JP6262723B2 - オキシステロールアナログoxy133は、骨発生及びヘッジホッグシグナル伝達を誘導し、脂肪生成を阻害する - Google Patents
オキシステロールアナログoxy133は、骨発生及びヘッジホッグシグナル伝達を誘導し、脂肪生成を阻害する Download PDFInfo
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Description
本発明者らは、種々の臨床的使用に十分適した造骨性オキシステロールOxy133を本明細書で同定し、インビトロで造骨性分化を促進し、ラットモデルにおいてインビボで脊椎固定を促進するその能力を記載する。合成及び試験した多数のオキシステロールアナログのうち、Oxy133は、予想外に特に有効であり、合成が容易であった。Oxy133は、C3H10T1/2マウス胚性線維芽細胞において、造骨性マーカーRunx2、オステリックス(OSX)、アルカリホスファターゼ(ALP)、骨シアロタンパク質(BSP)及びオステオカルシン(OCN)の顕著な発現を誘導した。8X−GliルシフェラーゼレポーターのOxy133誘導性の活性化、スムーズンド(Smoothened)に対するその直接的結合、及びヘッジホッグ(Hh)経路阻害剤シクロパミンによるOxy133誘導性の造骨性効果の阻害は、Oxy133に対する造骨性応答を媒介することにおけるHh経路の役割を実証した。さらに、Oxy133は、OSX、BSP及びOCNの発現を誘導し、初代ヒト間葉系幹細胞における強い石灰化を刺激した。固定部位における、Oxy133で処置した動物におけるインビボの両側脊椎固定が、たった4週間後にX線で観察され、骨形成タンパク質−2(BMP2,bone morphogenetic protein-2)と等しい効率で、8週間後に手動評価、micro−CT及び組織学で確認された。しかし、BMP2とは異なり、Oxy133は、固定塊において脂肪生成を誘導せず、より高いBV/TV比及びより小さい海綿分離によって明らかな、より高密度の骨の形成を生じた。従って、Oxy133は、例えば脊椎固定、骨折修復、骨再生/組織操作適用、歯科インプラントのための顎における骨密度の増強、骨粗鬆症などを含む、骨形成の局在化刺激から利益を得る状態を処置するために有用である。
1.Oxy133は、例えばコラーゲンIであるがこれに限定されない適合性の分子から構成される足場を使用して、局在化骨形成を刺激するために身体中に局所的に送達される造骨性薬剤として使用され、この足場は、Oxy133を吸収し、次いで身体の内側に配置される。例えば、脊椎固定、仮関節及び非癒合固定における、例えば、Oxy133を含み、2又は3以上の椎骨の固定が示される横突起間又は椎間板中に配置され得る足場。他の実施形態では、Oxy133を含む足場は、骨形成及び骨折の治癒を刺激するために骨折した骨中に配置され;Oxy133による骨再生が示される頭蓋冠若しくは顎顔面の骨欠損などの骨欠損に配置され;又は歯科インプラントなどの歯科手順の前に骨を再生させる手段として、骨形成を刺激するために顎の骨中に配置される。
[実施例]
細胞培養及び試薬
マウス多分化能骨髄間質細胞(MSC)株M2−10B4(M2)及び胚性線維芽細胞細胞株C3H10T1/2(C3H)を、American Type Culture Collection(Rockville、MD)から購入し、本発明者らが以前に報告したように培養した(Kim W-K, Meliton V, Amantea CM, Hahn TJ, Parhami F 2007 20(S)-hydroxycholesterol inhibits PPARgamma expression and adipogenic differentiation of bone marrow stromal cells through a Hedgehog-dependent mechanism. J Bone Miner Res 22:1711-9、Johnson JS, Meliton V, Kim WK, Lee K-B, Wang JC, Nguyen K, Yoo D, Jung ME, Atti E, Tetradis S, Pereira RC, Magyar C, Nargizyan T, Hahn TJ, Farouz F, Thies S, Parhami F 2011 Novel oxysterols have pro-osteogenic and anti-adipogenic effects in vitro and induce spinal fusion in vivo. J Cell Biochem 112:1673-84)。造骨性分化を誘導するための処置を、5%胎仔ウシ血清、50μg/mlアスコルベート及び3mM β−グリセロホスフェート(βGP,β-glycerophosphate)を含む、M2細胞のためにはRPMI中で及びC3H細胞のためにはDMEM(分化培地)中で実施した。シクロパミンは、EMD Biosciences, Inc.社(La Jolla、CA)から購入した。初代ヒト間葉系幹細胞(HMSC,human mesenchymal stem cell)を、Lonza社(Walkersville、MD)から購入し、StemCell Technologies社(Vancouver、Canada)製の増殖培地中で、製造業者の指示に従って培養及び継代した。HMSCの造骨性分化を、抗生物質並びに10%熱不活化FBS、10−8Mデキサメタゾン、10mM βGP及び0.2mMアスコルベートを含むDMEM低グルコース中で細胞を処置することによって誘導した。
全細胞抽出物に対するアルカリホスファターゼ(ALP)活性アッセイ(Kha HT, Basseri B, Shouhed D, Richardson J, Tetradis S, Hahn TJ, Parhami F 2004 Oxysterols regulate differentiation of mesenchymal stem cells: pro-bone and anti-fat. J Bone Miner Res 19:830-40、Kim W-K, Meliton V, Amantea CM, Hahn TJ, Parhami F 2007 20(S)-hydroxycholesterol inhibits PPARgamma expression and adipogenic differentiation of bone marrow stromal cells through a Hedgehog-dependent mechanism. J Bone Miner Res 22:1711-9)、及び石灰化についての細胞単層のvon Kossa染色(Parhami F, Morrow AD, Balucan J, Leitinger N, Watson AD, Tintut Y, Berliner JA, Demer LL 1997 Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible explanation for the paradox of arterial calcification in osteoporotic patients. Arterioscler Thromb Vasc Biol 17:680-7)を、以前に記載されたように実施した。
総RNAを、製造業者の指示に従って、Ambion, Inc.社(Austin、TX)製のRNA単離Trizol試薬を用いて抽出した。RNA(1μg)を、Bio-Rad社(Hercules、CA)製の逆転写酵素を使用して逆転写して、一本鎖cDNAを作製した。Q−RT−PCR反応を、iQ SYBR Green Supermix及びiCycler RT-PCR Detection System(Bio-Rad社)を使用して実施した。マウス遺伝子Gli−1、パッチト1(Ptch1,Patched1)、アルカリホスファターゼ(ALP)の骨−肝臓−腎臓アイソザイム、骨シアロタンパク質(BSP)、Runx2、オステリックス(OSX)、オステオカルシン(OCN)及びGAPDHのためのプライマー配列を、以前に記載されたように使用した(Kim W-K, Meliton V, Amantea CM, Hahn TJ, Parhami F 2007 20(S)-hydroxycholesterol inhibits PPARgamma expression and adipogenic differentiation of bone marrow stromal cells through a Hedgehog-dependent mechanism. J Bone Miner Res 22:1711-9)。ヒトプライマー配列は以下であった:GAPDH 5'-CCT CAA GAT CAT CAG CAA TGC CTC CT(配列番号1)及び3'-GGT CAT GAG TCC TTC CAC GAT ACC AA(配列番号2)、BSP 5'- AGA AGA GGA GGA GGA AGA AGA GG(配列番号3)及び3' - CAG TGT TGT AGC AGA AAG TGT GG(配列番号4)、OSX 5' - GCG GCA AGA GGT TCA CTC GTT CG(配列番号5)及び3' - CAG GTC TGC GAA ACT TCT TAG AT(配列番号6);相対的発現レベルを、以前に記載されたように2ΔΔCT法を使用して計算した(Johnson JS, Meliton V, Kim WK, Lee K-B, Wang JC, Nguyen K, Yoo D, Jung ME, Atti E, Tetradis S, Pereira RC, Magyar C, Nargizyan T, Hahn TJ, Farouz F, Thies S, Parhami F 2011 Novel oxysterols have pro-osteogenic and anti-adipogenic effects in vitro and induce spinal fusion in vivo. J Cell Biochem 112:1673-84)。
24ウェルプレート中の70%コンフルエンスにおける細胞に、本発明者らが以前に記載したように、Gli依存的ホタルルシフェラーゼ及びウミシイタケ(Renilla)ルシフェラーゼベクターを一過的にトランスフェクトした(Dwyer JR, Sever N, Carlson M, Nelson SF, Beachy PA, Parhami F 2007 Oxysterols are novel activators of the Hedgehog signaling pathway in pluripotent mesenchymal cells. J Biol Chem 282:8959-68、Kim W-K, Meliton V, Bourquard N, Hahn TJ, Parhami F 2010 Hedgehog signaling and osteogenic differentiation in multipotent bone marrow stromal cells are inhibited by oxidative stress. J Cell Biochem 111:1199-209)。FuGENE 6トランスフェクション試薬(Roche Applied Science社、Indianapolis、IN)を、ヌクレアーゼを含まない水と3:1の比で使用し、ウェル当たりの総DNAは、500ngを超えなかった。ルシフェラーゼ活性を、48時間にわたって細胞を処置した後に、製造業者の指示に従ってDual Luciferase Reporter Assay System(Promega Corporation社、Madison、WI)を使用して評価した。
材料を、商業的供給者から取得し、さらなる精製なしに使用した。空気又は湿気感受性の反応を、オーブン乾燥したガラス器具及び標準的なシリンジ/隔壁技術を使用して、アルゴン雰囲気下で実施した。これらの反応を、UV光(254nm)の下、シリカゲルTLCプレート上でモニタリングし、その後Hanessian染色溶液で可視化した。カラムクロマトグラフィーを、シリカゲル60上で実施した。1H NMRスペクトルを、CDCl3中で測定した。得られたデータは、内部標準(TMS、0.0ppm)からのppm:化学シフト(多重度、積分、カップリング定数Hz)で、以下のように報告される。合成プロトコル並びに中間体及び最終生成物の特徴付けの段階的な詳細な記載は、補足材料において提供される。
オキシステロールとスムーズンド(Smo,Smoothened)との相互作用を、本発明者らが以前に報告したように試験した(Nachtergaele S, Mydock LK, Krishnan K, Rammohan J, Schlesinger PH, Covey DF, Rohatgi R 2012 Oxysterols are allosteric activators of the oncoprotein Smoothened. Nat Chem Biol 8:211-20)。Smo−/−:YFP−Smo細胞(Rohatgi R, Milenkovic L, Corcoran RB, Scott MP 2009 Hedgehog signal transduction by Smoothened: pharmacologic evidence for a 2-step activation process. Proc Natl Acad Sci USA 106:3196-201)を、低張SEAT緩衝液(250mMスクロース、1mM EDTA、10mM酢酸、10mMトリエタノールアミン、10mg/mLロイペプチン−ペプスタチン−キモスタチン(LPC,leupeptin-pepstatin-chymostatin)プロテアーゼ阻害剤ミックス及びSigmaFast EDTA-Freeプロテアーゼカクテル)中で溶解させた。遠心分離(500×g、5分)による核の除去の後、膜を、95,000×gで30分間の遠心分離によってペレット化した。膜を、n−ドデシル−b−D−マルトピラノシド(DDM,n-dodecyl-b-D-maltopyranoside)抽出緩衝液(50mM Tris pH7.4、500mM NaCl、10%v/vグリセロール、0.1%w/v DDM及びSigmaFast EDTA-Freeプロテアーゼカクテル)中で4℃で4時間抽出し、その後、遠心分離(100,000×g、30分)によって不溶性材料を除去した。この膜抽出物を、50uMの遊離20(S)又はOxy133若しくはOxy16(Hhシグナル伝達を活性化しないオキシステロールアナログ;Parhami et al.の未公開の観察)と共に4℃で1時間インキュベートし、その後、nat−20(S)−yneにカップリングさせた磁気ビーズ又はコントロール磁気ビーズを添加した(Nachtergaele S, Mydock LK, Krishnan K, Rammohan J, Schlesinger PH, Covey DF, Rohatgi R 2012 Oxysterols are allosteric activators of the oncoprotein Smoothened. Nat Chem Biol 8:211-20)。結合反応を、4℃で一晩インキュベートした。全ての実験において、溶媒の量を、各試料において注意深く等しくした。徹底的な洗浄の後、ビーズ上に捕捉されたタンパク質を、還元性SDS試料緩衝液で溶出させた。これらの溶出液中のYFP−Smoの存在を、抗GFP抗体(Novus社、NB600-308、1:5000)を用いた定量的免疫ブロッティング及び赤外線画像化(Li-Cor Odysseyシステム)によって決定した。
38匹の8週齢雄性Lewisラットを、Charles River Laboratories社(Wilmington,MA)から購入し、UCLA研究対象保護局(UCLA Office of Protection of Research Subjects)によって示された規制に従って、UCLA生態動物園において維持及び収容した。この研究は、UCLA動物研究委員会(ARC,Animal Research Committee)によって承認されたプロトコルの下で実施した。全ての動物を、脊椎固定手順の8週間後に、標準的なCO2チャンバを使用して安楽死させ、その脊椎を切り出し、40%エチルアルコール中で貯蔵した。
動物に、手術前に30分間徐放性(sustained release)ブプレノルフィンを予め投薬し、酸素中で投与した2%イソフルラン(1L/分)で麻酔した。手術部位を剃毛し、Betadine及び70%エタノールで消毒した。L4〜L5における後外側横突間突起脊椎固定を、以前の研究と同様に実施した(Alanay A, Chen C, Lee S, Murray SS, Brochmann EJ, Miyazaki M, Napoli A, Wang JC 2008 The adjunctive effect of a binding peptide on bone morphogenetic protein enhanced bone healing in a rodent model of spinal fusion. Spine 33:1709-13、Miyazaki M, Sugiyama O, Tow B, Zou J, Morishita Y, Wei F, Napoli A, Sintuu C, Lieberman JR, Wang JC 2008 The effects of lentiviral gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats. J Spinal Disord Tech 21:372-9)。L6椎体を、目印として腸骨稜を使用して同定した。4cmの縦方向正中切開を、L4〜L5にわたる皮膚及び皮下組織を介して腰背筋膜に至るまで形成した。次いで、2cmの縦方向正中傍(paramedial)切開を、傍脊柱筋群において両側で形成して、L4〜L5の横突起を露出させ、これを高速バールで除皮質した。次いで、この手術部位を、無菌食塩水で洗浄し、ジメチルスルホキシド(DMSO)コントロール、rhBMP−2又はOxy133を含む5mm×5mm×13mm片のコラーゲンスポンジ(Helistat, Integra Life Sciences社)を両側に配置し、各インプラントは横突起にわたった。次いで、これらのインプラントを、覆っている傍脊柱筋群で被覆し、腰背筋膜及び皮膚を、4〜0のProlene縫合糸(Ethicon, Inc.社、Somerville、NJ)で閉鎖した。動物を、手術の直後に、自由裁量で歩き回らせ、摂食させ、飲水させた。
腰椎の前後方向X線写真を、Faxitron LX60キャビネットX線写真システムを使用して、手術の4週間後、6週間後及び8週間後に各動物について撮影し、以下の標準化尺度を使用して2人の独立した観察者が盲検で評価した:0、固定なし;1、片側固定;及び2、完全な両側固定。これらの観察者からのスコアを一緒に加算し、4のスコアのみを完全な固定とみなした。
手術の8週間後、動物を安楽死させ、脊椎を外科的に取り出し、レベル間の動きについて2人の盲検の独立した観察者が評価した。いずれかの側の椎間又は横突起間で動きが観察された場合、偽関節を記録した。動きが両側で観察されなかった場合、完全な固定を記録した。脊椎を、固定された又は固定されていないのいずれかとしてスコア付けした。完全な固定とみなすには、満場一致の同意が必要であった。
取り出された各脊椎を、20μmのボクセル等方性分解能並びに55kVp及び181mAのX線エネルギーでSkyScan 1172スキャナ(SkyScan社、Belgium)を使用して、高分解能マイクロコンピュータ断層撮影(micro−CT,micro-computed tomography)によって分析して、本発明者らが以前に報告したように、固定率をさらに評価し、固定塊を観察した(Johnson JS, Meliton V, Kim WK, Lee K-B, Wang JC, Nguyen K, Yoo D, Jung ME, Atti E, Tetradis S, Pereira RC, Magyar C, Nargizyan T, Hahn TJ, Farouz F, Thies S, Parhami F 2011 Novel oxysterols have pro-osteogenic and anti-adipogenic effects in vitro and induce spinal fusion in vivo. J Cell Biochem 112:1673-84)。360の投影を、220msec/スライスの露光時間で、0.5のステップで、180°の角度範囲にわたって獲得した。5つのフレームを、各回転ステップにおいて平均化して、より良いシグナルノイズ比を得た。ビーム硬化(beam hardening)アーチファクトを最小化するために、0.5mmのアルミニウムフィルターを使用して、X線ビーム周波数を絞り込んだ。仮想画像スライスを、Feldkampアルゴリズム(SkyScan社)に基づくコーンビーム再構成ソフトウェアバージョン2.6を使用して再構成した。これらの設定は、連続断面の1024×1024ピクセル画像を生じた。試料の再配向及び2D可視化を、DataViewer(SkyScan社)を使用して実施した。3D可視化を、Dolphin Imagingバージョン11(Dolphin Imaging & Management Solutions社、Chatsworth、CA)を使用して実施した。固定を、L4横突起とL5横突起との間の橋渡し骨の両側性の存在として規定した。再構成された画像を、2人の熟練の独立した観察者が、固定されているか固定されていないかについて判断した。各固定塊内に形成された骨の密度を定量するために、塊の組織容量(TV,tissue volume)、塊内の海綿骨容量(BV,bone volume)、BV/TV比、海綿厚及び海綿分離を計算した。L4〜5の椎間体(intervertebral body)のレベルを中心として、各固定塊内の501の軸方向スライス(1スライス当たり20um、10.02mmの長さ)にわたる測定値でDataViewerソフトウェアを使用して、これを実施した。
micro−CTを受けた後に、各手術群由来の2つの代表的標本を、本発明者らが以前に報告したように脱水によって脱灰しないで処理し、キシレン中で清浄化し、メタクリル酸メチル中に包埋した(Johnson JS, Meliton V, Kim WK, Lee K-B, Wang JC, Nguyen K, Yoo D, Jung ME, Atti E, Tetradis S, Pereira RC, Magyar C, Nargizyan T, Hahn TJ, Farouz F, Thies S, Parhami F 2011 Novel oxysterols have pro-osteogenic and anti-adipogenic effects in vitro and induce spinal fusion in vivo. J Cell Biochem 112:1673-84、Pereira RC, Stadmeyer LE, Smith DL, Rydziel S, Canalis E 2007 CCAAT/Enhancer-binding protein homologous protein (CHOP) decreases bone formation and causes osteopenia. Bone 40:619-26)。連続冠状切片を、5umの厚さで切断し、トルイジンブルーpH 6.4で染色した。切片の顕微鏡写真を、図7Aでは10×の倍率で、図7Bでは20×の倍率で、以前に報告されたようにScanScope XT System(Aperio Technologies, Inc.社、Vista、CA)を使用して取得した(Magyar CE, Aghaloo TL, Atti E, Tetradis S 2008 Ostene, a new alkylene oxide copolymer bone hemostatic material, does not inhibit bone healing. Neurosurgery 63:373-378; discussion 378)。
統計分析を、StatView 5プログラムを使用して実施した。全てのp値を、ANOVA及びFisher投影最小有意差(PLSD,projected least significant difference)有意性検定を使用して計算した。0.05未満のpの値を有意とみなした。
以下は、Oxy133の化学構造である:
公開された特許手順(Parhami et al.、国際公開第2009/07386号パンフレット、52頁)に従って調製した。1H NMR(CDCl3,400MHz)δ:3.47(1H,dddd,J=11.0,11.0,4.8,4,8Hz)、3.36(1H,ddd,J=10.4,10.4,4.4Hz)、2.53(1H,d,J=8.8,8.8Hz)、2.20〜2.14(1H,m)、2.10(3H,s)、2.01〜1.97(1H,m)、1.88〜1.82(1H,m)、1.73〜0.89(17H,m)、0.88(18H,s)、0.79(3H,s)、0.59(3H,s)、0.043(3H,s)、0.04(3H,s)、0.03(3H,s)、0.02(3H,s)。13C NMR(CDCl3,100MHz)δ:209.5、72.2、70.1、63.7、56.4、53.7、51.8、44.2、41.9、38.9、37.6、36.3、34.3、33.2、31.7、31.5、25.94、25.92、24.4、22.7、21.1、18.3、18.1、13.5、13.4、−4.1、−4.6、−4.7。
THF(6mL)中のn−ヘキシン(1.5mL、12mmol)の冷(0℃)溶液に、n−ブチルリチウムの1.6Mヘキサン溶液(3.75mL)を添加した。THF(10mL)中の化合物1(1.27g、2.2mmol)の溶液を、カニューレを介して添加するまで、得られた溶液を30分間撹拌した。この混合物を、3時間かけて室温まで昇温し、水(40mL)で希釈し、粗製生成物を、酢酸エチル抽出によって単離した(3×30mL)。合わせた有機層を、ブラインで洗浄し、Na2SO4で乾燥させた。濃縮により、油性生成物が得られ、これを、シリカゲル(ヘキサン、酢酸エチル、勾配)で精製して、1.30gの化合物2(92%)を得た。1H NMR(CDCl3,300MHz)δ:3.50(1H,ddd,J=15.9,11.0,4.8Hz)、3.36(1H,ddd,J=10.6,10.6,4.3Hz)、2.18(1H,t,J=6.9Hz)、2.10(1H,m)、1.91〜1.62(4H,m)、1.53〜1.31(2H,m),1.44(3H,s)、1.31〜0.93(22H,m)、0.93(3H,s)、0.92(3H,m)、0.90(18H,s)、0.88(3H,s)、0.61(1H,m)、0.04(6H,s)、0.03(6H,s)。13C NMR(CDCl3,75MHz)δ:85.9、83.9、72.4、71.4、70.3、60.5、55.8、53.8、51.8、43.5、36.3、33.7、33.0、30.7、25.9、22.0、18.4、18.3、18.1、13.6、13.5、−4.7、−4.7。
化合物2(1.3g、2.0mmol)を、酢酸エチル(5mL)及びメタノール(5mL)中に溶解させ、Pd/C(10%、0.1g)をこの溶液に添加した。この混合物を、真空下で繰り返し脱気し、次いで、大気圧下で水素ガスに曝露させた(バルーン)。室温で18時間の後、この混合物を酢酸エチル(20mL)で希釈し、セライト(Celite)でろ過して、触媒を除去した。このフィルターを酢酸エチルで洗浄し、合わせた濾液を蒸発させて1.3gの還元された生成物3を得、これをさらなる精製なしに使用した。1H NMR(CDCl3,300MHz)δ:3.50(1H,ddd,J=15.9,11.0,4.8Hz)、3.36(1H,ddd,J=10.6,10.6,4.3Hz)、2.1〜1.95(2H,m)、1.75〜1.35(10H,m)、1.32〜1.29(10H,m),1.24(3H,s)、0.91〜1.21(10H,m)、0.89(18H,s)、0.82(3H,s)、0.79(3H,s)、0.63(1H,m)、0.04(6H,s)、0.03(6H,s)13C NMR(CDCl3,75MHz)δ:75.2、72.3、57.6、56.4、53.8、51.8、42.9、37.6、36.3、33.7、31.9、30.0、25.9、22.6、18.3、18.1、14.1、13.8、13.5、−4.6、−4.7。
TBAFの1MTHF溶液(8mL、8mmol、4当量)を、化合物3(1.3g、2.0mmol、1.0当量)に直接添加し、得られた溶液をTHF(1mL)で希釈し、室温で72時間撹拌した。次いで、この混合物を水(50mL)で希釈し、酢酸エチルで繰り返し抽出した(4×40mL)。合わせた有機層をブラインで洗浄し、Na2SO4で乾燥させ、溶媒を蒸発させた。シリカゲルクロマトグラフィー(ヘキサン、酢酸エチル、勾配、次いで酢酸エチル中10%のメタノール)による粗製生成物の精製により、白色固体(0.6g、70%)が得られ、これを、水性アセトン(アセトン、水、3:1)中での粉砕に供し、純粋なOxy133、0.5gを得た。1H NMR(CDCl3,300MHz)δ:3.50(1H,ddd,J=15.9,11.0,4.8Hz)、3.36(1H,ddd,J=10.6,10.6,4.3Hz)、2.19(1H,m)、2.10〜1.90(3H,m)、1.85〜1.60(7H,m)、1.55〜1.38(7H,m)、1.25(11H,m)、1.20〜0.95(4H,m)、0.90(3H,m)、0.86(3H,s)、0.80(3H,s),0.62(1H,m)。13C NMR(CDCl3,75MHz)δ:75.1、71.1、69.3、57.5、56.2、53.6、51.6、44.0、42.8、41.4、40.1、37.2、36.2、33.5、32.1、31.8、30.9、29.9、26.3、24.2、23.6、22.5、22.2、20.9、14.0、13.6、13.3。MS:M+H=420.36。HRMS(ESI)m/z[M− 2 H2O H]+ C27H44OHの計算値:385.3470、実測値385.3478。
Oxy133は、骨髄間質細胞、胚性線維芽細胞及びヒト間葉系幹細胞の造骨性分化を誘導する
骨前駆細胞の造骨性分化を誘導することが可能な分子を開発するという目的を達成するために、100を超える以前に合成されたアナログにおいて観察された構造活性関連性の本発明者らの理解に基づいて、本発明者らは、最も強力な造骨性の天然に存在するオキシステロール、20(S)−ヒドロキシコレステロール(20S)の分子構造を改変した。本発明者らは、強い造骨性分化が、20Sの2つの構造アナログOxy34及びOxy49で達成されたことを以前に報告している(Johnson JS, Meliton V, Kim WK, Lee K-B, Wang JC, Nguyen K, Yoo D, Jung ME, Atti E, Tetradis S, Pereira RC, Magyar C, Nargizyan T, Hahn TJ, Farouz F, Thies S, Parhami F 2011 Novel oxysterols have pro-osteogenic and anti-adipogenic effects in vitro and induce spinal fusion in vivo. J Cell Biochem 112:1673-84)。これらの分子は、Oxy34及びOxy49の両方においては炭素6(C6)上にαヒドロキシル(OH)基を付加し、Oxy49においてはC25とC27との間の二重結合を付加することによって、形成した(図1)(Johnson JS, Meliton V, Kim WK, Lee K-B, Wang JC, Nguyen K, Yoo D, Jung ME, Atti E, Tetradis S, Pereira RC, Magyar C, Nargizyan T, Hahn TJ, Farouz F, Thies S, Parhami F 2011 Novel oxysterols have pro-osteogenic and anti-adipogenic effects in vitro and induce spinal fusion in vivo. J Cell Biochem 112:1673-84)。本明細書で報告した研究において、本発明者らは、それぞれ大型動物及びヒトにおける将来の前臨床研究及び臨床研究のために大量までスケールアップするのに適した、より合成が容易でより強力なアナログを開発することによって、これら2つの分子をさらに改善することを試みた。この分子は、脊椎固定及び骨折治癒の刺激のために局所的に骨形成を増加させるための、並びにおそらくは骨減少症及び骨粗鬆症などの障害に対処するために全身的にさえ骨形成を増加させるための、治療剤開発及び臨床使用の候補であり得る。構造活性関連性研究を通じて、新規アナログOxy133を、実施例2に記載されたプロトコルに従って合成し、骨誘導活性について試験した。Oxy133は、C27の欠失及び側鎖の長さの1炭素分の増加により、Oxy34及びOxy49とは異なっている(図1)。重要なことに、Oxy133は、Oxy34及びOxy49と比較して顕著にコストがかからない生成物を生じる安価な市販の出発材料に起因して、大規模でより容易に調製され得る。さらに、Oxy133の調製に使用したアルキン付加は、収率、生成物の純度(ジアステレオ選択性)及びスケーラビリティに関して、Oxy34及びOxy49の合成において使用されるGrignard化学よりも優れている。
以前の研究により、20S並びにその構造アナログOxy34及びOxy49が、Hh経路シグナル伝達の活性化を介して造骨性分化を誘導することが実証されている(Johnson JS, Meliton V, Kim WK, Lee K-B, Wang JC, Nguyen K, Yoo D, Jung ME, Atti E, Tetradis S, Pereira RC, Magyar C, Nargizyan T, Hahn TJ, Farouz F, Thies S, Parhami F 2011 Novel oxysterols have pro-osteogenic and anti-adipogenic effects in vitro and induce spinal fusion in vivo. J Cell Biochem 112:1673-84)。しかし、Hh経路シグナル伝達の造骨性オキシステロール媒介性の活性化に関する分子機構は、以前には知られていなかった。そのより高い造骨性活性を考慮すると、Oxy133は、Hh経路活性化及び骨発生が半合成オキシステロールによって達成される分子機構を同定するための有用なツールである。Oxy133がHh経路を介して造骨性分化を誘導するか否か、及びどのように誘導するかを決定するために、Oxy133誘導性のALP活性並びに造骨性分化マーカーALP、BSP及びOSXの発現に対する、選択的Hh経路阻害剤シクロパミンの影響を試験した。シクロパミンは、C3H細胞(図4A)及びM2細胞(データ示さず)において、Oxy133誘導性のALP活性並びに造骨性マーカーALP、BSP及びOSXの発現を完全に阻害し、Oxy133がHhシグナル伝達経路を介して作用することが示唆された。Oxy133によるHhシグナル伝達の活性化をさらに分析するために、C3H細胞中にトランスフェクトされたGli依存的ルシフェラーゼレポーターの活性化を、以前に報告された方法を使用して試験した(Johnson JS, Meliton V, Kim WK, Lee K-B, Wang JC, Nguyen K, Yoo D, Jung ME, Atti E, Tetradis S, Pereira RC, Magyar C, Nargizyan T, Hahn TJ, Farouz F, Thies S, Parhami F 2011 Novel oxysterols have pro-osteogenic and anti-adipogenic effects in vitro and induce spinal fusion in vivo. J Cell Biochem 112:1673-84、Dwyer JR, Sever N, Carlson M, Nelson SF, Beachy PA, Parhami F 2007 Oxysterols are novel activators of the Hedgehog signaling pathway in pluripotent mesenchymal cells. J Biol Chem 282:8959-68)。Oxy133は、Gli依存的レポーターの活性における用量依存的増加を誘導し、これは、100nMのOxy133で5倍の誘導及び1μMのOxy133で17倍の誘導に達した(図4B)。
本発明者らは、20Sが、Smo受容体に対する結合によってHhシグナル伝達を選択的に活性化することを以前に報告している(Nachtergaele S, Mydock LK, Krishnan K, Rammohan J, Schlesinger PH, Covey DF, Rohatgi R 2012 Oxysterols are allosteric activators of the oncoprotein Smoothened. Nat Chem Biol 8:211-20)。Oxy133が同じ機構によってHhシグナル伝達を活性化するか否かを決定するために、本発明者らは、磁気ビーズにカップリングされた20SアナログとのYFPタグ化Smo(YFP−Smo)の結合について競合するOxy133の能力を試験した。本発明者らが以前に報告したように、このアナログnat−20S−yneは、イソ−オクチル鎖上にアルキン部分を含み、クリックケミストリー媒介性の、磁気ビーズに対するカップリングを可能にする(20S−ビーズ)(Nachtergaele S, Mydock LK, Krishnan K, Rammohan J, Schlesinger PH, Covey DF, Rohatgi R 2012 Oxysterols are allosteric activators of the oncoprotein Smoothened. Nat Chem Biol 8:211-20)。ステロール結合アッセイのためにこれらのビーズを使用して、競合物なしの試料と比較した、ビーズ上に残留しているYFP−Smoの量を、ウエスタンブロッティングによって測定する。20Sと同じ部位においてSmoを結合する化合物は、20S−ビーズと競合し、溶出液中のタンパク質の量を低減させる。本発明者らは、Smo結合アッセイ及びHhシグナル伝達アッセイの両方において、多数の他のステロールを試験したが、全ての場合において、Smoに対する結合は、Hh経路活性における変化と相関した(Nachtergaele S, Mydock LK, Krishnan K, Rammohan J, Schlesinger PH, Covey DF, Rohatgi R 2012 Oxysterols are allosteric activators of the oncoprotein Smoothened. Nat Chem Biol 8:211-20)。陽性コントロールであるOxy133及び20Sは共に、20Sカップリングビーズ上に捕捉されたYFP−Smoの量を低減させた(図4C)。重要なコントロールにおいて、Hhシグナル伝達も骨発生も活性化できなかった(Parhami et al.の未公開の観察)、構造的に関連するアナログOxy16は、YFP−Smoと20S−ビーズとの間の相互作用を防止できなかった(図4C)。遊離Oxy133の存在下での、20S−ビーズにより捕捉されたYFP−Smoの量におけるこの低減は、Oxy133が、Smo上の、20Sと同じ部位に結合することを示唆している。主に本発明者らが、抽出物中のYFP−Smoの濃度及びビーズ上に生産的に固定化された20Sの量を知らないことに起因して、本発明者らのアッセイが半定量的であり、相互作用についてKdを導出するためには使用できないことを強調することが重要である。
8週齢のLewisラットを、手術部位におけるコラーゲンスポンジ内に含まれる試薬だけが異なる5つの処置群に分割した:群1−コントロール媒体(DMSO)のみ(n=7)、群II−5μg rhBMP−2(n=8)、群III−20mg Oxy133(n=7)、群IV−2mg Oxy133(n=8)及び群V−0.2mg Oxy133(n=8)。骨形成及び脊椎固定を、X線撮影分析を介して術後の種々の時点で評価し、並びに手動評価、マイクロコンピュータ断層撮影及び組織学を使用して屠殺の時点で評価した。屠殺の時点の固定率を表1にまとめる。
第1のセットのX線写真を、手術の4週間後に実施した。この時点では、両側固定が、BMP2群中の8/8の動物、Oxy133−20mg群中の6/7の動物、Oxy133−2mg群中の3/8の動物で観察され、コントロール群及びOxy133−0.2mg群では固定は観察されなかった。片側固定が、残りのOxy133−20mg処置した動物及びOxy133−2mgで処置した3匹の動物において観察された。これは、4週目の時点で固定が観察されなかった、Oxy34及びOxy49を用いた以前の研究とは対照的である(Johnson JS, Meliton V, Kim WK, Lee K-B, Wang JC, Nguyen K, Yoo D, Jung ME, Atti E, Tetradis S, Pereira RC, Magyar C, Nargizyan T, Hahn TJ, Farouz F, Thies S, Parhami F 2011 Novel oxysterols have pro-osteogenic and anti-adipogenic effects in vitro and induce spinal fusion in vivo. J Cell Biochem 112:1673-84)。6週目までには、Oxy133−20mg群中の全ての動物が、両側固定されていた。8週目に、固定は、BMP2群及びOxy133−20mg群中の全ての動物並びにOxy133−2mg群の4/8において再度注目された(図5)。最後の8週目のX線写真では、固定塊はDMSO群でもOxy133−0.2mg(データ示さず)群でも観察されなかった(図5)。
屠殺後、本発明者らが以前に記載したように、脊椎を各動物から外植し、手動評価に供した(Johnson JS, Meliton V, Kim WK, Lee K-B, Wang JC, Nguyen K, Yoo D, Jung ME, Atti E, Tetradis S, Pereira RC, Magyar C, Nargizyan T, Hahn TJ, Farouz F, Thies S, Parhami F 2011 Novel oxysterols have pro-osteogenic and anti-adipogenic effects in vitro and induce spinal fusion in vivo. J Cell Biochem 112:1673-84、Sintuu C, Simon RJ, Miyazaki M, Morishita Y, Hymanson HJ, Taghavi C, Brochmann EJ, Murray SS, Wang JC 2011 Full-length spp24, but not its 18.5-kDa proteolytic fragment, inhibits bone-healing in a rodent model of spine fusion. J Bone Joint Surg Am 93:1022-32、Miyazaki M, Morishita Y, He W, Hu M, Sintuu C, Hymanson HJ, Falakassa J, Tsumura H, Wang JC 2009 A porcine collagen-derived matrix as a carrier for recombinant human bone morphogenetic protein-2 enhances spinal fusion in rats. Spine J 9:22-30、Zhu W, Rawlins BA, Boachie-Adjei O, Myers ER, Arimizu J, Choi E, Lieberman JR, Crystal RG, Hidaka C 2004 Combined bone morphogenetic protein-2 and -7 gene transfer enhances osteoblastic differentiation and spine fusion in a rodent model. J Bone Miner Res 19:2021-32)。肉眼評価及び手動評価の結果は、8週目におけるX線撮影的知見と類似していた。DMSO群でもOxy133−0.2mg群でも、片側固定も両側固定も観察されなかった。いくらかの骨形成が、Oxy133−0.2mg群中の2匹の動物において注目された。両側固定は、BMP2群中の全ての動物及びOxy133−20mg群中の6/7の動物において観察された。Oxy133−20mg群中の残りの動物は、顕著な両側固定塊にもかかわらず、片側で動きを有した。Oxy133−2mg群中の半分(4/8)の動物は、手動触診で両側固定が確認されたが、2匹のさらなる動物は片側固定を有し、2匹の動物は固定の証拠を有さなかった。
micro−CT分析による橋渡し海綿骨の評価により、X線写真、肉眼観察及び手動触診で観察された結果が確認された(図6)。いくらかの骨形成が、Oxy133−0.2mg群中の2匹の動物において見られたが、両側固定は、この群でもDMSO群でも観察されなかった。両側の橋渡し海綿骨は、BMP2群及びOxy133−20mg群中の全ての動物において見られた。両側固定は、Oxy133−2mg群中の4/8の動物においても観察され、2匹のさらなる動物においては片側固定が観察された。micro−CT画像からの微細構造分析の結果を表2に示す。BMP2固定塊の総容量は、Oxy133−2mg試料及び20−mg試料の両方よりも有意に高かった。しかし、Oxy133−2mg及び20−mg固定塊の平均BV/TV比は、BMP2群よりも有意に高く、これは、塊内のより高密度の骨を示す。海綿厚は、BMP2とOxy133−2mg又はOxy133−20mgのいずれかとの間で、有意に異ならなかった。海綿分離は、Oxy133−2mg及びOxy133−20mgと比較して、BMP2固定塊において有意に大きく、これもまた、BMP2固定塊におけるより低い密度の骨を示す。
従来の手順を使用して、C3H10T1/2骨−脂肪前駆細胞を、脂肪細胞形成を誘導することが報告されている、PPARγアクチベータのトログリタゾン(「Tro」,Troglitazone)で処置した。2週間後、Tro処置したウェルにおける脂肪生成を、Oil Red O染色によって可視化し、脂肪細胞を光学顕微鏡によって定量した。造骨性分化及びHhシグナル伝達の両方を誘導する用量におけるOxy133は、脂肪細胞形成を完全に阻害したことが明らかであった。以下はデータである(1視野当たりの平均脂肪細胞≠SDとして):
視野≠SD):
コントロール:2.5≠2
Tro:28≠4
Tro+Oxy133(5uM):0.5≠1
Oxy133(5uM):0.8≠1
コントロール対Tro対Tro+Oxy133についてのp値は、0.01未満で有意性に達した
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Claims (11)
-
の構造を有する化合物又はその薬学的に許容される塩若しくは溶媒和物。 - 薬学的に許容される担体、及び
の構造を有する化合物又はその薬学的に許容される塩若しくは溶媒和物を含む、組成物。 - 副甲状腺ホルモン、フッ化ナトリウム、インスリン様増殖因子I(ILGF−I)、インスリン様増殖因子II(ILGF−II)、トランスフォーミング増殖因子β(TGF−β)、チトクロムP450阻害剤、造骨性プロスタノイド、BMP2、BMP4、BMP7、BMP14及びそれらの組合せからなる群から選択される少なくとも1つのさらなる薬剤をさらに含む、請求項2に記載の組成物。
- 請求項2に記載の組成物を含む、骨障害、骨粗鬆症又は骨折を有する対象の処置剤。
- 選択された間隔で有効な剤形で対象に投与されて、骨量を増加させる、請求項4に記載の処置剤。
- 選択された間隔で有効な剤形で対象に投与されて、骨粗鬆症の症状を改善させる、請求項4に記載の処置剤。
- 請求項2に記載の組成物を含む、骨形態形成及び/又は骨増殖における増加を必要とする対象の処置剤。
- 請求項2に記載の組成物を含む、対象における骨形成の誘導剤であって、選択された間隔で有効な剤形で投与されて、骨量を増加させる、前記誘導剤。
- 対象中の細胞、組織又は臓器に局所的に投与される、請求項4に記載の処置剤。
- ヒト又は動物の身体における使用のためのインプラントであって、表面を有する基材を含み、前記インプラントの前記表面又は内側が、周囲の骨組織において骨形成を誘導するのに十分な量で、請求項2に記載の組成物を含むインプラント。
- 基材が、ピン、ネジ、プレート又は人工関節の形状へと形成される、請求項10に記載のインプラント。
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PCT/US2013/032693 WO2013169399A1 (en) | 2012-05-07 | 2013-03-15 | Oxysterol analogue oxy133 induces osteogenesis and hedgehog signaling and inhibits adipogenesis |
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JP (1) | JP6262723B2 (ja) |
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WO2011103175A2 (en) | 2010-02-16 | 2011-08-25 | The Regents Of The University Of California | Oxysterols that activate liver x receptor signaling and inhibit hedgehog signaling |
WO2012024583A2 (en) * | 2010-08-20 | 2012-02-23 | Fate Therapeutics, Inc. | Oxysterol compounds |
WO2012024581A2 (en) | 2010-08-20 | 2012-02-23 | Fate Therapeutics, Inc. | Oxysterol compounds |
WO2012024584A2 (en) * | 2010-08-20 | 2012-02-23 | Fate Therapeutics, Inc. | Oxysterol compounds |
CN104507951B (zh) | 2012-05-07 | 2016-09-07 | 加利福尼亚大学董事会 | 诱导骨生成和hedgehog信号传导且抑制脂肪形成的氧固醇类似物氧固醇化合物149 |
KR20150013232A (ko) | 2012-05-07 | 2015-02-04 | 더 리전트 오브 더 유니버시티 오브 캘리포니아 | 골형성 및 헤지호그 신호전달을 유도하고 지방생성을 억제하는 옥시스테롤 유사체 oxy133 |
WO2014179756A1 (en) * | 2013-05-02 | 2014-11-06 | The Regents Of The University Of California | Bone-selective osteogenic oxysterol-bone targeting agents |
JP6530056B2 (ja) | 2014-05-02 | 2019-06-12 | ザ・リージエンツ・オブ・ザ・ユニバーシテイ・オブ・カリフオルニア | 骨選択的骨形成オキシステロールビスホスホネート類似体 |
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KR20150013232A (ko) | 2015-02-04 |
RU2632191C2 (ru) | 2017-10-03 |
CN104395331A (zh) | 2015-03-04 |
RU2014149164A (ru) | 2016-07-10 |
AU2013260059A1 (en) | 2014-11-27 |
EP2847206A1 (en) | 2015-03-18 |
CA2872751A1 (en) | 2013-11-14 |
WO2013169399A1 (en) | 2013-11-14 |
US20150118277A1 (en) | 2015-04-30 |
JP2015518493A (ja) | 2015-07-02 |
AU2013260059B2 (en) | 2017-05-18 |
IN2014DN09805A (ja) | 2015-07-31 |
CN104395331B (zh) | 2016-11-02 |
EP2847206A4 (en) | 2016-01-20 |
HK1207649A1 (en) | 2016-02-05 |
US9717742B2 (en) | 2017-08-01 |
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