JP3750983B2 - Treatment for bone diseases - Google Patents

Description

【００１３】
β-ADには不斉炭素が存在するが、その不斉炭素の立体配置は特に限定されず、S-又はR-配置（α位又はβ位と呼ぶ場合もある）のいずれであってもよい。本発明で用いるβ-ADは、不斉炭素に基づく異性体として存在可能であり、立体化学的に純粋な形態の任意の異性体（光学異性体、ジアステレオ異性体など）や、任意の異性体の混合物又はラセミ体などを用いることができる。
【００１４】
また、β-AD又はその機能性誘導体は、生理学的に許容される塩の形態で本発明の薬剤に配合してもよい。さらに、遊離形態又は塩の形態のβ-ADの水和物又は溶媒和物を本発明の薬剤に有効成分として配合して用いてもよい。
【００１５】
生理学的に許容される塩には酸付加塩、塩基付加塩、及びアミノ酸付加塩などが包含される。酸付加塩としては、例えば、塩酸塩、臭化水素酸塩、硫酸塩、リン酸塩等の無機酸塩、ギ酸塩、酢酸塩、シュウ酸塩、安息香酸塩、メタンスルホン酸塩、ｐ−トルエンスルホン酸塩、マレイン酸塩、フマル酸塩、酒石酸塩、クエン酸塩、コハク酸塩、乳酸塩等の有機酸塩を挙げることができる。塩基付加塩としては、例えば、ナトリウム塩、カリウム塩、マグネシウム塩、カルシウム塩などの金属塩、アンモニウム塩、メチルアミン塩、トリエチルアミン塩などのアミン塩を用いることができる。アミノ酸付加塩としては、例えば、グリシン、フェニルアラニン、アスパラギン酸、グルタミン酸等の付加塩を用いることができる。
【００１６】
本発明の薬剤で有効成分として用いるβ−アラニル−３，４−ジヒドロキシフェニルアラニン（β-AD）の製造方法は特に限定されず、有機化学合成分野の当業者であれば、適宜合成することができ、例えば、具体的には、特開平８−３３７５９４号公報、Leem JY, et al. J Biol Chem 1996, 271, 13573-13577、並びにIto S, et al. J Med Chem 1981, 124, 673-677等に記載の製造方法に準じて、当業者ならば容易に製造することができる。
【００１７】
β-ADは、はアミノ酸の縮合合成法及び酵素処理法の両方を利用して合成することもできる。具体的には、ｔ−ブトキシカルボニル−β−アラニル−Ｎ−ヒドロキシスクシンイミドとＬ−β−３，４−ジヒドロキシフェニルアラニン（ｄｏｐａ）とをジクロロメタン、トリエタノールアミン、シアン化メチル及びジメチルフルオリドの存在下に反応させ、反応混合物に１Ｎ塩酸を添加して酸性条件下で酢酸エチルを用いて抽出処理を行う。酢酸エチル層について減圧下で濃縮して析出する結晶を回収し、溶解後、ＨＰＬＣにより精製してβ−アラニル−３，４−ジヒドロキシフェニルアラニン（β-AD）を得ることができる。また、β−アラニル−３，４−ジヒドロキシフェニルアラニン（β-AD）は、液相ペプチド合成法により調製することもできる。
【００１８】
本発明ではβ-ADの機能性誘導体を有効成分として使用することもできる。β-ADの機能性誘導体とは、β-ADに存在する１以上の官能基を適当な置換基で修飾した誘導体であって、β-ADと同等以上の破骨細胞の骨吸収抑制作用、又は破骨細胞の分化阻害作用を有する誘導体を言う。修飾される官能基としては、例えば、β-ADにおける１つのカルボキシル基、１つのアミノ基または２つの水酸基の何れか１つ以上が挙げられる。カルボキシル基の修飾の例としては、アルコールと反応させてエステル体としたもの、又はアミン化合物と反応させてアミド体としたもの等が挙げられる。また、アミノ基の修飾の例としては、カルボン酸と反応させてアミド体としたもの、アミノ基中の片方又は両方の水素原子を置換基（好ましくは低級アルキル基など）で置換したもの等が挙げられる。また、水酸基の修飾の例としては、カルボン酸と反応させてエステル体としたもの、水酸基中の水素原子を置換基（好ましくは低級アルキル基など）で置換したもの等が挙げられる。これらのβ-ADの機能性誘導体は、上記した5-S-GADの製造方法に準じて通常の有機化学合成法に従って入手することができる。
【００１９】
本発明の一つの態様によれば、β-AD、その機能性誘導体、又は生理学的に許容されるそれらの塩を有効成分として含む、骨疾患の治療及び／又は予防のための医薬が提供される。本発明の別の態様によれば、5-S-GAD、その機能性誘導体、又は生理学的に許容されるそれらの塩を有効成分として含む、破骨細胞の骨吸収作用の抑制剤並びに破骨細胞の分化阻害剤が提供される。
【００２０】
破骨細胞は、直径２０〜１００μｍの大きさを有し、約５０個ほどの核を含む大型多核細胞で、骨細胞を吸収及び除去する作用を有する。健常な生体においては、骨芽細胞による骨形成と破骨細胞による骨吸収はバランスよく保たれ、その結果、骨組織は基本的形状を変えることなく常に新生骨と置換されて健常な状態が保たれている。また、このバランスは、血中のカルシウム濃度を一定に保つなど生体の恒常性維持のためにも重要な役割を果たしている。一方、このバランスが崩れた場合、特に骨吸収量が骨形成量を上回るような場合には、骨関連の疾患のみならずその他の種々の疾患が惹き起こされることとなる。
【００２１】
破骨細胞の形成の機構は、例えば、促進因子としてインターロイキン−１や阻害因子としてインターロイキン−４等が確認されているにも拘わらず、未だ完全に解明されたと言えない。これは、生体における破骨細胞の形成も、他の生体内での種々の現象と同様に、数多くの促進因子や阻害因子が複雑に且つ密接に関わり合って制御されているためであると考えられている。
【００２２】
本発明の薬剤は、骨疾患の治療及び／又は予防のために使用することができる。骨疾患としては、骨量減少等の内因性骨疾患、及び物理的骨折等の外因性骨疾患の両方を含み、本発明の薬剤は上記骨疾患の治療及び／又は予防、あるいは上記骨疾患の治療期間の短縮のために使用することができる。内因性骨疾患としては、生体内での破骨細胞の過剰な形成及び／又は過剰な機能を伴う全ての疾患が包含される。骨疾患の具体例としては、骨粗鬆症、高カルシウム血症、骨ページェット病、破骨細胞腫、骨肉腫、関節症、慢性関節リウマチ、変形性骨炎、原発性甲状腺機能亢進症、骨減少症、骨多孔症、骨軟化症、外傷性骨折、疲労骨折、又は栄養障害、悪性腫瘍など他の疾病が原因による骨組織の脆弱化及び骨折などが挙げられるが、これらに限定されるわけではない。
【００２３】
上記の骨疾患の一例として挙げた骨粗鬆症は、骨の量的減少により骨折しやすくなる疾患である。骨の量は、骨芽細胞による骨形成と破骨細胞による骨吸収という一連の過程により維持されているが、骨形成と骨吸収は、通常、量的に平衡が保たれるように厳密にコントロールされている。しかしながら、内分泌異常および栄養障害等の原因により骨吸収が骨形成を上回ると骨粗鬆症となる。本発明の薬剤は、好ましくは、骨粗鬆症の治療及び／又は予防のために用いられる。
【００２４】
本発明の薬剤は、β−アラニル−３，４−ジヒドロキシフェニルアラニン（β-AD）、その機能性誘導体、又は生理学的に許容されるそれらの塩を有効成分として含有するものであり、インビトロ、インビボを問わず、破骨細胞の骨吸収作用の抑制、又は破骨細胞の分化阻害のために用いられる全ての形態を包含する。本発明の薬剤は、例えば、（１）破骨細胞の骨吸収作用を抑制して、所望の細胞の維持、増殖及び／又は分化を良好にするための動物細胞等の細胞培養用の培地成分として、（２）骨疾患の治療剤又は予防剤のスクリーニング用キットの構成物として、（３）骨吸収調節剤として、また、（４）骨疾患の治療及び／又は予防のために用いることができる。ここでいう骨吸収調節剤とは、生体内での破骨細胞の形成を阻害することにより骨吸収を正常な域に調節して、比較的軽微な関節痛などの体調不良を改善する薬剤及び健康食品等を包含する。
【００２５】
破骨細胞の骨吸収作用の抑制は、例えば、ピットフォーメーションアッセイと称されるアッセイ系によって評価することができる。ピットフォーメーションアッセイでは、適当な大きさの象牙にマウスやウサギなどから取り出した破骨細胞を移植する。本発明の薬剤の存在下でこの破骨細胞を培養すると、時間の経過とともに、破骨細胞の骨吸収作用に応じて骨基質が吸収され、ピットと呼ばれる小さな穴（吸収窩）が形成される。従って、染色を施し、それを顕微鏡で観察し、ピット（吸収窩）の個数、面積、形状などを測定することにより、破骨細胞の骨吸収作用を測定することができる。
【００２６】
破骨細胞の分化阻害は、例えば、以下の方法で評価することができる。先ず、破骨細胞の前駆細胞である骨髄細胞を採取し、適当な培養下で培養した後、破骨細胞分化促進因子（Osteoclast differentiation factor）と本発明の薬剤を添加してさらに一定時間培養する。一定時間培養後、ＴＲＡＰ染色を行い、多核（３核以上）の成熟破骨細胞数を測定することにより、破骨細胞の分化阻害を評価することができる。
【００２７】
本発明の薬剤としては、β−アラニル−３，４−ジヒドロキシフェニルアラニン（β-AD）、その機能性誘導体、又は生理学的に許容されるそれらの塩から選択される少なくとも１種をそのまま単独で用いてもよいが、通常は製剤学的に許容される製剤用添加物を用いて医薬組成物の形態で供給することが好ましい。本発明の薬剤は経口的又は非経口的に投与することができる。経口投与に適する薬剤としては、例えば、錠剤、顆粒剤、カプセル剤、散剤、溶液剤、懸濁剤、シロップ剤などを挙げることができ、非経口投与に適する薬剤としては、例えば、注射剤、点滴剤、坐剤、経皮吸収剤などを挙げることができるが、本発明の薬剤はこれらの製剤に限定されることはない。
【００２８】
本発明の薬剤の製造に用いられる製剤用添加物の種類は特に限定されず、当業者が適宜選択可能である。例えば、賦形剤、結合剤、滑沢剤、分散剤、懸濁剤、乳化剤、緩衝剤、抗酸化剤、防腐剤、等張化剤、pH調節剤、溶解剤、安定化剤などを用いることができ、これらの目的で使用される個々の具体的成分は当業者に周知されている。
【００２９】
本発明の薬剤はヒトを含む哺乳動物に投与することができる。本発明の薬剤の投与量は患者の年齢、性別、体重、症状、及び投与経路などの条件に応じて適宜増減されるべきであるが、一般的には、成人一日あたり1μg／ｋｇから１，０００mg／ｋｇ程度の範囲であり、好ましくは１０μｇ／ｋｇから１００mg／ｋｇ程度の範囲である。上記投与量の薬剤は、毎日投与してもよいしあるいは数日間隔で投与してもよく、例えば１〜４日毎に投与される。
以下の実施例により本発明をさらに具体的に説明するが、本発明の範囲は実施例によって限定されることはない。
【００３０】
【実施例】
実施例１：定量的吸収窩形成アッセイ（ピットフォーメーションアッセイ）を用いたβ-ADによる破骨細胞の骨吸収作用の抑制の評価
（１）全骨細胞培養物の調製
生後１１〜１２週齢のＩＣＲマウス（Charles River）をエーテル麻酔により殺処分した後、直ちに７０％エタノールに浸し消毒した。アルコール消毒した眼科バサミ及びピンセットを用いて、マウスの両足の踵に切り込みを入れ、足の付け根まで裂いた。皮膚をめくりとり、大腿部を露呈させた。眼科バサミを用いて筋肉を切り取った。関節部分を脱臼させ、大腿骨近位を胴体より分離した。分離した骨は、氷冷下、１００Ｕ／ｍｌペニシリン、１００ｍｇ／ｍｌストレプトマイシンを含むα−ＭＥＭ（Flow Laboratories）培養液（以下、培養液と略す）中に入れた。同様に、全てのマウスの両足の骨を分離した。大腿骨と脛骨の間（膝関節）をハサミで切断し、骨に付着した筋肉及び軟組織を取り除いた。
【００３１】
得られた骨を全て５％ＦＣＳを含む培養液２ｍｌを含む３５ｍｌ培養用シャーレへ移し、約５分間眼科バサミを用いて細かく切断した。５％ＦＣＳを含む培養液２ｍｌをシャーレに加え、よく懸濁した後、上清を５０ｍｌの遠沈チューブに移した。上記切断及び懸濁の操作を２回繰り返した後、２５ｍｌのピペットを用いて骨切片全てを５０ｍｌチューブに移した。細胞及び骨切片懸濁液を５％ＦＣＳを含む培養液を用いて５０ｍｌにメスアップした。２５ｍｌのピペット（Sumilon）を用いて５回懸濁した後、３分間静置し、骨切片を沈殿させた。静かに上清を取り、メッシュ（セルストレーナー７０ｉμｍ、Ｆａｌｃｏｎ）に通しながら別の５０ｍｌの遠沈チューブに移した。一部を分取し、細胞数をカウントした。１２０００ｒｐｍで５分間遠心した後、５％ＦＣＳを含む培養液を用いて懸濁し、細胞濃度を１×１０⁷細胞／ｍｌに調整した。この細胞をラット副甲状腺ホルモン１０^-8Ｍ（ｒａｔＰＴＨ、合成１〜３４残基、BACHEM）及び５％ＦＣＳを含む培養液を用いて、細胞濃度４×１０⁶細胞／ｍｌに調整し、７５ｃｍ²の培養用フラスコ（Sumilon）１本当たり１５ｍｌ入れ（６×１０⁷細胞／ボトル）、３７℃で５％ＣＯ₂インキュベーター内で７日間培養した。
【００３２】
（２）象牙切片の調製
象牙片を厚さ１５０μｍに精密低速切断機を用いて切断した。切断した象牙片を直径６ｍｍの円状に１穴パンチを用いて切り抜いた。象牙片を７０％エタノールに浸し、５分間ずつ２回超音波で処理した。象牙片を滅菌ＰＢＳ（リン酸緩衝生理食塩水）で３回、培養液で２回洗浄した後、以下の定量的吸収窩形成アッセイで使用した。
【００３３】
（３）定量的吸収窩形成アッセイ
上記（２）で調製した象牙片を９６穴プレート（Falcon）に入れ、種々の濃度（０．０１、０．１、１、１０及び１００μｇ／ｍｌ）のβ−アラニル−３，４−ジヒドロキシフェニルアラニン（β-AD）の入った培養液１００ｍｌを添加した。次いで、上記（１）で調製したラット副甲状腺ホルモン１０^-8Ｍの存在下で１週間培養した全骨細胞１×１０⁵細胞を含む培養液１００μｌを各穴に添加した。
アッセイ系を１０％ＣＯ₂インキュベーター内で３７℃で２日間培養した。培養後、象牙片上の細胞を２Ｎ水酸化ナトリウム液内でゴムへらを用いて除き、水洗し、メタノールに浸した後、象牙片を４８穴培養プレートの１穴へ入れた。クマシーブリリアントブルー２％液約０．５ｍｌを象牙片の入った各穴に入れ、冷風下で２時間乾燥させた。クマシーブリリアントブルー液が完全に乾燥したことを確認し、象牙片上の余分なクマシーブリリアントブルーを２Ｎ水酸化ナトリウム水溶液に浸し、表面を滑らかな紙上で擦することによって除去した。
顕微鏡下で形成された吸収窩の数を測定した。
【００３４】
結果を以下の表１に示す。表１中の数値は、対照（１１例）の吸収窩の平均に対する百分率で示す。即ち、表１に示した値は、各実験における吸収窩の数（測定値）を対照（１１例）の吸収窩の数（測定値）の平均値で割り、１００倍した値である。また、表１の結果を図１にグラフとして示した。
【００３５】
【表１】

【００３６】
上記表１の結果から分かるように、β-AD濃度を増加するにつれて吸収窩の数は減少している。従って、β-ADは破骨細胞の骨吸収作用を抑制する作用を有することが実証された。なお、吸収窩の数を５０％減少させるＩ₅₀は約６４μｇ／ｍｌであった。
【００３７】
実施例２：β-ADによる破骨細胞の分化阻害作用の評価
骨髄細胞は、６週齢のｄｄｙマウス（雄）の大腿骨より採取し、α−ＭＥＭ（１０％ＦＣＳ、１０％Ｍ−ＣＳＦ culture sup）培地中にて培養した。マウス骨髄細胞を２×１０⁴細胞／穴の濃度で４８穴プレートにまき、翌日１００ｎｇ／ｍｌのヒト可溶性ＯＤＦ（破骨細胞分化促進因子；Osteoclast differentiation factor）を添加し、また１、１０、５０、１００、２００及び５００μｇ／ｍｌのβ-ADの存在下または非存在下で、５％ＣＯ₂インキュベーター内で３７℃で培養した。４８時間後に培地を交換し、同一の薬物を添加し、さらに４８時間培養した後、ＴＲＡＰ染色を行い、多核（３核以上）の成熟破骨細胞数を測定した。
得られた結果を図２に示す。図２中の縦軸は、コントロール（β-ADの非存在下）の場合の多核成熟破骨細胞数に対する百分率を示す。
【００３８】
図２の結果から分かるように、β-ADの濃度が増大するにつれて、多核成熟破骨細胞数の数は減少した。従って、β-ADは破骨細胞の分化阻害作用を有することが実証された。
【００３９】
【発明の効果】
本発明の薬剤は、破骨細胞の骨吸収作用を抑制し、また破骨細胞の分化を阻害することができ、骨疾患の治療及び／又は予防のための医薬として有用である。
【図面の簡単な説明】
【図１】図１は、β-ADを用いた定量的吸収窩形成アッセイの結果を示すグラフである。
【図２】図２は、β-ADによる破骨細胞の分化阻害作用の評価の結果を示すグラフである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a medicament for the treatment and / or prevention of bone diseases, an inhibitor of osteoclastic bone resorption, and an osteoclast differentiation inhibitor. More specifically, the present invention relates to the above drug comprising β-alanyl-3,4-dihydroxyphenylalanine (β-AD) or a derivative thereof as an active ingredient.
[0002]
[Prior art]
Insects are known to produce a variety of protective molecules, such as antibacterial proteins, antibacterial proteins and lectins, in response to body damage or microbial infection (Boman, HG (1991) Cell 65, 205-207; Natori, S (1994) Antibacterial Peptides, Chiba Foundation Symposium, Vol.186, pp.123-134, Wiley, Chichester; Natori, S. (1998) in: Molecular Mechanisms of Immune Responses in Insects (Brey, PT and Hultmark, D., Eds.), Pp. 245-260, Chapman and Hall, London; and Hoffmann, JA (1995) Curr. Opin. Immunol. 7, 4-10).
[0003]
During the study of insect immunity using Sarcophaga peregrina, we isolated a new substance in the lysate of the immunized adult, which was N-β-alanyl-5-S-glutathionyl-3, Identified as 4-dihydroxyphenylalanine (5-S-GAD) (Leem JY, et al. J Biol Chem 1996, 271, 13573-13577; Natori S. Molecular Mechanisms of Immune Responses in Insects. London, Chapman & Hall, 1998, 245-260; and JP-A-8-337594). Although 5-S-GAD was first discovered as an antibacterial substance, the antibacterial activity of 5-S-GAD was inhibited by catalase that degrades H ₂ O _{2, and} thus the antibacterial activity of 5-S-GAD It was suggested that it was due to H ₂ O ₂ derived from the catechol component.
[0004]
5-S-GAD is a complex of glutathione and β-alanyl-dihydroxyphenylalanine (dopa). When insects infect bacteria, tyrosinase is activated and catalyzes 5-S-GAD synthesis from glutathione and β-alanyl-3,4-dihydroxyphenylalanine (β-AD). The hydroxyl group of 3,4-dihydroxyphenylalanine in 5-S-GAD is unstable and is easily converted to orthoquinone which produces hydrogen peroxide. The antibacterial activity of 5-S-GAD was found to be due to hydrogen peroxide derived from orthoquinone (Leem JY, et al. J Biol Chem 1996, 271, 13573-13577).
There have been several reports on the physiological activity of 5-S-GAD, including antitumor activity (JP-A-8-337594). Although there are some reports on the physiological activity of β-alanyl-3,4-dihydroxyphenylalanine (β-AD), it has not been fully elucidated.
[0005]
[Problems to be solved by the invention]
The first problem to be solved by the present invention is the effect of β-alanyl-3,4-dihydroxyphenylalanine (β-AD) on the bone resorption of osteoclasts and the effect on osteoclast differentiation. It is to elucidate.
The second problem to be solved by the present invention is a drug useful for the treatment and / or prevention of a disease associated with excessive osteoclast formation and / or excessive function in vivo such as bone disease, particularly osteoporosis. Is to provide.
[0006]
[Means for Solving the Problems]
In order to solve the above problems, the present inventors evaluated the bone resorption effect of osteoclasts by performing a pit formation assay using osteoclasts and β-AD, and β-AD was converted to osteoclasts. As a result of studying the influence on the differentiation of bone marrow, it was found that β-AD can enhance the bone resorption effect of osteoclasts and inhibit the differentiation of osteoclasts in this system. The present invention has been completed based on these findings.
[0007]
That is, according to the present invention, treatment of bone diseases comprising β-alanyl-3,4-dihydroxyphenylalanine (β-AD), a functional derivative thereof, or a physiologically acceptable salt thereof as an active ingredient and / or Alternatively, a medicament for prevention is provided.
Preferably, the bone disease is a disease accompanied by the bone resorption effect of osteoclasts or an increase in the number of cells, and particularly preferably osteoporosis.
[0008]
According to another aspect of the present invention, osteoclasts comprising β-alanyl-3,4-dihydroxyphenylalanine (β-AD), a functional derivative thereof, or a physiologically acceptable salt thereof as an active ingredient. An inhibitor of the bone resorption action of is provided.
According to another aspect of the present invention, osteoclasts comprising β-alanyl-3,4-dihydroxyphenylalanine (β-AD), a functional derivative thereof, or a physiologically acceptable salt thereof as an active ingredient. Of differentiation inhibitors.
[0009]
According to another aspect of the present invention, there is provided a method for the treatment and / or prevention of bone diseases, comprising a pharmacologically effective amount of β-AD, a functional derivative thereof, or a physiologically acceptable one thereof. Comprising the step of administering to a mammal, including a human, a salt of
A method for suppressing the bone resorption effect of osteoclasts, wherein a pharmacologically effective amount of β-AD, a functional derivative thereof, or a physiologically acceptable salt thereof is applied to mammals including humans. And a method for inhibiting osteoclast differentiation, comprising a pharmacologically effective amount of β-AD, a functional derivative thereof, or a physiologically acceptable salt thereof. There is provided a method comprising administering to a mammal, including a human.
[0010]
According to yet another aspect of the present invention, the use of β-AD, a functional derivative thereof, or a physiologically acceptable salt thereof in the manufacture of a medicament for the treatment and / or prevention of bone diseases;
The use of β-AD, functional derivatives thereof, or physiologically acceptable salts thereof in the manufacture of an inhibitor of bone resorption of osteoclasts; and β-AD in the manufacture of osteoclast differentiation inhibitors; Use of functional derivatives thereof, or physiologically acceptable salts thereof;
Is provided.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
Medicament for treatment and / or prevention of bone disease of the present invention, inhibitor of osteoclastic bone resorption, and osteoclast differentiation inhibitor (in the present specification, these are collectively referred to as agents of the present invention) Active ingredient) may be a compound selected from β-alanyl-3,4-dihydroxyphenylalanine (β-AD), a functional derivative thereof, or a physiologically acceptable salt thereof. The chemical structure of β-AD is shown below.
[0012]
[Chemical 1]

[0013]
β-AD has an asymmetric carbon, but the configuration of the asymmetric carbon is not particularly limited, and any of S- or R-configuration (sometimes referred to as α-position or β-position). Good. Β-AD used in the present invention can exist as an isomer based on an asymmetric carbon, and is stereoisomerically pure in any isomer (optical isomer, diastereoisomer, etc.) or any isomer. Mixtures of racemates or racemates can be used.
[0014]
Further, β-AD or a functional derivative thereof may be blended with the agent of the present invention in the form of a physiologically acceptable salt. Furthermore, a hydrate or solvate of β-AD in a free form or a salt form may be used as an active ingredient in the drug of the present invention.
[0015]
Physiologically acceptable salts include acid addition salts, base addition salts, amino acid addition salts and the like. Examples of the acid addition salt include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, and phosphate, formate, acetate, oxalate, benzoate, methanesulfonate, p- Examples thereof include organic acid salts such as toluenesulfonate, maleate, fumarate, tartrate, citrate, succinate and lactate. As the base addition salt, for example, metal salts such as sodium salt, potassium salt, magnesium salt and calcium salt, and amine salts such as ammonium salt, methylamine salt and triethylamine salt can be used. As the amino acid addition salt, for example, addition salts such as glycine, phenylalanine, aspartic acid, glutamic acid and the like can be used.
[0016]
The production method of β-alanyl-3,4-dihydroxyphenylalanine (β-AD) used as an active ingredient in the drug of the present invention is not particularly limited, and can be appropriately synthesized by those skilled in the field of organic chemical synthesis. For example, specifically, JP-A-8-337594, Leem JY, et al. J Biol Chem 1996, 271, 13573-13577, and Ito S, et al. J Med Chem 1981, 124, 673-677 According to the manufacturing method described in the above, it can be easily manufactured by those skilled in the art.
[0017]
β-AD can also be synthesized utilizing both amino acid condensation synthesis methods and enzyme treatment methods. Specifically, t-butoxycarbonyl-β-alanyl-N-hydroxysuccinimide and L-β-3,4-dihydroxyphenylalanine (dopa) in the presence of dichloromethane, triethanolamine, methyl cyanide and dimethyl fluoride. 1N hydrochloric acid is added to the reaction mixture and extraction is performed with ethyl acetate under acidic conditions. Crystals deposited by concentrating the ethyl acetate layer under reduced pressure are collected, dissolved, and purified by HPLC to obtain β-alanyl-3,4-dihydroxyphenylalanine (β-AD). Β-alanyl-3,4-dihydroxyphenylalanine (β-AD) can also be prepared by a liquid phase peptide synthesis method.
[0018]
In the present invention, a functional derivative of β-AD can also be used as an active ingredient. A functional derivative of β-AD is a derivative obtained by modifying one or more functional groups present in β-AD with an appropriate substituent, and has an osteoclast bone resorption inhibitory activity equal to or higher than that of β-AD. Or a derivative having an osteoclast differentiation inhibitory action. Examples of the functional group to be modified include one or more of one carboxyl group, one amino group, and two hydroxyl groups in β-AD. Examples of the modification of the carboxyl group include those obtained by reacting with an alcohol to give an ester, or those obtained by reacting with an amine compound to give an amide. Examples of modification of the amino group include those obtained by reacting with a carboxylic acid to form an amide, and those obtained by substituting one or both hydrogen atoms in the amino group with a substituent (preferably a lower alkyl group). Can be mentioned. Examples of modification of the hydroxyl group include those obtained by reacting with a carboxylic acid to form an ester, and those obtained by substituting a hydrogen atom in the hydroxyl group with a substituent (preferably a lower alkyl group). These functional derivatives of β-AD can be obtained according to a general organic chemical synthesis method in accordance with the above-described method for producing 5-S-GAD.
[0019]
According to one aspect of the present invention, there is provided a medicament for the treatment and / or prevention of bone diseases comprising β-AD, a functional derivative thereof, or a physiologically acceptable salt thereof as an active ingredient. The According to another aspect of the present invention, an inhibitor of osteoclastic bone resorption and an osteoclast comprising 5-S-GAD, a functional derivative thereof, or a physiologically acceptable salt thereof as an active ingredient. Cell differentiation inhibitors are provided.
[0020]
Osteoclasts are large multinucleated cells having a diameter of 20 to 100 μm and including about 50 nuclei, and have an action of absorbing and removing bone cells. In a healthy living body, bone formation by osteoblasts and bone resorption by osteoclasts are maintained in a balanced manner, and as a result, bone tissue is always replaced with new bone without changing its basic shape and remains healthy. I'm leaning. This balance also plays an important role in maintaining the homeostasis of the living body, such as keeping the calcium concentration in the blood constant. On the other hand, when this balance is lost, especially when the amount of bone resorption exceeds the amount of bone formation, not only bone-related diseases but also various other diseases are caused.
[0021]
The mechanism of osteoclast formation, for example, has not yet been fully elucidated despite the fact that interleukin-1 as an promoting factor and interleukin-4 as an inhibiting factor have been confirmed. This is thought to be because the formation of osteoclasts in the living body is controlled by a number of intricate and closely related factors, as well as various other in vivo phenomena. It has been.
[0022]
The agent of the present invention can be used for treatment and / or prevention of bone diseases. Bone diseases include both endogenous bone diseases such as bone loss and exogenous bone diseases such as physical fractures. The agent of the present invention can treat and / or prevent the above bone diseases, or prevent the above bone diseases. Can be used to shorten the treatment period. Endogenous bone diseases include all diseases involving excessive formation of osteoclasts and / or excessive function in vivo. Specific examples of bone diseases include osteoporosis, hypercalcemia, Paget's disease, osteoclastoma, osteosarcoma, arthropathy, rheumatoid arthritis, osteoarthritis, primary hyperthyroidism, osteopenia Bone bone weakness and fractures due to other diseases such as osteoporosis, osteomalacia, traumatic fracture, fatigue fracture, or nutritional disorders, malignant tumors, but are not limited to these .
[0023]
The osteoporosis mentioned as an example of said bone disease is a disease which becomes easy to fracture by the quantitative reduction | decrease of a bone. The amount of bone is maintained by a series of processes: bone formation by osteoblasts and bone resorption by osteoclasts, but bone formation and bone resorption are usually strictly followed to be quantitatively balanced. Controlled. However, osteoporosis occurs when bone resorption exceeds bone formation due to endocrine abnormalities and nutritional disorders. The medicament of the present invention is preferably used for the treatment and / or prevention of osteoporosis.
[0024]
The drug of the present invention contains β-alanyl-3,4-dihydroxyphenylalanine (β-AD), a functional derivative thereof, or a physiologically acceptable salt thereof as an active ingredient, and is used in vitro and in vivo. Regardless of the above, all forms used for suppressing osteoclastic bone resorption or inhibiting osteoclast differentiation are included. The drug of the present invention includes, for example, (1) a medium component for cell culture such as animal cells for suppressing the bone resorption effect of osteoclasts and improving the maintenance, proliferation and / or differentiation of desired cells. (2) as a component of a screening kit for a therapeutic or prophylactic agent for bone diseases, (3) as a bone resorption regulator, and (4) used for the treatment and / or prevention of bone diseases. it can. The term “bone resorption regulator” as used herein refers to a drug that adjusts bone resorption to a normal range by inhibiting the formation of osteoclasts in vivo, and improves relatively poor physical condition such as joint pain, Includes health foods.
[0025]
Inhibition of the bone resorption effect of osteoclasts can be evaluated by, for example, an assay system called a pit formation assay. In the pit formation assay, osteoclasts taken from mice or rabbits are transplanted into ivory of an appropriate size. When this osteoclast is cultured in the presence of the agent of the present invention, the bone matrix is absorbed according to the bone resorption action of the osteoclast with the passage of time, and a small hole (absorption fossa) called a pit is formed. . Therefore, the bone resorption effect of osteoclasts can be measured by applying staining, observing it with a microscope, and measuring the number, area, shape, etc. of pits (absorption pits).
[0026]
Inhibition of osteoclast differentiation can be evaluated, for example, by the following method. First, bone marrow cells that are progenitor cells of osteoclasts are collected and cultured in an appropriate culture, and then osteoclast differentiation promoting factor (Osteoclast differentiation factor) and the agent of the present invention are added and further cultured for a certain period of time. . After culturing for a certain period of time, TRAP staining is performed, and the number of mature osteoclasts with multiple nuclei (3 nuclei or more) is measured to evaluate osteoclast differentiation inhibition.
[0027]
As the drug of the present invention, at least one selected from β-alanyl-3,4-dihydroxyphenylalanine (β-AD), a functional derivative thereof, or a physiologically acceptable salt thereof is used alone as it is. However, it is usually preferable to supply the pharmaceutical composition in the form of a pharmaceutical composition using pharmaceutically acceptable pharmaceutical additives. The agent of the present invention can be administered orally or parenterally. Examples of drugs suitable for oral administration include tablets, granules, capsules, powders, solutions, suspensions, syrups, etc. Examples of drugs suitable for parenteral administration include injections, Examples thereof include drops, suppositories, and transdermal absorption agents, but the drug of the present invention is not limited to these preparations.
[0028]
There are no particular limitations on the type of formulation additive used in the production of the drug of the present invention, and those skilled in the art can appropriately select it. For example, excipients, binders, lubricants, dispersants, suspending agents, emulsifiers, buffers, antioxidants, preservatives, isotonic agents, pH adjusters, solubilizers, stabilizers, etc. are used. The specific components used for these purposes are well known to those skilled in the art.
[0029]
The agent of the present invention can be administered to mammals including humans. The dose of the drug of the present invention should be appropriately increased or decreased depending on conditions such as the age, sex, weight, symptoms, and administration route of the patient. Generally, the dose is 1 μg / kg to 1 per day for an adult. , 000 mg / kg, preferably 10 μg / kg to 100 mg / kg. The above dose of drug may be administered daily or at intervals of several days, for example, every 1 to 4 days.
The following examples further illustrate the present invention, but the scope of the present invention is not limited by the examples.
[0030]
【Example】
Example 1: Evaluation of inhibition of osteoclastic bone resorption by β-AD using quantitative resorption pit formation assay (pit formation assay) (1) Preparation of whole bone cell culture After ICR mice (Charles River) were killed by ether anesthesia, they were immediately immersed in 70% ethanol for disinfection. Using an alcohol-sterilized ophthalmic scissors and tweezers, a cut was made in the heel of both feet of the mouse, and it was split to the base of the foot. The skin was removed and the thigh was exposed. Muscles were cut out using ophthalmic scissors. The joint was dislocated and the proximal femur was separated from the trunk. The separated bone was placed in an α-MEM (Flow Laboratories) culture solution (hereinafter abbreviated as a culture solution) containing 100 U / ml penicillin and 100 mg / ml streptomycin under ice cooling. Similarly, the bones of both feet of all mice were separated. The area between the femur and the tibia (knee joint) was cut with scissors to remove muscles and soft tissues attached to the bone.
[0031]
All of the obtained bones were transferred to a 35 ml culture dish containing 2 ml of a culture solution containing 5% FCS, and finely cut with an ophthalmic scissors for about 5 minutes. 2 ml of a culture solution containing 5% FCS was added to the petri dish and suspended well, and then the supernatant was transferred to a 50 ml centrifuge tube. After the above cutting and suspending operations were repeated twice, all bone sections were transferred to 50 ml tubes using a 25 ml pipette. The cell and bone slice suspension was diluted to 50 ml using a culture solution containing 5% FCS. After suspending 5 times using a 25 ml pipette (Sumilon), the mixture was allowed to stand for 3 minutes to precipitate a bone section. The supernatant was gently removed and transferred to another 50 ml centrifuge tube while passing through a mesh (cell strainer 70 iμm, Falcon). A part was taken and the number of cells was counted. After centrifugation at 12000 rpm for 5 minutes, the suspension was suspended using a culture solution containing 5% FCS, and the cell concentration was adjusted to 1 × 10 ⁷ cells / ml. The cells were adjusted to a cell concentration of 4 × 10 ⁶ cells / ml using a culture solution containing rat parathyroid hormone 10 ⁻⁸ M (ratPTH, synthetic 1 to 34 residues, BACHEM) and 5% FCS, and 75 cm ² 15 ml per culture flask (Sumilon) (6 × 10 ⁷ cells / bottle) was cultured at 37 ° C. in a 5% CO ₂ incubator for 7 days.
[0032]
(2) Preparation of ivory slice An ivory piece was cut to a thickness of 150 μm using a precision low-speed cutter. The cut ivory piece was cut out into a circle with a diameter of 6 mm using a one-hole punch. The ivory pieces were immersed in 70% ethanol and treated with ultrasonic waves twice for 5 minutes. The ivory pieces were washed 3 times with sterile PBS (phosphate buffered saline) and twice with the culture solution, and then used in the following quantitative absorption fossa formation assay.
[0033]
(3) Quantitative Absorption Cavity Formation Assay The ivory pieces prepared in (2) above are placed in a 96-well plate (Falcon) and various concentrations (0.01, 0.1, 1, 10 and 100 μg / ml) of β -100 ml of a culture solution containing alanyl-3,4-dihydroxyphenylalanine (β-AD) was added. Next, 100 μl of a culture solution containing 1 × 10 ⁵ whole bone cells cultured for 1 week in the presence of rat parathyroid hormone 10 ⁻⁸ M prepared in (1) above was added to each well.
The assay system was cultured for 2 days at 37 ° C. in a 10% CO ₂ incubator. After culturing, the cells on the ivory pieces were removed with a rubber spatula in 2N sodium hydroxide solution, washed with water, immersed in methanol, and the ivory pieces were put into one hole of a 48-well culture plate. About 0.5 ml of Coomassie Brilliant Blue 2% solution was placed in each hole containing ivory pieces and dried for 2 hours under cold air. After confirming that the Coomassie brilliant blue solution was completely dried, excess Coomassie brilliant blue on the ivory pieces was soaked in 2N aqueous sodium hydroxide solution and removed by rubbing the surface on smooth paper.
The number of absorption fossa formed under a microscope was measured.
[0034]
The results are shown in Table 1 below. The numerical values in Table 1 are shown as a percentage of the average of the absorption fossa of the control (11 cases). That is, the values shown in Table 1 are values obtained by dividing the number of absorption pits (measured value) in each experiment by the average value of the number of absorption pits (measured value) of the control (11 cases) and multiplying by 100. The results of Table 1 are shown as a graph in FIG.
[0035]
[Table 1]

[0036]
As can be seen from the results in Table 1 above, the number of resorption pits decreases with increasing β-AD concentration. Therefore, it was demonstrated that β-AD has an action of suppressing the bone resorption action of osteoclasts. In addition, I _{50 which} reduces the number of absorption fossa by 50% was about 64 μg / ml.
[0037]
Example 2 Evaluation of Osteoclast Differentiation Inhibitory Action by β-AD Bone marrow cells were collected from the femur of 6-week-old ddy mice (male) and α-MEM (10% FCS, 10% M-CSF). culture sup) was cultured in a medium. Mouse bone marrow cells are seeded in 48-well plates at a concentration of 2 × 10 ⁴ cells / well, and the next day 100 ng / ml human soluble ODF (Osteoclast differentiation factor) is added, and 1, 10, 50 Incubated at 37 ° C. in a 5% CO ₂ incubator in the presence or absence of 100, 200 and 500 μg / ml β-AD. After 48 hours, the medium was changed, and the same drug was added. After further incubation for 48 hours, TRAP staining was performed, and the number of multinucleated (three or more nuclei) mature osteoclasts was measured.
The obtained results are shown in FIG. The vertical axis | shaft in FIG. 2 shows the percentage with respect to the number of multinucleated mature osteoclasts in the case of control (in the absence of β-AD).
[0038]
As can be seen from the results of FIG. 2, the number of multinucleated mature osteoclasts decreased as the concentration of β-AD increased. Therefore, it was demonstrated that β-AD has an osteoclast differentiation inhibitory action.
[0039]
【The invention's effect】
The agent of the present invention can suppress the bone resorption effect of osteoclasts and inhibit osteoclast differentiation, and is useful as a medicament for the treatment and / or prevention of bone diseases.
[Brief description of the drawings]
FIG. 1 is a graph showing the results of a quantitative absorption cavity formation assay using β-AD.
FIG. 2 is a graph showing the results of evaluation of osteoclast differentiation inhibitory action by β-AD.

Claims (5)

  A medicament for the treatment and / or prevention of bone diseases comprising β-alanyl-3,4-dihydroxyphenylalanine (β-AD) or a physiologically acceptable salt thereof as an active ingredient.   The medicament according to claim 1, wherein the bone disease is a disease accompanied by bone resorption of osteoclasts or an increase in the number of cells.   The medicament according to claim 1 or 2, wherein the bone disease is osteoporosis.   An inhibitor of osteoclastic bone resorption, comprising β-alanyl-3,4-dihydroxyphenylalanine (β-AD) or a physiologically acceptable salt thereof as an active ingredient.   An osteoclast differentiation inhibitor containing β-alanyl-3,4-dihydroxyphenylalanine (β-AD) or a physiologically acceptable salt thereof as an active ingredient.

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