitro: Effects of Progestin and Relaxin on the Ultrastructure and Production of Decidual Secretory Proteins, Human Reproduction 9: 259-66 (1994); Lanzafame et al. , Pharmacological Stimulation of Sperm Mobility, Human Reproduction 9: 192-99 (1994); Petersen et al., Normal Serum Relaxin. n Women with Disabling Pelvic Pain During Pregnancy, Gynecol. Obstet. Invest. 38: 21-23 (1994); Tashima et al., Human Relaxins in Normal, Benign and Neoplastic Breast Tissue, J. Am. Mol. Endocrinology 12: 351-64 (1994); Winn et al., Individual and Combined Effects of Relaxin, Estrogen, and Progestone in Ovarietorialized Gilts. I. Effects on the Growth, Softening, and Histological Properties of the Cervix, Endocrinology 135: 1241-49 (1994); Winn et al., Individual and Combined Effects of Relaxin, Estrogen, and Progesterone on Ovariectomized Gilts. II. Effects on Mammary Development, Endocrinology 135: 1250-55 (1994); Bryant-Greenwood et al, Human Relaxins: Chemistry and Biology, Endocrine Reviews 15: 5-26 (1994); Johnson et al., Relationship Between Ovarian Steroids, Gonadotrophins and Relaxin During the Menstrual Cycle, Acta Endocrinilogica 129: 121-25 (1993).
[0124]
In yet another aspect of the invention, the activity of the agonist or antagonist is determined by measuring the ability of the agonist or antagonist to compete with a wild-type relaxin polypeptide or a relaxin receptor polypeptide for binding to an anti-relaxin antibody. ,It is determined. Various immunoassays known in the art can be used. Such assays include, but are not limited to, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assays), “sandwich” immunoassays, immunoradiometric assays, gel diffusion sedimentation reactions, immunodiffusion assays, In situ immunoassay (using colloidal gold, enzyme or radioisotope labeling, etc.), western blot, sedimentation reaction, agglutination assay (eg, gel or hemagglutination assay), complement binding assay, immunofluorescence assay, protein A assay Competitive and non-competitive assay systems using techniques such as immunoelectrophoresis assays. Antibody binding can be detected by measuring the amount of label on the primary antibody that binds the substrate or has been prevented from binding to the substrate. Alternatively, binding of the primary antibody is detected by measuring the binding of a secondary antibody or reagent to the primary antibody. This secondary antibody can also be directly labeled. Many means for detecting binding in an immunoassay are known in the art and are considered to be within the scope of the present invention.
[0125]
The functional activity of an agonist or antagonist can also be determined in an in vivo system. For example, the ability of a relaxin agonist or relaxin antagonist to bind to or compete for binding to the relaxin receptor, or to modulate apoptosis in a cell population and / or tissue can be measured. The above assay can be used to determine the activity resulting from the expression of a relaxin agonist or relaxin antagonist in vertebrate cells. Alternatively, a relaxin agonist or relaxin antagonist can be expressed in a heterologous system, and the activity of the relaxin agonist or relaxin antagonist can be assayed as a modulator of a physiological change in the system. For example, the ability of a relaxin agonist or relaxin antagonist to modulate apoptosis can be tested in vertebrate cells (eg, transfected mammalian cells).
[0126]
(Administration of relaxin agonist and relaxin antagonist)
The present invention provides a method for administering to a subject an effective amount of a relaxin agonist or relaxin antagonist (also collectively referred to as an "active agent"). Typically, the active agent is substantially purified prior to formulation. The subject can be a human or non-human vertebrate animal, and is typically an animal, including but not limited to cows, pigs, horses, chickens, cats, dogs, and the like. More typically, the subject is a mammal, and in certain embodiments, a human.
[0127]
Various delivery systems (eg, by infusion, by injection (eg, intradermal, intramuscular or intraperitoneal), oral, nasal, pulmonary, rectal, transdermal, interstitial or subcutaneous) can be used. It is known and can be used to administer active agents. In certain embodiments, it may be desirable to administer the active agent locally to the area in need of treatment; such administration includes, but is not limited to, by local injection, by local application, by injection (eg, Intratesticular or intraprostatic), by a catheter, or by an implant, which is, for example, a porous, non-porous, gelatinous or polymeric material, such as a silastic membrane. Including membranes or fibers. In one embodiment, administration can be by direct injection at the target site.
[0128]
Pharmaceutical compositions containing the active agent can be formulated according to the desired delivery system. Such pharmaceutical compositions typically include a therapeutically effective amount of the active agent and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" refers to a vertebrate (typically a vertebrate (typically, (Animals, more typically humans). The term "carrier" refers to a diluent, adjuvant, excipient, stabilizer, preservative, viscogen or vehicle with which the active agent is formulated for administration. Pharmaceutical carriers can be sterile liquids, such as water and oils, including petroleum, animal, vegetable or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, Ethanol and the like can be mentioned. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
[0129]
Suitable preservatives include, for example, sodium benzoate, quaternary ammonium salts, sodium azide, methyl paraben, propyl paraben, sorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, chlorobutanol, dehydroacetic acid, Ethylenediamine, potassium benzoate, potassium metabisulfite, potassium sorbate, sodium bisulfite, sulfur dioxide, organic mercury salts, phenol and ascorbic acid. Suitable viscosity modifiers include, for example, carboxymethyl cellulose, sorbitol, dextrose, and polyethylene glycol. Other examples of suitable pharmaceutical carriers are, for example, Remington's Pharmaceutical Sciences (edited by Gennaro), Mack Publishing Co. , Easton, Pennsylvania (1990).
[0130]
Activators can also be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with free amino groups (eg, salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like), and salts formed with free carboxyl groups ( Examples thereof include salts derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.
[0131]
In one embodiment, the active agent is formulated as a pharmaceutical composition adapted for intravenous administration to humans according to conventional procedures. For intravenous delivery, water is a typical carrier. Saline solutions, aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent or a local anesthetic to ease pain at the site of the injection. Generally, the components are administered separately or in a single dosage form (eg, as a dry lyophilized powder or in a water-free concentrate in a sealed container such as an ampoule or pouch indicating the quantity of active agent). ) Or mixed together. If the composition is to be administered by infusion, the composition can be dispersed in an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
[0132]
Orally deliverable compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like.
[0133]
For rectal administration, the compositions are formulated according to standard pharmaceutical procedures. Typically, the composition is formed as a dissolvable composition (eg, a suppository). Suppositories may include adjuvants to provide a desired consistency to the composition. Suppositories may also contain water-soluble carriers, such as polyethylene glycol, polypropylene glycol, glycerogelatin, methylcellulose or carboxymethylcellulose. Suppositories may also include wetting agents, such as fatty acids, fatty acid glycerides, polyethylene sorbitan fatty acid esters, polyethylene sorbital fatty acid esters, polyethylene fatty acid esters, and higher alcohol esters of polyoxyethylene, and esters of lower alkyl sulfonic acids. . Suppositories may also contain suitable emulsifying and dispersing ingredients, as well as ingredients for adjusting the viscosity and coloring substances.
[0134]
Nasal administration is typically performed using the solution as a nasal spray and can be formulated by various methods known to those skilled in the art. Systems for intranasal administration of liquids as a spray are well known (see, for example, US Pat. No. 4,511,069, which is incorporated herein by reference). Preferred nasal spray solutions comprise an active agent in a liquid carrier, which optionally increases absorption of the drug and one or more buffers or other additives to minimize nasal irritation. A nonionic surfactant. In some embodiments, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution is typically between about pH 6.8 and pH 7.2.
[0135]
For intranasal administration, ingredients that improve the absorption of nasal administered active agents and reduce nasal irritation are desirable, particularly when used in chronically administered treatment protocols. In this case, the use of surfactants is preferred in order to increase the absorption of the active agent (for example, Hirai et al., Int. J. Pharmaceutics 1: 173-84 (1981); British Patent Specification 1 527 605 (these are incorporated herein by reference). Each of which is incorporated herein by reference)). However, intranasal administration of drugs augmented by surfactants can cause irritation of the nasal cavity, including stinging, congestion and rhinorrhea. Thus, compositions that increase absorption through the nasal mucosa while reducing irritation (eg, nonionics such as nonoxynol-9, lauret-9, poloxamer-124, octoxynol-9, and lauramide DEA) Surfactants). Nonoxynol-9 (N-9) is an ethoxylated alkylphenol, which is a polyethyleneoxy condensate of nonylphenol and 9 moles of ethylene oxide. This surfactant has been used in surfactant products and is available in SURFONIC® N-95 (Jefferson), NEUTRONYX® 600 (Onyx) and IGEPAL® (CO-630 (GAF)). ). N-9 is considered a hard surfactant and has been used as a spermatocide (see The Merck Index, 10th edition, Entry 6518). To minimize irritation due to the use of surfactants, one or more anti-irritant additives are included in the emulsion. In one example, polysorbate-80 has been shown to reduce irritation caused by intranasally administered drugs, the delivery of which is facilitated by using non-ionic surfactants (e.g., See U.S. Patent No. 5,902,789, which is incorporated herein by reference.
[0136]
The pulmonary dosage form containing the active agent can be administered intranasally into the respiratory tract, or can be administered to the respiratory tract by inhaling a spray or aerosol containing the active agent. The active agent is typically delivered directly into the lungs in the form of small particle aerosols, which are specifically targeted to minimal air pathways and alveoli.
[0137]
Pulmonary dosage forms are typically formed as particulate, dispersed forms. This can be accomplished by preparing an aqueous aerosol of solid particles containing the composition. Typically, aqueous aerosols are made by formulating an aqueous solution or suspension of the composition together with conventional pharmaceutically acceptable carriers and stabilizers. These carriers and stabilizers are typically nonionic surfactants (eg, Tween, Pluronic or polyethylene glycol), harmless proteins (eg, serum albumin), sorbitan esters, oleic acid, amino acids (eg, glycine ), Buffers, salts, sugars or sugar alcohols. The formulation may also include a mucolytic agent, such as those described in US Pat. No. 4,132,803, which is incorporated herein by reference, and a bronchodilator. The formulation is preferably sterile. Aerosols are generally prepared from isotonic solutions. The particles optionally contain normal lung surfactant proteins.
[0138]
Aerosols of the particles can be formed in aqueous or non-aqueous suspensions, such as fluorocarbon propellants. The aerosol is preferably free of pulmonary irritants (ie, surfactants that cause acute bronchoconstriction, cough, pulmonary edema or tissue destruction). Non-irritating absorption enhancers are also suitable for use herein.
[0139]
To prepare an aerosol, a sonic nebulizer may be used. Sonic nebulizers minimize exposure of the composition to shear, which can cause degradation. A suitable device is a Bird Micronebulizer. Other suitable automation or nebulization systems, or intratracheal delivery systems, include, for example, as long as they are compatible with the composition to be administered and can deliver particles of the desired size: U.S. Pat. No. 3,915,165; system disclosed in EP 0 116 476; jet nebulizer described by Newman et al. (Thorax 40: 671-76 (1985)); measured dose inhalers (e.g., Berenberg, J. Asthma-USA 22: 87-92 (1985)); Braunner (US Pat. No. 5,803,078) intratracheal catheter assembly or other device (see, eg, Sears et al., NZ. Med. J. 96: 743 II (1983); O'Reilly et al., Br. d.J.286: 6377 (1983); or Stander et al, Respiration 44: 237-40 see (1982)). (The disclosures of these references are incorporated herein by reference).
[0140]
Particulate aerosol suspensions are typically fine, dry powders containing the active agent. Particulate aerosol suspensions are prepared by a number of conventional procedures. The simplest method of preparing such a suspension is to micronize the active agent (eg, as crystals or lyophilized cake) and disperse the particles in a dry fluorocarbon propellant. In these formulations, the active agent is preferably suspended in the fluorocarbon. In an alternative embodiment, the active agent is stored in a separate compartment from the propellant. Upon firing of the propellant, a predetermined dose is dispensed from the storage compartment. Devices used to deliver active agents in this manner are known as metered dose inhalers (MDIs) (eg, Byron, Drug Development and Industrial Pharmacy 12: 993 (1986), which is hereby incorporated by reference). , Incorporated herein by reference).
[0141]
Aerosol or particle sizes generally range from about 0.5 μm to about 5 μm, typically about 2 μm to 5 μm, preferably about 2 μm to about 4 μm, or about 4 μm to about 5 μm. In some aspects, smaller particles become less acceptable. This is because smaller particles do not accumulate and instead tend to diverge. Similarly, in other aspects, larger particles are less preferred. Because larger particles are less likely to accumulate in the alveoli and are removed by intrusion in the nasopharyngeal or oral cavity (see, eg, Byron, J. Pharm. Sci. 75: 433 (1986)). That). Aerosol or particulate compositions can be heterogeneous in size distribution, but heterogeneity can be reduced by known methods (eg, the screening unit described in EP 0 135 390). Heterogeneity is not disadvantageous unless the fraction of particles having an average diameter typically greater than about 4 μm is not large enough to prevent delivery of a therapeutic dose by pulmonary inhalation. Suspensions containing more than about 15% of particles in the range of 0.5 to 5 μm can be used, but generally the particle portion having an average diameter of more than 5 μm will instead be replaced by about It is less than 25%, preferably 10% or less. The indicated diameter refers to the diameter of the particle when introduced into the respiratory tract.
[0142]
The amount of active agent that is effective in treating a particular subject is based on the particular disorder being treated and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose of active agent used in the formulation will also depend on the route of administration, and the seriousness of the condition, and must be decided according to the judgment of the practitioner and each subject's circumstances. Suitable dosage ranges for administration are generally about 0.001 mg / kg to about 100 mg / kg of active agent per kg of body weight. Effective doses can also be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations typically contain 10% to 95% active ingredient.
[0143]
The present invention also provides a pharmaceutical pack or kit containing one or more containers filled with one or more components of the pharmaceutical composition of the present invention. A notice in the form prescribed by an administrative body regulating the manufacture, use or sale of a pharmaceutical or biological product is optionally associated with such a container, and the notice may refer to the manufacture, use or use for human administration. Or affect the authorization by the distributor of sale.
[0144]
In yet another embodiment, the active agent can be delivered in a sustained release system. In one embodiment, a pump may be used (eg, Langer, supra; Sefton, Crit. Ref. Biomed. Eng. 14: 201-40 (1987); Buchwald et al., Surgary 88: 507-16 (1980). Saudek et al., N. Engl. J. Med. 321: 574-79 (1989)). In another embodiment, a polymeric material may be used (Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Biologia Digital Pharma Biologica Pharmaceuticals, Canada). and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228: 190-92 (1985). See also During et al., Ann. l.25: 351-56 (1989); Howard et al., J.Neurosurg.71: 105-12 (1989)) (the disclosures of which are incorporated herein by reference).
[0145]
Thus, in yet another embodiment, a sustained release system can be placed near a therapeutic target that requires only a systemic dose fraction (eg, Goodson, Medical Applications of Controlled Release, supra, supra). 2, 115-38 (1984)). Other sustained release systems are discussed, for example, in the review by Langer (Science 249: 1527-33 (1990), which is incorporated herein by reference).
[0146]
(Administration of nucleic acid)
The invention relates to nucleic acids encoding nucleic acids (eg, relaxin agonists or relaxin antagonists (relaxin, relaxin analogs, soluble relaxin receptors, relaxin binders, relaxin receptor binders), relaxin antisense nucleic acids and / or (Eg, relaxin receptor antisense nucleic acid) to provide additional methods of modulating apoptosis. Nucleic acids (both sense and antisense) can be used in gene therapy processes. Gene therapy refers to a process that provides for the expression of a nucleic acid of exogenous origin, including the antisense of a subject to modulate apoptosis (eg, to treat tissue abnormalities) in the subject. Examples include nucleic acids or antisense nucleic acids encoding relaxin agonists or relaxin antagonists. In certain embodiments, a nucleic acid encoding relaxin or a relaxin analog is administered into cells having a relaxin receptor to reduce apoptosis. In other specific embodiments, a nucleic acid encoding a relaxin binding agent (eg, relaxin, a soluble relaxin receptor) or a relaxin receptor binding agent (eg, a relaxin analog) activates the relaxin receptor to increase apoptosis. Administered to the expressing cell aggregate. Any of the methods for gene therapy that are useful in the art can be used according to the present invention. An exemplary method is described below.
[0147]
For a general review of methods of gene therapy, see Steinberg and Raso (J. Pharm. Pharm. Sci. 1: 48-59 (1998)); Pantuck et al. (World J. Urol. 18: 143-47 (2000)). Prince (Pathology 30: 335-47 (1998)); Ledley (Curr. Opin. Biotechnol. 5: 626-36 (1994)); Goldspiel et al. (Clin. Pharm. 12: 488-505 (1993)); Wu. and Wu (Biotherapy 3: 87-95 (1991)); Tolstoshev (Ann. Rev. Pharmacol. Toxicol. 32: 573-96 (1993)); Mulligan (Science 260). 926-32 (1993)); Morgan and Anderson (Ann.Rev.Biochem.62: 191-217 (1993)); and May (TIBTECH 11: 155-215 (1993)) to see.
[0148]
Methods that are commonly known in the art of recombinant DNA technology and that can be used include Ausubel et al. (1996, supra) and Kriegler (Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990)). Method. In one embodiment, the nucleic acid comprises a sense nucleic acid that is part of a vector that expresses the nucleic acid in a suitable host (eg, a nucleic acid encoding a relaxin analog, a soluble relaxin receptor, etc.). In particular, such nucleic acids have a promoter operably linked to a coding region (eg, relaxin or relaxin receptor) in a sense orientation, wherein the promoter is inducible or constitutive, and optionally , Tissue-specific. In another embodiment, the nucleic acid comprises an antisense nucleic acid (eg, a relaxin antisense nucleic acid or a relaxin receptor antisense nucleic acid) that is part of a vector that expresses the nucleic acid in a suitable host. In particular, such nucleic acids have a promoter operably linked to the coding region (eg, relaxin or relaxin receptor) in an antisense orientation, wherein the promoter is inducible or constitutive, and optionally And tissue-specific.
[0149]
In another specific embodiment, the nucleic acid (eg, a sense nucleic acid encoding a relaxin analog, a soluble relaxin receptor, or the like, or an antisense nucleic acid encoding a relaxin antisense nucleic acid, a relaxin receptor antisense nucleic acid, etc.) This nucleic acid and any other desired sequence are used when flanked by regions that promote homologous recombination at the desired site in the genome, thereby providing intrachromosomal expression of the nucleic acid (eg, Koller and Smiths, Proc. Natl. Acad. Sci. USA 86: 8932-35 (1989); Zijlstra et al., Nature 342: 435-38 (1989); U.S. Patent Nos. 5,631,153; No. 059; 5,487,992 No. 5,464,764; (the disclosures of which are incorporated herein by reference).
[0150]
For any of these embodiments, delivery of the nucleic acid into the subject can be direct (if the subject is directly exposed to the nucleic acid or nucleic acid transport vector) or indirect (if the cell is First transformed with the nucleic acid in vitro, and then transplanted into the subject). These two approaches are known, respectively, as in vivo gene therapy or ex vivo gene therapy. In certain embodiments, the nucleic acid is directly administered in vivo and expresses the nucleic acid to produce the encoded product. This can be accomplished by any of a number of methods known in the art: for example, a method of making and administering a nucleic acid within a cell as part of a suitable nucleic acid expression vector; Methods by infection using retroviruses or attenuated retroviruses or other viral vectors (infra), direct injection of naked DNA (eg, Asahara et al., Semin. Interv. Cardiol. 1: 225-32 (1996)). Prazeres et al., Trends Biotechnol. 17: 169-74 (1999), the disclosures of which are incorporated herein by reference), electroporation (e.g., Muramatsu et al., Int. J .; Mol.Med. 1: 55-6. 2 (1998), (the disclosures of which are incorporated herein by reference), or by the gene gun (BIOLISTIC). ^TM (See, eg, Biewenga et al., J. Neurosci. Methods 71: 67-75 (1997), the disclosures of which are incorporated herein by reference). By way. Nucleic acids can also be inserted into cells by coating of naked nucleic acids with lipids or cell surface receptors or transformants, encapsulation in liposomes, derivatized liposomes, microparticles, or microcapsules (eg, De Smedt et al.). 17: 113-26 (2000); Maurer et al., Mol. Membr. Biol. 16: 129-40 (1999); Tarahovsky and Ivanitsky, Biochemistry 63: 607-18 (1998); 16: 307-21 (1998); Gao and Huang, Gene Ther. 2: 710-22 (1995); (the disclosures of which are incorporated herein by reference). Nucleic acids linked to peptides known to enter cells can also be administered, or nucleic acids linked to ligands affected by receptor-mediated endocytosis, can be used to specifically bind the receptor. Expressing cell types (eg, Liang et al., Pharmazie 54: 559-66 (1999); Cristiano, Front. Biosci. 15: D1161-70 (1998); Guy et al., Mol. Biotechnol. 3: 237. -48 (1995); Wu and Wu, J. Biol. Chem. 262: 4429-32 (1987), the disclosures of which are incorporated herein by reference). Nucleic acid-ligand complexes are formed by the spindle It may be in a form that contains the viral peptide of the cell and avoids lysosomal degradation of the nucleic acid (eg, Phillips, Biologicals 23: 13-16 (1995), (the disclosures of which are incorporated herein by reference). ), See).
[0151]
In yet another embodiment, antisense nucleic acids can be targeted in vivo for specific uptake and expression of cells by targeting specific receptors (eg, Phillips, Biologicals 23: 13-16 (1995); See International Patent Publications WO 92/06180; WO 92/22635; WO 92/20316; WO 93/14188, and WO 93/20221; the disclosures of which are incorporated herein by reference).
[0152]
In certain embodiments, the viral vector comprises a nucleic acid (eg, a sense nucleic acid encoding a relaxin analog, a soluble relaxin receptor, etc., or an antisense nucleic acid encoding a relaxin antisense nucleic acid, a relaxin receptor antisense nucleic acid). Etc.) are used. For example, retroviral vectors may be used (eg, Palu et al., Rev. Med. Virol. 10: 185-202 (2000); Buchschacher and Wong-Staal, Blood 15: 2499-504 (2000); Miller et al., Meth. Enzymol. 217: 581-99 (1993), the disclosures of which are incorporated herein by reference). These retroviral vectors are typically modified to remove retroviral sequences that are not necessary for packaging and integrating the viral genome into host cell DNA. Antisense nucleic acids used in gene therapy are cloned into vectors to facilitate delivery of the antisense nucleic acid into a subject. Lentiviral vectors can also be used. (See, e.g., Buchschacher and Wong-Staal, supra; Naldini et al., Science 272: 263-67 (1996), the disclosures of which are incorporated herein by reference). Other references illustrating the use of viral vectors in gene therapy are Lundstrom (J. Recept. Signal. Transduct. Res. 19: 673-86 (1999)); Clowes et al. (J. Clin. Invest. 93: 644-51 (1994)); Kiem et al., (Blood 83: 1467-73 (1994)); Salmons and Gunzberg (Hum Gene Ther. 4: 129-41 (1993)); and Grossman and Wilson (Curr. Opin. Genet Dev. 3: 110-14 (1993), the disclosures of which are incorporated herein by reference.
[0153]
Adenoviruses can also be used in gene therapy. Adenoviruses are particularly attractive vehicles for delivering genes to the prostate, liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson (Curr. Opin. Genet Dev. 3: 499-503 (1993), the disclosures of which are incorporated herein by reference) provide a review of adenovirus-based gene therapy. I do. Herman et al. (Human Gene Therapy 10: 1239-49 (1999), the disclosures of which are incorporated herein by reference) describe a replication deficiency involving the herpes simplex thymidine kinase gene in the human prostate. Intraprostatic infusion of adenovirus followed by intravenous administration of the prodrug ganciclovir in a Phase I clinical experiment is described. Other examples of the use of adenoviruses in gene therapy are Rosenfeld et al. (Science 252: 431-34 (1991)); Rosenfeld et al. (Cell 68: 143-55 (1992)); Mastrangeli et al. (J. Clin. Invest. 91: 225-34 (1993)); and Thompson (Oncol. Res. 11: 1-8 (1999)). Adeno-associated virus (AAV) can also be used in gene therapy (eg, Rabinowitz and Samulski, Curr. Opin. Biotechnol. 9: 475-85 (1988); Carter and Samulski, Int. J. Mol. Med. 6). Tal, J. Biomed. Sci. 7: 279-91 (2000); Ali et al., Gene Therapy 1: 367-84 (1994); U.S. Patent No. 4,797,368 and the same. No. 5,139,941; Walsh et al., Proc. Soc. Exp. Biol. Med. 204: 289-300 (1993); Grimm et al., Human Gene Therapy 10: 2445-50 (1999)) (these disclosures). , Which is incorporated by reference herein).
[0154]
Another approach to gene therapy involves transferring nucleic acids to cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Typically, the method of transfer involves the transfer of a selectable marker to the cell. The cells are then harvested and placed under selection to isolate those cells that express the nucleic acid. The selected cells are then delivered to a subject.
[0155]
In one embodiment, the nucleic acid is introduced into the cells prior to in vivo administration of the resulting recombinant cells. Such introduction may be performed by any method known in the art, including but not limited to: transfections, electroporations, microinjections, viral or bacteriophage vectors containing nucleic acids. Infection, cell fusion, chromosome-mediated gene transfer, micronucleoid-mediated gene transfer, and the like. Numerous techniques for introducing foreign genes into cells are known in the art (eg, Muramatsu et al., Int. J. Mol. Med. 1: 55-62 (1998); Liang et al., Pharmazie 54: 559-66 (1999); Loeffer and Behr, Meth. Enzymol. 217: 599-618 (1993); Cotten et al., Meth. Enzymol. 217: 618-44 (1993); Cline, Pharmacol. Ther. 29: 69-. 92 (1985); these disclosures are incorporated herein by reference) and can be used in accordance with the present invention. The technique is typically provided for stable transfer of nucleic acid to a cell, such that the nucleic acid is expressible by the cell and is genetic and expressible by its cell progeny.
[0156]
The resulting recombinant cells can be delivered to a subject by various methods known in the art. Typically, cells are injected subcutaneously. In another embodiment, the recombinant skin cells can be applied to the subject as a skin graft. The amount of cells required for use depends on the desired effect, the condition of the subject, etc., and can be determined by one skilled in the art.
[0157]
Cells into which the nucleic acid can be introduced for gene therapy purposes encompass any desired useful cell type and include, but are not limited to: the male reproductive tract (eg, prostate cells, testis cells, spermatids) Cells, or epididymis cells), female reproductive tract (eg, uterus, cervix, interpubic ligament, connective tissue in lower limb girdle, etc.), liver, kidney, spleen, thymus, brain, heart, intestinal tract, skin A cell or collection of cells, such as the lungs. Suitable cells further include epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, and stem or progenitor cells. The cells used for gene therapy are generally autologous to the subject, but allogeneic cells that can be classified for compatibility with the subject can be used.
[0158]
In another aspect, the nucleic acid (eg, a relaxin analog, a sense nucleic acid encoding a soluble relaxin receptor, or the like, or a relaxin antisense nucleic acid, an antisense nucleic acid encoding a relaxin receptor antisense nucleic acid, etc.) is directly transferred to the cell. Is administered. The nucleic acid is at least 6 nucleotides and typically an oligonucleotide (ranging from 6 to about 50 or more nucleotides). In certain aspects, the oligonucleotide can be at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 200 nucleotides. The oligonucleotide may be DNA or RNA or a chimeric mixture or derivative thereof or an analog thereof, and may be single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide may be a peptide or an agent that promotes translocation across cell membranes (eg, Nielsen, Pharmacol. Toxicol. 86: 3-7 (2000); Soomets et al., Front. Biosci. 1: D782-86 (1999); Galderisi et al., J. Cell Physiol. 181: 251-57 (1999); Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553-56 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. USA. 84: 648-52 (1987); International Patent Publication WO 88/09810, and hybridization-triggered cleavage agents (e.g., Kroll et al., BioTechniques 6: 958-76 (198). 8)) or intercalating agents (see, eg, Zon, Pharm. Res. 5: 539-49 (1988)). (These disclosures are incorporated herein by reference).
[0159]
In one embodiment, the antisense oligonucleotide is provided as single-stranded DNA. The oligonucleotide can be modified at any position in its structure using substituents generally known in the art. The oligonucleotide may include at least one modified base moiety such as, for example, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetyl. Cytosine, 5- (carboxy-hydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylamino-methyluracil, dihydrouracil, β-D-galactosyl queosine, inosine, N6- Isopentenyl adenine, 1-methyl guanine, 1-methyl inosine, 2,2-dimethyl guanine, 2-methyl adenine, 2-methyl guanine, 3-methyl cytosine, 5-methyl cytosine, N6-adenine, 7-methyl guanine, 5- Tylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylcuosin, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil- 5-oxyacetic acid (V), pseudouracil, cuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5 -Oxyacetic acid (V), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, 2,6-diaminopurine, and the like. In another embodiment, the oligonucleotide comprises at least one modified sugar moiety (eg, arabinose, 2-fluoroarabinose, xylulose, and hexose).
In yet another embodiment, the antisense oligonucleotide comprises at least one of the following modified phosphate backbones: for example, phosphorthionate, phosphordithionate, phosphoramidothionate, phosphoramidate, phosphoamidate, phosphoamidate, Ludiamidate, methyl phosphonate, alkyl phosphotriester, and formacetal or analogs thereof.
[0160]
In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. α-anomeric oligonucleotides form specific double-stranded hybrids with complementary RNA (the strands of which, contrary to the normal β-unit, are parallel to each other) (Gautier et al., Nucl. Acids Res. 15: 6625-41 (1987)). The oligonucleotide can be conjugated to another molecule, such as a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, and the like.
[0161]
In certain embodiments, the antisense oligonucleotide comprises a catalytic RNA, or ribozyme (eg, Welch et al., Curr. Opin. Biotechnol. 9: 486-96 (1998); Norris et al., Adv. Exp Med. Biol. 465: 293-301 (2000); International Patent Publication WO 90/11364; Sarver et al., Science 247: 1222-25 (1990)). In another embodiment, the oligonucleotide is a 2'-0-methyl ribonucleotide (Inoue et al., Nucl. Acids Res. 15: 6131-48 (1987)), or a chimeric RNA-DNA analog (Inoue et al., FEBS Lett). .215: 327-30 (1987)).
[0162]
In another specific embodiment, double-stranded RNA directs sequence-specific degradation of mRNA by RNA interference (Hunter, Curr. Biol. 10: R137-40 (2000); Bosher and Labouesse, Nat. Cell. Biol .: e31-36 (2000); (the disclosures of which are incorporated herein by reference). Briefly, double-stranded nucleic acids are introduced into cells to selectively inhibit gene expression by causing mRNA degradation. (See, for example, Zamore et al., Cell 101: 25-33 (2000), the disclosures of which are incorporated herein by reference).
Nucleic acids according to the present invention can be synthesized by standard methods known in the art. Enzymatic methods for nucleic acid synthesis frequently use Klenow, T7, T4, Taq or Escherichia coli DNA polymerase, as described in Sambrook et al. (Supra). Enzymatic methods for RNA nucleic acids frequently use SP6, T3, or T7 RNA polymerase, as described in Sambrook et al., Supra. Reverse transcriptase can also be used for the synthesis of DNA from RNA (Sambrook et al., Supra). Nucleic acids are typically prepared using template nucleic acids enzymatically. The nucleic acid is either chemically synthesized or obtained as mRNA, genomic DNA, genomic DNA to be cloned, cDNA to be cloned, or other nucleic acid. Some enzymatic methods of DNA nucleic acid synthesis may require additional primers that can be chemically synthesized. Finally, linear nucleic acids can be prepared by polymerase chain reaction (PCR) techniques, such as those described by Saiki et al. (Science 239: 487 (1988)).
Chemical methods can also be used to synthesize nucleic acids (eg, antisense oligonucleotides), such as by using a commercially available automated DNA synthesizer. For example, phosphorthioate nucleic acids can be synthesized by the method of Stein et al. (Nucl. Acids Res. 16: 3209-21 (1988)), and methylphosphonate nucleic acids can be synthesized using a controlled pore glass polymer support ( For example, see Sarin et al., Proc. Natl. Acad. Sci. USA 85: 7448-51 (1988)), the disclosures of which are incorporated herein by reference. Other methods include those disclosed by Usman et al. (J. Am. Chem. Soc. 109: 7845-54 (1987)), Scaringe et al. (Nucleic Acid Res. 18: 5433-41 ( Caruthers (Oligonucleotides: Antisense Inhibitors of Gene Expression, p. 7-24, Cohen, (eds.), CRC Press, Inc. Boca Ratton, Fla, 1989); Oligonucleotides and Analogues, A Practi., IRL Press, 1984); al Approach (Eckstein, IRL Press, 1991); and U.S. Patent Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; Nos. 4,973,679; 5,026,838; 5,132,418; and Re. 34,069. All of the aforementioned references are incorporated herein by reference.
[0163]
(Small molecule effector or small molecule antagonist)
Relaxin nucleic acids, polypeptides, analogs and fragments, and relaxin receptor nucleic acids, polypeptides, analogs and fragments and analogs are also screened to detect candidate compounds that specifically bind to relaxin polypeptide or relaxin receptor Used in assays to modulate apoptosis. Such candidate compounds are typically small molecule effectors (agonists) or antagonists, and can be identified by in vitro and / or in vivo assays. Such assays can be used to identify small molecule effectors or antagonists that are therapeutically effective as relaxin agonists or antagonists or as lead compounds for drug development. Accordingly, the present invention provides an assay for detecting a compound. The compound specifically affects the activity or expression of relaxin nucleic acid, relaxin polypeptide, relaxin receptor nucleic acid, relaxin receptor, and the like.
[0164]
In a typical in vivo assay, recombinant cells expressing relaxin or relaxin receptor nucleic acid can be used to screen candidate compounds for compounds that affect relaxin or relaxin receptor nucleic acid expression. The effects of agonists or antagonists on relaxin or relaxin receptor expression may include stimulation or inhibition (eg, up-regulation or down-regulation) of mRNA transcription, increased or decreased mRNA stability, translation of mRNA, translation of relaxin or relaxin. Effects may be on synthesis of a toxin receptor polypeptide, relaxin or relaxin receptor polypeptide function (eg, binding to a relaxin receptor), and / or stability or localization of a relaxin or relaxin receptor polypeptide. Such effects on expression can be identified as physiological changes, such as changes in relaxin-responsive tissue growth rate, division, viability, collagen deposition, apoptosis, and the like. In one embodiment, the candidate compounds express relaxin or a relaxin receptor polypeptide to identify those compounds that produce a physiological change (eg, stimulation or inhibition of relaxin or relaxin receptor polypeptide function). Administered to recombinant cells.
[0165]
Candidate compounds can also be identified by in vitro screening methods. For example, a recombinant cell expressing relaxin or a relaxin receptor nucleic acid can be used to identify a candidate compound that binds to the relaxin or relaxin receptor polypeptide. Can be used to produce A candidate compound (such as a small molecule) is contacted with the polypeptide (or a fragment or analog thereof) under conditions leading to binding, and candidate compounds that specifically bind to the polypeptide are identified. Methods that can be used to carry out the preceding description are generally known in the art, and can screen for random or combination peptides or non-peptides that can screen for candidate compounds that specifically bind to relaxin or relaxin receptor polypeptide. Contains diverse libraries such as peptide libraries. Many libraries that can be used are known in the art, for example, chemically synthesized libraries, recombinant phage display libraries, and in vitro translation-based libraries.
[0166]
Examples of chemically synthesized libraries are described below: Fodor et al. (Science 251: 767-73 (1991)), Houghten et al. (Nature 354: 84-86 (1991)), Lam et al. (Nature). 354: 82-84 (1991)), Medynski (BioTechnology 12: 709-10 (1994)), Gallop et al. (J. Med. Chem. 37 (9): 1233-51 (1994)), Ohlmeyer et al. (Proc. Natl. Acad. Sci. USA 90: 10922-26 (1993)), Erb et al. (Proc. Natl. Acad. Sci. USA 91: 11422-26 (1994)), Houghten et al. (BioTechniques 13: 412-21 (1)). 992)), Jayawickreme et al. (Proc. Natl. Acad. Sci. USA 91: 1614-18 (1994)), Salmon et al. (Proc. Natl. Acad. Sci. USA 90: 11708-12 (1993)), international patent. Published WO 93/20242, and Brenner and Larner (Proc. Natl. Acad. Sci. USA 89: 5381-83 (1992)). (The disclosures of these references are incorporated herein.)
Examples of phage display libraries include Scott and Smith (Science 249: 386-90 (1990)), Devlin et al. (Science 249: 404-06 (1990)), Christian et al. (J. Mol. Biol. 227: 711-). 18 (1992)), Lenstra (J. Immunol. Meth. 152: 149-57 (1992)), Kay et al. (Gene 128: 59-65 (1993)) and International Patent Publication WO 94/18318 (the disclosures of Which are incorporated herein by reference).
[0167]
Libraries based on in vitro translation include those described in International Patent Publication WO 91/05058, and Mattheakis et al. (Proc. Natl. Acad. Sci. USA 91: 9022-26 (1994)). It is not limited to. As an example of a non-peptide library, a benzodiazepine library (see, for example, Bunin et al., Proc. Natl. Acad. Sci. USA 91: 4708-12 (1994)) may be adapted for use. Peptide libraries (see, for example, Simon et al., Proc. Natl. Acad. Sci. USA 89: 9367-71 (1992)) may also be used. Another example of a library that can be used, where the amide function in the peptide has been palmitylated to generate a chemically transformed combinatorial library, is described by Ostresh et al. (Proc. Natl. Acad. USA 91: 11138-42 (1994)).
[0168]
Screening the library can be accomplished by any of a variety of commonly known methods. See, for example, the following references, which disclose the screening of peptide libraries: Palmley and Smith (Adv. Exp. Med. Biol. 251: 215-18 (1989)); Scott and Smith (1990, supra). Fowlkes et al. (BioTechniques 13: 422-28 (1992)); Oldenburg et al. (Proc. Natl. Acad. Sci. USA 89: 5393-97 (1992)); Yu et al. (Cell 76: 933-45 (1994)). (Staudt et al. (Science 241: 577-80 (1988)); Bock et al. (Nature 355: 564-66 (1992)); Turk et al. (Proc. Natl. Acad. Sci. USA 89: 6988-). Ellington et al. (Nature 355: 850-52 (1992)); U.S. Pat. Nos. 5,096,815, 5,223,409 and 5,198,346; Rebar and Pabo (Science 263: 671-73 (1994)); and International Patent Publication WO 94/18318, the disclosures of which are incorporated herein by reference.
[0169]
In certain embodiments, the screening comprises contacting a member of the library with relaxin, a relaxin analog or relaxin receptor immobilized on a solid phase, and a library of libraries that bind to the relaxin, relaxin analog or relaxin receptor. This can be done by retrieving members. Examples of such screening methods, called "panning" techniques, are described, for example, in Parmley and Smith (Gene 73: 305-18 (1988)); Fowlkes et al. (1992, supra); International Patent Publication WO 94/18318; Described by references cited therein.
[0170]
(Identification of subjects with relaxin-related abnormalities)
Relaxin nucleic acids (both sense and antisense), and fragments and analogs thereof, and anti-relaxin antibodies also have utility for identifying subjects with relaxin-related abnormalities. Such molecules can be used in assays (hybridization or immunoassays) to detect, prognosticate, diagnose or monitor various abnormalities, or affect relaxin expression or response to relaxin or relaxin analogs. Or not. Similarly, such molecules have utility for monitoring the treatment of cellular or tissue abnormalities. In particular, methods such as immunoassays involve contacting a sample from a subject with an anti-relaxin antibody under conditions that lead to immunospecific binding, and detecting any immunospecific binding of proteins by the antibody. It can be done by a process that involves detecting or measuring the amount. Detection of abnormal levels of relaxin and / or relaxin receptor polypeptide (eg, reduced, absent or elevated levels) using antibody binding to relaxin or relaxin receptor polypeptide in tissue sections or from semen I can do it. In certain embodiments, an antibody against relaxin or a relaxin receptor polypeptide can be used to assay a subject's tissue or semen for the presence of the relaxin or relaxin receptor polypeptide, wherein the abnormal level of relaxin Is an indicator of relaxin-related abnormalities. An "abnormal level" is an increase or a level that is present in a similar sample from a part of the body or a subject that does not have an abnormality, or a standard level that indicates their presence in a similar sample. Mean reduced level.
[0171]
Immunoassays that can be used include, but are not limited to: competitive and non-competitive assay systems (using techniques such as Western blot), radioimmunoassays, ELISAs (enzyme linked immunosorbent assays) ), “Sandwich” immunoassays, immunoprecipitation assays, precipitin assays, gel diffusion sedimentation reactions, immunodiffusion assays, agglutination assays, complement fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, and the like.
[0172]
Relaxin and relaxin receptor nucleic acids (both sense and antisense), including fragments and analogs thereof, can also be used in hybridization assays. Such a nucleic acid comprising or consisting of at least 8 contiguous nucleotides can be used as a hybridization probe or for polymerase chain reaction detection. Hybridization assays can be used to detect, prognose, diagnose or monitor diseases or conditions associated with aberrant relaxin or relaxin receptor expression and / or activity (as described above). In particular, the hybridization assay comprises contacting a sample comprising the polynucleotide with a nucleic acid probe capable of hybridizing to the relaxin or relaxin receptor DNA or RNA under conditions that allow hybridization to occur, and any resulting hybridization step. And detecting or measuring the hybridization.
[0173]
In certain embodiments, abnormalities associated with relaxin overexpression or underexpression may be diagnosed or suspected by detecting a decrease or increase in the level of relaxin polypeptide, relaxin RNA or relaxin functional activity. The presence can be screened or a predisposition to develop such an abnormality can be identified. In addition, overexpression of relaxin or increased relaxin functional activity results in a mutation in relaxin RNA or DNA or relaxin polypeptide (eg, translocation in relaxin nucleic acid, relaxin, respectively) that results in increased expression or activity of relaxin polypeptide. (A shortening of a gene or relaxin polypeptide, a change in nucleotide sequence or amino acid sequence relative to wild-type relaxin).
[0174]
For example, levels of relaxin polypeptide during biopsy or from semen can be detected by a tissue immunoassay; levels of relaxin RNA can be detected by a hybridization assay (eg, Northern blot or dot blot). Translocations or point mutations in relaxin or relaxin receptor nucleic acids can be performed by Southern blot, RFLP analysis, PCR using primers to detect point mutations, deletions or insertions, the sequence of relaxin genomic DNA or cDNA obtained from a sample. It can be detected by a determination or the like.
[0175]
In another embodiment, the level of relaxin or relaxin receptor mRNA or polypeptide in a sample of tissue isolated from a subject is detected or measured, wherein the elevated level indicates that the subject has relaxin Indicates having or having a predisposition to an associated tissue abnormality.
[0176]
In yet another embodiment, abnormal relaxin receptor activity in a tissue is detected or measured using any of the functional assays described above. Abnormal relaxin receptor activity includes, for example, an increase or decrease in the number of relaxin receptors on relaxin responsive cells, the presence of relaxin receptors on cells that are not normally responsive to relaxin, an increase in relaxin receptor response time. Increasing or decreasing, increasing or decreasing the binding affinity for relaxin or the relaxin receptor, or altering the dissociation constant of relaxin from the relaxin receptor, and the like can be mentioned.
[0177]
Also provided are kits for diagnostic and / or prognostic use, which comprise, in one or more containers, an agonist or antagonist of relaxin and, optionally, a labeled binding partner for the antibody. Alternatively, the antibodies can be labeled with a detection marker (eg, a chemiluminescent moiety, an enzymatic moiety, a fluorescent moiety, a radioactive moiety, etc.). Kits are also provided that include a nucleic acid probe capable of hybridizing to relaxin or relaxin receptor mRNA or DNA in one or more containers.
[0178]
In another embodiment, the kit may include, in one or more containers, a primer pair (e.g., each ranging in size from 6 to 30 nucleotides or more), wherein the primer pair comprises at least a portion of the relaxin nucleic acid. The amplification can be primed under appropriate reaction conditions such that it is amplified (see, eg, the polymerase chain reaction (see, eg, Innis et al., PCR Protocols, Academic Press, Inc., San Diego, CA (1989)). Ligase chain reaction (see, eg, EP 0 320 308), use of Qβ replicase, cyclic 5 ′ probe reaction, or other methods known in the art). The kit can optionally further comprise a predetermined amount of purified relaxin nucleic acid or relaxin receptor nucleic acid in a container, for example, for use as a standard or control.
[0179]
The following examples are merely provided as illustrations of various aspects of the invention and should not be construed as limiting the invention in any way.
[0180]
(Example 1)
The effect of inactivating one or both alleles of the mouse RLX gene was tested. The following methods and materials were followed.
[0181]
(animal)
Wild-type, heterozygous and homozygous relaxin knockout mice were obtained from the Howard Florey Institute of Experimental Physiology and Medicine (Parkville, Victoria, 3052, Australia) and bred using these programs and established. Subsequent generations of RLX + / + (wild type), RLX +/− (heterozygous) and rlx − / − (null mutant) mice were generated from RLX +/− parents. All animals are housed in a controlled environment and given access to Labdiet rodent experimental samples (Deans Animal Feed, San Bruno, CA) and water for 14 hours in the dark and 10 hours in the dark. Maintained on schedule. These experiments were approved by the Institute's Animal Experimental Ethics Committee, which adheres to the NIH Code of Practice for the care and use of laboratory animals.
[0182]
(Genotyping by PCR)
Mouse DNA was incubated in 400 μl of PCR lysis buffer containing 50 mM Tris-HCl, pH 8.0, 0.5% SDS, 0.1 M EDTA and 1 mg / ml proteinase K (Gibco BRL, Gaithersburg, MD). , 50-55 ° C overnight, by dissolving tail tissue (5-7 mm). The digested sample is then mixed with 3 M sodium acetate (40 μl), buffer saturated phenol (200 μl) and chloroform (200 μl) in a serum vacutainer tube, after which the sample is centrifuged at 3000 rpm for 10 minutes. did. The DNA (contained in the upper aqueous phase) was decanted into another microcentrifuge tube containing isopropyl alcohol (240 μl) to precipitate the DNA, after which the sample was vortexed, centrifuged and the supernatant was removed. . The remaining DNA pellet was dissolved in 40-50 μl of sterile water. For PCR, each DNA template (1 μl) was used in a 30 μl reaction mixture containing: PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl) ₂ ), 2.5 mM dNTPs, 2.5 U Taq polymerase (PGC Scientific, Gaithersburg, MD), and 150 ng of each RLX + / + and rlx − / − primers (Zhao and coworkers (Zhao Endocrinology 140: 445-53 ( The amplification protocol consists of a starting denaturation step at 94 ° C. (3 minutes) followed by 35 consecutive cycles of 94 ° C. (60 seconds), 55 ° C. (60 seconds) and 72 ° C. (90 seconds). And terminated with an additional 10 minutes of extension at 72 ° C. 15 μl of each sample was then analyzed by electrophoresis on a 2% (w / v) agarose gel and stained with ethidium bromide. Products designed from wild-type alleles Primers generated a 170 bp product from the mutant allele primer (Zhao (1999), supra).
[0183]
(Tissue collection and histology)
Male and female wild type, heterozygous and homozygous for relaxin (n> 20 in each of the six groups) were obtained at 1 week, 1 month, 2 months and 3 months of age and weighed. . RLX + / + and rlx − / − male mice were then sacrificed under carbon dioxide anesthesia for tissue collection. Male genital tracts (including testes, epididymis, prostate, seminal vesicles and associated fat) were obtained from each animal at 1 week of age (n = 10 RLX + / + male, n = 11 − / − male); 1 month of age (N = 10 RLX + / + male, n = 10 − / − male); and at 3 months of age (n = 10 RLX + / + male, n = 10 rlx − / − male). After weighing each tissue, they were placed in 10% formalin for histological analysis.
[0184]
Harvested tissue was processed from 70% alcohol to paraffin (sequential dehydration), then embedded, cut using an AO Spencer 820 microtome (4 μm sections), and poly-L-lysine coated Placed on glass slide. Serial sections from each tissue were stained with H & E (hematoxylin and eosin) and for collagen with Masson trichrome (staining kit, Richard-Allan Scientific, Kalamazoo, MI) as described by the manufacturer. . Stained slides were viewed on a Zeiss Axioplan-2 microscope, images were supplemented by a digital camera (Hamamatsu), and saved for retrieval and analysis. The images were digitally enhanced for maximum contrast and brightness using Adobe Photoshop (Adobe Systems Inc, Mountain View, CA).
[0185]
(Antibody staining of paraffin-embedded tissue sections)
Tissues from the genital tract of 1- and 3-month-old male mice are mounted on pre-coated slides and heated at 58 ° C. (about 30 minutes) to remove paraffin, then washed three times with xylene, anhydrous Washed twice with ethanol and twice with 95% ethanol, then briefly immersed in water. These samples were then stained in a humidified environment using an Immunocruz staining system utilizing horseradish peroxidase (HRP) -streptavidin complex (Santa Cruz Biotechnology Inc, Santa Cruz, CA). Tissue sections were first treated with a peroxidase blocker (to quench endogenous peroxidase activity) (5 minutes) and then pre-blocked in goat serum (20 minutes). Serial sections from each RLX + / + and rlx − / − tissue samples were then used to isolate Bax monoclonal IgG primary antibody (4 μg / ml) (Santa Cruz Biotechnology Inc), caspase-9 polyclonal IgG antibody (4.5 μg / ml). ) (Santa Cruz Biotech.) Or proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotech.) Monoclonal IgG antibody (4 μg / ml) (2 hours). Depending on the type of antibody used, either a mouse IgG control or a rabbit IgG control (Santa Cruz Biotech.) Of the tissue (2 hours) was also used in all experiments performed. Samples are washed with PBS (2 minutes), subjected to an appropriate secondary antibody (goat anti-mouse IgG or goat anti-rabbit IgG) (30 minutes), washed as above (2 minutes), and HRP-streptavidin. The conjugate was treated (30-45 minutes) and incubated (2-10 minutes) with a diaminobenzidine chromogenic substrate prepared according to the manufacturer's instructions. The slides were then washed with distilled water (2 minutes), then dehydrated from 95% alcohol to xylene, mounted, and photographed as described above.
[0186]
(Statistical analysis)
The results were analyzed using the one-ANOVA test. All data in this document are presented as mean ± SEM, with p <0.05 considered statistically significant.
[0187]
(Example 2)
(Effect of relaxin gene knockout on mouse growth)
Male and female relaxin wild-type (+ / +), heterozygous (+/-) and null mutant (-/-) mice weighed at 1 week, 1 month, 2 months and 3 months of age. The measurement was performed (n = 20 to 21 for each group). At 1 week of age, both male mice (RLX + / +: 3.87 ± 0.09 g; rlx − / −: 3.6 ± 0.13 g) and female mice (RLX + / +: 3.52 ± 0) 0.09 g; rlx − / −: 3.37 ± 0.08 g), no significant difference in mean body weight was noted. However, in mice at 1 month of age, the average body weight of both rlx − / − male (17.05 ± 0.65 g) and female (14.77 ± 0.42 g) was greater than the respective RLX wild-type matched mice. Was also significantly lower (p <0.05) (RLX + / + M: 18.92 ± 0.61 g; RLX + / + F: 16.34 ± 0.51 g). Male and female null mutant mice remained significantly smaller (p <0.05) than RLX wild-type animals at 2 months of age (RLX + / + M: 25.42 ± 0.41 g; rlx− / -M: 24.23 ± 0.42 g; RLX + / + F: 20.96 ± 0.32 g; rlx − / − F: 19.94 ± 0.22 g); however, the size difference between the two groups is It was smaller than the difference observed at one month of age. By adulthood (3 months of age), the mean body weight of rlx − / − mice (rlx − / − M: 26.57 ± 0.47 g; rlx − / − F: 22.54 ± 0.31 g) was equivalent to adult RLX + This difference was no longer significant, although still slightly less than the average body weight of RLX + / + M mice (RLX + / + M: 27.30 ± 0.32 g; RLX + / + F: 23.03 ± 0.71 g). The average body weight of RLX +/− mice was between the average body weight of RLX + / + and rlx − / − mice. No significant differences were observed between the mean body weight of RLX + / + and RLX +/− animals, nor between RLX +/− and rlx − / − mice.
[0188]
(Effect of relaxin gene knockout on male reproductive tract size)
At one week of age, no significant differences were observed in the weight or size of the male reproductive tract. This is mainly indicated by the testes (Table 1). By one month of age, the collected male genital tract was composed of testis, epididymis, prostate, seminal vesicles, vas deferens and associated fat. At one month of age, no differences were observed in the weight of the entire genital tract, but the differences in the size of testes and prostate from one-month-old null mutant mice were approximately, respectively, greater than the corresponding tissues of RLX wild-type animals. 20% and about 30% smaller. On the other hand, the size of epididymis derived from rlx − / − mice was significantly smaller (p <0.05) than epididymis obtained from RLX + / + animals. However, no difference was noted in seminal vesicle size between tissue samples collected from RLX + / + and rlx − / − mice.
[0189]
By the time the mice reached adulthood (3 months of age), significant differences (p <0.05) were observed in the overall weight of the male reproductive tract and the size of individual organs (Table 1). The weight of the genital tract in normal mice increased 248.6% from one month of age to adulthood, and represented 3.37% of the total weight. During this time, the weight of the genital tract from the male RLX null mutant mouse increased only 187.9% and represented 2.33% of the total weight. This latter finding implies that the reproductive tract of male rlx − / − mice represents a 31% decrease in% body weight. The size of testis, epididymis, prostate and seminal vesicles from rlx − / − mice were all smaller than their respective counterparts from RLX + / + mice (p <0.05).
[0190]
(Table 1.1 Weight and size of male reproductive tract from week old to adult (6.5 months))
[0191]
[Table 1]

¹ Testis and ³ The size of each prostate was measured by its area multiplied by the length of the tissue by its width. Since the size (area) of the epididymis and seminal vesicle could not be measured accurately, a close approximation of each tissue was calculated as follows: epididymis (length of tissue, epididymis, Multiplied by head width ² ); Seminal vesicles (measured by multiplying the length of each organ by the average width of the tissue) ⁴ ). ^* Indicates p <0.05.
[0192]
(Effect of relaxin gene knockout on male reproductive tract histology)
H & E & Masson trichrome staining: Male genital tract tissue sections from RLX + / + and rlx − / − mice were first observed for differences in sperm maturation, duct size / compactness (testis, epididymis) and collagen.
[0193]
Testes: At one week of age, the seminiferous tubules (compartment containing germ cells / spermatocytes, Sertoli cells and sperm maturation) of relaxin wild-type animals are smaller and more cylindrical in shape (Table 2). ) And is primarily supported by a thin layer of collagen surrounding the white membrane of each organ (the membrane covering the oval body). In contrast, tubules from testis of rlx − / − mice were larger and enlarged, but were completely surrounded and supported by collagen in the testes. This immediately suggested a difference in the internal tissues of the testes, even in the absence of relaxin, even shortly after birth. However, at one week of age, immature sperm was not detected in any of the groups of tissue sections. By one month of age, testicular tubules from RLX + / + mice were larger in size (compared to one week old tubule size), slightly less compact, and mainly contained immature sperm. In contrast, tubules from 1 month old RLX homozygous mice did not differ from the tubule size measured from 1 week old knockout mice and were less immature compared to samples from RLX + / + mice But not sperm. This indicates that relaxin null mutant mice undergo a process of delayed sperm maturation, further confirmed when compared to 3-month-old tissue sections; sections from RLX + / + mouse tissues show that predominantly mature sperm It was further suggested to include larger tubules (as compared to one month old tubule size). Conversely, sections from rlx − / − mice show slightly smaller tubules (compared to 3 month old RLX + / + tissue sections) with less mature sperm and still some immature sperm. Did not differ from the tubule size from 1 week / 1 month old rlx − / − mice). Sperm maturation levels in testis from RLX + / + mice were shown to be significantly (p <0.05) higher than the sperm maturation levels observed in testis of rlx − / − mice (grading used). See Table 2 for the scale of). It was also noted that sperm maturation levels in the testes of adult RLX knockout mice were similar to those observed in immature (1 month old) RLX wild type mouse tissue. At 1-3 months of age, the testes of normal mice remained surrounded by a thin film of collagen overlying the white membrane; however, in some tissues, a thin lining of collagen also supports the seminiferous tubules. Was observed. However, the inner layer of collagen was found to decrease with age. In rlx − / − mice, collagen detected in the middle of the tubule structure was lost due to the larger tubule size structure in the testes, but compared to that observed in tissue samples from wild-type animals. He was even more dense and solid. It is expected that the increased collagen observed in the genital tract of RLX null mutant mice at very young ages could make the tissue harder and firmer, indicating that tubule composition and sperm maturation discussed above May be related to change. Interestingly, the presence of relaxin in normal adult mice also induced a relaxation of the tubular structures of the seminiferous tubules (increased interstitial spacing) compared to structures observed from normal immature (1 month old) mice. did. In contrast, there were no differences in the organization (size / compactness) of testis-derived tubules from 1 month and 3 month old rlx − / − mice. In addition, by 1 month and up to 3 months, testicular tubules from rlx − / − mice appeared to contain an increased number of dead cells compared to tubule cells from RLX wild type animals. The increased number of dead cells found in immature tissues varied between animals but was consistently detected as mice matured with age.
[0194]
Epididymis: No significant differences in tubule size were noted in epididymis of RLX + / + and rlx − / − genital tracts at 1 and 3 months of age. However, in adults, tubule compactness was more apparent in tissues from RLX homozygous mice as compared to tissues from RLX wild-type animals. Epididymal tubules of rlx − / − mice were also supported to a greater extent by connective tissue. Tubule structures from 3-month-old RLX + / + mouse tissue loosened in shape and were only partially maintained by collagen. Conversely, tissue sections obtained from RLX knockout animals were shown to have more compact tubules very well surrounded by a thin layer of collagen. In the testes, tissue from rlx − / − mice was supported by increased concentrations of collagen at all ages observed, as compared to tissue samples from RLX + / + mice. As shown in Table 2, the density of mature spermatozoa (in the epididymal tubules) was higher in each age group at 1 month and 3 months compared to tissue sections obtained from RLX wild-type mice. , Lower in tissue sections obtained from RLX null mutant mice. However, this difference was not statistically significant. This decrease in mature sperm in the epididymis of mice lacking relaxin is probably due to the delayed sperm maturation process that occurs in the testes of these animals (Table 2).
[0195]
[Table 2]

Table 2: Tissue sections were stained with H & E and graded as follows:
^a Capillary size -1 = all capillaries are small and in annular form; 2 = most capillaries are slightly elongated compared to 1, but some smaller capillaries are observed; 3 = The capillaries are significantly stretched compared to 1, but some smaller annular capillaries and medium-sized capillaries are observed; 4 = all capillaries are significantly stretched compared to 1 and 2.
^b Capillary compactness-1 = all capillaries are loose; 2 = percentage of loose capillaries is greater than percentage of compact capillaries; 3 = percentage of compact capillaries is shape Is greater than the percentage of loose capillaries; 4 = all capillaries are compact.
^c Sperm maturity-0 = no sperm detected; 1 = only immature sperm detected; 2 = immature sperm percentage higher than mature sperm percentage; 3 = mature sperm percentage The percentage is higher than the percentage of immature sperm; 4 = only mature sperm was detected.
The values shown are the mean ± SE (n) of the grading scales used for each parameter measured. ^* Indicates p <0.05.
[0196]
[Table 3]

Table 3: Tissue sections were stained by Masson's trichrome stain and graded as follows:
^a Collagen-1 = only a thin inner layer of collagen surrounding the outer layer of the structure; 2 = a further inner layer of collagen surrounding the inner components of the tissue; 3 = a thicker inner layer of collagen surrounding the outer and inner components of the tissue; 4 = 3+ Space between tubules / components filled with collagen.
[0197]
The values shown are the mean ± SE (n) of the grading scale. ^* Indicates p <0.05.
[0198]
Prostate: At one month of age, no significant differences in prostate structure between RLX + / + and rlx − / − derived tissues were noted. However, the space between the prostate ducts from normal animals was associated with only trace amounts of collagen. In contrast, prostate ducts from rlx − / − mice were intervened by a layer of loose connective tissue. In adults (3 months), rlx − / − mice contained smaller prostates with smaller glands and ducts. Tissue tubing from RLX knockout mice was more compact and completely supported by connective tissue, whereas tubing from RLX wild-type mice was more widespread (loose in shape) and still bound by collagen. Little support. In addition, adult prostate ducts obtained from rlx − / − mice appear to have smaller epithelium (contains less cells), while the RLX + / + prostate sample ducts have larger epithelial layers / glandular tissue. Was included. These results, in addition to our initial findings on the testis and epididymis, indicate that the delayed maturation process of male reproductive tissues occurs in mice lacking a functionally active relaxin gene. And is associated with the accumulation of collagen in these tissues.
[0199]
(Detection of cell apoptosis by antibody staining)
Based on observations made from H & E staining, which show that tissue from rlx − / − mice causes reduced sperm maturation and increased cell death (testis) and includes a reduced epithelial (cell) layer (prostate) It was decided to investigate whether the observed increased cell death was the result of an apoptotic pathway.
[0200]
Bax antibody staining: Overexpression of Bax accelerates apoptotic death induced by cytokine blockade and also counteracts the death repressor activity of Bcl-2 (Krajewski et al., Am. J. Pathol. 145: 1323-). 36 (1994)). Using the Bax monoclonal antibody, the in vivo distribution of the Bax protein was evaluated in the male reproductive tract. Testis: seminiferous tubules from immature (1 month) wild type animals showed weak immunostaining for Bax (dark brown cell staining), which was mainly observed in germinal cells near the basement membrane. These findings are consistent with previous reports (Ben-Hur et al., Calcif. Tiss. Int. 53: 91-96 (1996)). In comparison, an increased number of cells stained positive for Bax in testis from rlx − / − mice. The number of Bax positive cells was counted using a haemocytometer and compared to the number of apoptotic cells observed from tissue from RLX + / + mice (13.1 ± 3.5 cells / testis (n = 6)). Showed significantly more (32.7 ± 4.8 cells / testis (n = 6)) in testis of rlx − / − mice. Increased Bax staining was consistently observed in rlx − / − tissues, but the number of tubules that stained positive for Bax varied between tissue samples, and that some tubules also did not contain Bax Was also observed. In the adult male reproductive tract, testes of adult RLX wild-type mice showed reduced staining for Bax (3.4 ± 1.4 cells / testis (n = 6)), indicating that these animals Correlates with increased levels of sperm and tissue maturation experienced. Conversely, the slower rate of tissue maturation observed in the rlx − / − mouse genital tract was attributed to a further increase in Bax staining in 3 month old RLX knockout mice (44.4 ± 8.1 cells / testis (n = 6)). Many of the cells observed appeared to be in the last stages of apoptosis, possibly showing apoptotic bodies. However, Bax staining was not associated with mature sperm cells. Epididymis: More intense Bax staining was present in the inner membrane of epithelial cells of epididymal tubules obtained from 1 month RLX + / + mouse tissues. Tubules of epididymal tubules obtained from 1 month rlx − / − mouse tissues contain relatively stronger levels of Bax staining, which is further maintained even if not increased in 3 month tissue sections. Was done. In contrast, the epididymis of adult wild-type mice contained weaker staining for Bax expression as this tissue matured with age, as compared to tissue from 1 month normal mice. Prostate: Prostate epithelial cells of normal 1 and 3 month animals showed weak positive staining for Bax. With respect to the other tissues studied, especially cells in the epithelial layer (of the prostate tract) of rlx − / − animals show increased staining for Bax in both immature (1 month) and adult (3 months) tissues. Indicated. These findings suggest that relaxin may play a new role in regulating cell apoptosis in the male genital tract, but further studies with another antibody to detect cell apoptosis were performed to confirm the effects of relaxin. Was carried out.
[0201]
Caspase-9, a central death protease, belongs to a unique family of cysteine proteases that differ in sequence, structure, and substrate specificity from the other described protease families. Members of this caspase family, which are usually involved in a cascade of proteolytic cleavage events, act by destroying certain target proteins that are important for cellular activity, thereby forming a key component of the apoptotic machinery. Functions as an ingredient. Testis: Moderate caspase-9 staining is observed in 1 month RLX + / + testes (28.1 ± 5.6 cells / testis (n = 7)), which indicates spermatogonia (spermatagonia) or sperm It appears to be primarily associated with embryonic cells within the seminiferous tubules rather than cells. For Bax, significantly (p <0.05) increased levels of caspase-9 staining were found in testis (76.6 ± 10.2 cells / testis (n = 6)) of 1 month rlx − / − mice. Although detected, severe caspase-9 staining was observed to be poorly associated with tubules, while many tubules contained little or no caspase-9 protein. With respect to age, the level of caspase-9 detected in normal immature tissue was consistently found in 3 month older tissue (26.9 ± 4 cells / testis (n = 6)) ). However, as testis size increased, the number of apoptotic cells detected indicated a smaller fraction of aged tissue. Testicular tubules from rlx − / − mice show slightly fewer apoptotic cells at 3 months (61.6 ± 9.8 cells / testis (n = 5) compared to the density of caspase-9 stained cells in tissues at 1 month. )). Despite the number of positively stained apoptotic cells from the tissues of RLX knockout mice, the remainder was significantly (p <0.05) larger than the wild-type counterpart of all ages studied. Epididymis: Caspase-9 staining was not detected from 1 month RLX + / + epididymal tubules, while traces of positive cells were observed in rlx − / − tissue sections. However, cells that stained positively were sparsely scattered and were detected in the epithelial layer of the tubules. With respect to age, no clear staining for caspase-9 was detected in 3 month old RLX + / + and rlx − / − mouse tissues. Prostate: Caspase-9 staining was not identified in RLX + / + and rlx − / − tissue prostates at all ages examined (1-3 months). Nevertheless, the results obtained initially suggest that this relaxin is involved in regulating cell apoptosis. However, further studies were needed to establish whether relaxin is involved in the cell growth pathway.
[0202]
(Detection of cell proliferation by antibody staining)
PCNA staining: Proliferating cell nuclear antigen (PCNA) antibodies were used to detect cell proliferation in the male genital tract based on the ability of DNA synthesis to associate with the nuclear region where it occurs. Testis: PCNA was detected in immature and mature tubules, but significant differences in PCNA staining were found in testis tracts from 1 and 3 month old RLX + / + and rlx − / − mice. Not detected. In immature tissues, PCNA staining is usually highly expressed in mitotically active spermatogonia, and sometimes highly in some Sertoli cells, and these stains correspond to proliferative activity. In this study, these cells were better and more evenly and constantly labeled for PCNA in adult mice, which likely both could initiate regeneration via the constant activation of these proliferating cells. Reflect the ability of the group. Epididymis: No staining for PCNA was detected in epididymis from either 1 and 3 month old RLX + / + or rlx − / − mice. These findings are consistent with the role of the epididymis acting as a reservoir for mature sperm. Prostate: Weak staining for PCNA was associated with prostate duct epithelial cells from 1 month old RLX + / + and rlx − / − mice. However, no staining for PCNA was detected in any of the 3-month-old groups. Leukocyte proliferation studies are limited to some reproductive tissues, and this accumulated finding confirms that relaxin probably plays a more influential role in regulating cell apoptosis than on cell proliferation. Was done.
[0203]
(Effect of relaxin gene knockout on histology of other body cells)
Male and female RLX wild-type (RLX + / +) and male and female RLX gene knockouts (rlx-/-) were generated from parents of RLX heterozygotes (RLX +/-) as described above. Was. Mice were weighed and sacrificed for one week, one month and / or three months for tissue recovery. The following tissues (including brain, heart, liver, kidney, thymus, spleen and male genital tract) were collected, weighed, and placed in 10% formalin for detailed histological analysis. A summary of these tissue weights is shown in Tables 4-8.
[0204]
At one week of age, male rlx − / − mice contained significantly (p <0.05) smaller liver, kidney and spleen compared to RLX + / + animals. However, these weight differences were no longer apparent at one month of age. Instead, the thymus of 1 month old male rlx − / − mice was smaller than their RLX + / + counterparts (p <0.05). This difference in weight was no longer apparent at 3 months of age. For the male reproductive tract, no significant differences were noted between the RLX + / + and rlx − / − groups at 1 week or 1 month of age. However, by 3 months of age, the genital tract of rlx − / − mice was significantly (p <0.05) smaller than that obtained from RLX + / + animals.
[0205]
No notable differences were observed between female one week old RLS + / + and rlx − / − mice. However, by one month of age, rlx − / − female mice had significantly smaller livers than their RLX + / + counterparts. By three months of age, this weight difference was no longer observed, and other significant differences in other organs were no longer noticeable.
[0206]
To determine if organ and tissue differences in RLX − / − mice (derived from RLX +/− parents) are further accentuated when rlx − / − progeny are obtained from RLX − / − parents In the meantime, RLX + / + and rlx − / − mice were obtained from the corresponding set of parents and sacrificed at 1 week, 1 month and 3 months. Brain, heart, liver, kidney, thymus, spleen, male genital tract, intestine, and lung were collected and weighed. The weights of these organs are shown in FIGS.
[0207]
It is further noted that while organ or tissue weights appear to normalize as mice age, normal weight alone is not equal to normal organs or normal tissues. This data shows that the ratio of organ (tissue) weight to body weight at any point in the growth cycle indicates a potential change in organ or tissue development or cell composition. In one example, a small liver infiltrated (altered) by fibrosis is the same weight or heavier than a larger normal liver or a liver filled with fat. Thus, the brain, heart, liver, kidney, thymus, spleen, intestine and lungs (developed in the absence of relaxin) of female and male RLX − / − mice have increased apoptosis and extracellular matrix (collagen). Provides evidence of accumulation.
[0208]
(Table 4: Tissue comparison of 1 week old mice from RLX + / + and rlx − / − parents, respectively, shown as mean ± SE (n))
[0209]
[Table 4]

(Table 5: Tissue comparison of 1 month old mice from RLX +/− parents, shown as mean ± SE (n))
[0210]
[Table 5]

(Table 6: Tissue comparison of 3 month old male mice from RLX + / + and rlx − / − parents, respectively, shown as mean ± SE (n))
[0211]
[Table 6]

(Table 7: Tissue comparison of 1 month old mice from RLX + / + and RLX − / − parents, respectively, shown as mean ± SE (n))
[0212]
[Table 7]

(Table 8: Tissue comparison of 3 month old mice from RLX + / + and rlx − / − parents, respectively, shown as mean ± SE (n))
[0213]
[Table 8]

(Effect of relaxin gene knockout on skin histology)
The role of relaxin in regulating tissue remodeling was investigated by studying skin histology in rlx-null mice (rlx-/-). These mice were offspring of rlx − / − male and female parents. Serial skin samples from the ears and back of at least 5 male and 5 female mice were stained by H & E staining and Masson's Trichrome stain and examined at each time point. Similar samples at the same time point were obtained from the same line of Rlx + / + mice. Upon histological examination of rlx null mice, the dermis was found to become thicker over time and to have increased fibrosis throughout the dermis. Skin samples from rlx null mice were normal at 1 week of age; however, by 1 month, early cutaneous fibrosis was evident. By 3 months of age there was a marked increase in dermal fibrosis, which had increased in density by 6 months of age. Findings for these skins were similar in male and female rlx null mice.
[0214]
In these mice, the epidermis was normal and hair did not change except for the initial lighter coat color in rlx null mice, which became indistinguishable from Rlx + / + mice by one month of age. Serum chemistry, hematological parameters and urine tests from rlx mice were not notable.
[0215]
These findings support previous observations that relaxin affects matrix turnover in vitro and in vivo by altering key matrix molecules, matrix-degrading enzymes and growth factors. Many stromal cells that produce interstitial collagen have relaxin receptors, whether from male or female sources. An antibody-based assay can detect serum relaxin at low picogram levels during the luteal phase of the female menstrual cycle, and these levels can be detected during pregnancy, when tissue remodeling is most evident ) To rise. In contrast, serum relaxin in males has been reported to be undetectable.
[0216]
Rlx null mice provide the first direct evidence linking relaxin to the universal alteration of collagen turnover in normal skin. These results indicate that relaxin can be produced or circulate at biologically relevant concentrations below the detected flux levels in males and females. Relaxin, in cooperation with other matrix regulatory molecules, is involved in the regular maintenance of matrix turnover. Relaxin can affect tissue remodeling, promoting angiogenesis and stimulating vasodilation. These results also indicate that the relaxin synthesis pathway, and the relaxin receptor, are associated with fibrotic conditions (eg, scleroderma).
[0219]
The preceding examples are provided to illustrate and not to limit the scope of the claimed invention. Other modifications of the present invention will be readily apparent to one skilled in the art and are encompassed by the appended claims. All publications, patents, patent applications, and other references cited therein are hereby incorporated by reference.

Claims (68)

A method of regulating apoptosis, comprising:
Administering to a subject in need thereof an effective amount of a relaxin agonist for a time sufficient to reduce apoptosis in cells expressing the relaxin receptor,
A method comprising: 2. The method of claim 1, wherein the cells are from liver, kidney, spleen, thymus, brain, heart, intestine, skin, lung, female or male genital tract. 3. The method of claim 2, wherein the male genital tract tissue is a prostate, epididymis, seminal vesicle, or testis. 3. The method of claim 2, wherein the female genital tract tissue is a uterus, cervix, pubis, or connective tissue of the lower extremity girdle. 3. The method of claim 2, wherein the number of apoptotic cells is reduced. 2. The method of claim 1, wherein tissue maturation is stimulated. 7. The method of claim 6, wherein fibrosis is reduced. 7. The method of claim 6, wherein said tissue is male genital tract tissue. 9. The method of claim 8, wherein the tissue is prostate tissue, epididymal tissue, seminal tissue, testicular tissue or semen. 10. The method of claim 9, wherein said tissue is testis tissue. 11. The method of claim 10, wherein maturation is an increase in the number of cells in a testis with poor development. 10. The method of claim 9, wherein the number of live sperm cells is increased. 2. The method of claim 1, wherein said subject has a relaxin deficiency condition. 14. The method of claim 13, wherein the relaxin deficiency condition is immature tissue, excessive collagen deposition or low sperm count. 15. The method of claim 14, wherein the immature tissue is immature male genital tract tissue. 16. The method of claim 15, wherein the immature male genital tract tissue is a testis of poor development. 2. The method of claim 1, wherein excessive collagen deposition is reduced. 2. The method of claim 1, wherein the relaxin agonist is relaxin, a relaxin analog, a small relaxin effector, or a relaxin nucleic acid. 19. The method of claim 18, wherein said relaxin is vertebrate relaxin. 20. The method of claim 19, wherein said relaxin is human relaxin. 2. The method of claim 1, wherein said administering is by infusion, injection, oral delivery, nasal delivery, pulmonary delivery, rectal delivery, transdermal delivery, intratissue delivery, or subcutaneous delivery. 22. The method of claim 21, wherein said administering is by delayed release delivery. 22. The method of claim 21, wherein said subcutaneous delivery is by infusion or injection. 22. The method of claim 21, wherein said administering is by pulmonary, subcutaneous or transdermal delivery. 2. The method of claim 1, wherein said administering comprises delivering a vector encoding said relaxin agonist. 2. The method of claim 1, wherein said vector is an expression vector, whereby said relaxin agonist is expressed in said cells. 2. The method of claim 1, wherein said administering comprises delivering a relaxin or relaxin analog nucleic acid. The method of claim 1, wherein the subject is a post-pubertal subject. The method of claim 1, wherein the subject is a prepubertal subject. A method of regulating apoptosis in a population of cells that express the relaxin receptor, comprising:
Administering to a subject in need thereof an effective amount of a relaxin antagonist for a time sufficient to increase apoptosis in the population of cells expressing the relaxin receptor;
A method comprising: 31. The method of claim 30, wherein said relaxin antagonist inhibits binding of relaxin to a relaxin receptor. 31. The method of claim 30, wherein said relaxin antagonist reduces relaxin-related tissue remodeling. 31. The method of claim 30, wherein the cell population is from heart, brain, liver, kidney, spleen, thymus, skin, female genital tract tissue, or male genital tract tissue. 34. The method of claim 33, wherein the male genital tract tissue is prostate, epididymis, seminal vesicle or testis. 35. The method of claim 34, wherein said tissue is prostate tissue. 34. The method of claim 33, wherein the male genital tract tissue is mature. 34. The method of claim 33, wherein the female genital tract tissue is uterine, cervical, pubic, or lower limb girdle connective tissue. 31. The method of claim 30, wherein said cell population comprises fibroblasts, osteoblasts, monocytes, epithelial cells or endothelial cells. 31. The method of claim 30, wherein said relaxin antagonist is a relaxin binding agent, a relaxin receptor binding agent, or a relaxin antisense nucleic acid. 40. The method of claim 39, wherein the relaxin binding agent is an anti-relaxin antibody, a soluble relaxin receptor, or a small molecule relaxin antagonist. 41. The method of claim 40, wherein the antibody is a monoclonal antibody, polyclonal antibody, single chain antibody, Fab, Fab ', F (ab') ₂ , Fv, single heavy chain, or chimeric antibody. 40. The method of claim 39, wherein the relaxin receptor binding agent is an anti-relaxin receptor antibody, a relaxin analog, or a small molecule relaxin receptor antagonist. 43. The method of claim 42, wherein said antibody is a monoclonal antibody, polyclonal antibody, single chain antibody, Fab, Fab ', F (ab') ₂ , Fv, single heavy chain, or chimeric antibody. 31. The method of claim 30, wherein said administering is by infusion, injection, oral delivery, nasal delivery, pulmonary delivery, rectal delivery, transdermal delivery, intratissue delivery or subcutaneous delivery. 47. The method of claim 44, wherein said administering is by delayed release delivery. 46. The method of claim 44, wherein the subcutaneous delivery is by infusion or injection. 46. The method of claim 44, wherein said administering is by pulmonary, subcutaneous or transdermal delivery. 31. The method of claim 30, wherein said administering comprises delivering a vector encoding said relaxin antagonist. 31. The method of claim 30, wherein said vector is an expression vector, whereby said relaxin antagonist is expressed in said cell population. 31. The method of claim 30, wherein said administering comprises delivering relaxin or a relaxin receptor antisense nucleic acid. 31. The method of claim 30, wherein the increased apoptosis reduces unwanted cell accumulation. 52. The method of claim 51, wherein said unwanted cell accumulation is hyperplasia, hypertrophy, cancer or neoplasia. Use of a therapeutically effective amount of a relaxin antagonist for the manufacture of a medicament for reducing or preventing a relaxin-related disorder. 54. The use of claim 53, wherein the relaxin-related disorder is a relaxin-responsive cell accumulation, hyperplasia, hypertrophy, cancer or neoplasia in the subject. 55. The use according to claim 53 or 54, wherein the relaxin antagonist is a relaxin binding agent, a relaxin receptor binding agent, or a relaxin antisense nucleic acid. 56. The use according to claim 55, wherein said relaxin binding agent is an anti-relaxin antibody, a soluble relaxin receptor, or a small molecule relaxin antagonist. 57. The use according to claim 56, wherein said antibody is a monoclonal antibody, polyclonal antibody, single chain antibody, Fab, F (ab ') ₂ , Fv, single heavy chain, or chimeric antibody. 56. The use according to claim 55, wherein said relaxin receptor binding agent is an anti-relaxin receptor antibody, a relaxin analog, or a small molecule relaxin receptor antagonist. 59. The use according to claim 58, wherein the antibody is a monoclonal, polyclonal, single chain, Fab, F (ab ') ₂ , Fv, single heavy chain, or chimeric antibody. 55. Use according to claim 53 or 54, wherein the relaxin antagonist inhibits the binding of relaxin to the relaxin receptor. 55. The use according to claim 53 or 54, wherein the medicament is formulated for infusion, injection, oral delivery, nasal delivery, pulmonary delivery, rectal delivery, transdermal delivery, tissue delivery or subcutaneous delivery. 62. The use of claim 61, wherein the medicament is formulated for delayed release delivery. 55. Use according to claim 53 or 54, wherein the medicament comprises a vector encoding the relaxin antagonist. 64. The use according to claim 63, wherein said vector is an expression vector, whereby said relaxin antagonist is expressed in said cell population. 65. The use according to claim 64, wherein said vector comprises relaxin or a relaxin receptor antisense nucleic acid. 55. The use according to claim 53 or 54, wherein the tissue is prostate tissue, epididymis tissue, insemination tissue or testis tissue. 55. The use according to claim 53 or 54, wherein the subject is a post-pubertal subject. 55. The use according to claim 53 or 54, wherein the subject is a pre-pubertal subject.