JP2021519811A - 象牙質知覚過敏症緩和のための歯磨き剤組成物 - Google Patents
象牙質知覚過敏症緩和のための歯磨き剤組成物 Download PDFInfo
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- 210000003296 saliva Anatomy 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012128 staining reagent Substances 0.000 description 1
- 229910001631 strontium chloride Inorganic materials 0.000 description 1
- AHBGXTDRMVNFER-UHFFFAOYSA-L strontium dichloride Chemical compound [Cl-].[Cl-].[Sr+2] AHBGXTDRMVNFER-UHFFFAOYSA-L 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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- 210000004357 third molar Anatomy 0.000 description 1
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- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
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Abstract
Description
前記一般式1において、
R1は、アルギニン(R)、リジン(K)、またはグルタミン(Q)であり、
R2は、アルギニン(R)またはグルタミン(Q)であり、
R3、R4およびR5は、それぞれアルギニン(R)またはリジン(K)であり、
R6は、アスパラギン(N)またはセリン(S)であり、
R7およびR8は、リジン(K)またはチロシン(Y)である。
本発明を説明するに当たり、象牙芽細胞(または象牙質芽細胞)(odontoblast)とは、象牙質の基質を構成するタンパク質、多糖体を細胞内で合成、分泌する細胞のことを意味し得る。また、象牙前質(未石灰化の象牙質)に接して、歯髄の周辺部に一層の細胞層を構成する円柱状の細胞として、歯乳頭の細胞の中でエナメル基に面しているもの(外胚葉系の中間葉に由来する細胞となる)が分化した細胞であり、また、象牙質の石灰化にも関与する細胞のことを意味し得る。
[象牙芽細胞分化促進のためのペプチドの合成]
ペプチド(配列番号1)をFmoc(9-fluorenylmethyloxycarbonyl)法により合成し、前記合成されたペプチドのアミノ酸を置換して、各グループのペプチドを合成した(表1〜表12)。
まず、グループ1のペプチドは、配列番号1のペプチドまたは前記配列番号1のペプチドの5番目〜7番目のアミノ酸をリジンまたはアルギニンに置換して合成した(表1)。
[象牙芽細胞を利用した象牙質の再生促進効果検証]
<実施例2−1:DSPP(dentin sialophosphoprotein)プロモーター活性に及ぼすペプチドの影響検証>
まず、マウス由来象牙芽細胞であるMDPC−23細胞を、10%FBSを含むDMEM培地において、5%のCO2および37℃の条件で培養した。
前記実施例2−1で培養したMDPC−23細胞に、前記実施例1で合成された各グループのペプチドを処理して48時間培養した後、前記MDPC−23細胞にて発現される象牙芽細胞分化マーカー遺伝子であるDSPP遺伝子のmRNAレベルを測定し、前記測定された各DSPP遺伝子のmRNAレベルを、対照群から測定されたDSPP遺伝子のmRNAレベルに対する相対的比率で換算した(表13〜24)。また、各グループのペプチドにより測定されたDSPP遺伝子のmRNAレベルの平均値を各グループ別に比較した(図1b)。この際、対照群としては、象牙芽細胞分化促進ペプチドを処理していないMDPC−23細胞を使用した。
Dspp_R:5’-CTGTTGCTAGTGGTGCTGTT-3’(配列番号98)
Gapdh_F:5’-AGGTCGGTGTGAACGGATTTG-3’(配列番号99)
Gapdh_R:5’-TGTAGACCATGTAGTTGAGGTCA-3’(配列番号100)
前記実施例2−2の結果から、象牙芽細胞分化促進ペプチドは、DSPP遺伝子のmRNAレベルを増加させることができ、特に、グループ11および12のペプチドは、DSPP遺伝子のmRNAレベルを少なくとも3倍以上増加させ得ることを確認した。
Dmp1_R:5’-TGTGGTCACTATTTGCCTGTG-3’(配列番号102)
Nestin_F:5’-CCCTGAAGTCGAGGAGCTG-3’(配列番号103)
Nestin_R:5’-CTGCTGCACCTCTAAGCGA-3’(配列番号104)
まず、ヒト歯髄幹細胞は、ソウル大学歯科病院にて成人10人(18〜22歳)の親知らずから歯髄組織細胞を分離した。具体的に、すべての実験は、臨床研究審査委員会(hospital’s Institutional Review Board)から承認を受けた後、患者の同意を得て実験を行い、Jung HS et al(J Mol Histol.(2011))の方法に基づいて親知らずを切断し、歯髄を露出させて鉗子で歯髄組織を分離した。前記分離された歯髄組織を両面刀で細かく細切して60mmシャーレに入れ、カバースリップで覆った後、DMEM培地で培養して、培養された歯髄組織細胞を得た。
[象牙質知覚過敏症緩和のための歯磨き剤組成物の調製]
−段階1:精製水とD−ソルビトール液を混合する。
各段階は、減圧条件(‐760mmHg)下で撹拌を実施した。
<比較例3−1>
実施例3と同じ体積の精製水を準備した。
実施例3の成分のうち精製水以外に、象牙芽細胞分化促進ペプチド(配列番号96)が含まれないようにしたこと以外の成分は、すべて同様に含有されるように準備した。
イ.実施例3により提供された歯磨き剤組成物の試料約0.5gを蒸留水約1mlに溶かして歯磨き剤組成物溶液を製造する。
イ.象牙細管が露出するように歯を切断
抜歯した人の歯の歯冠部をダイヤモンドソーにより横に切断して象牙細管を露出した後、リン酸塩緩衝溶液により約5分間2回洗浄。
先に切断した歯を、0.5Mのエチレンジアミン四酢酸(ethylenediaminetetraacetic acid、EDTA、pH7.4)溶液に約5分間反応させた後、リン酸塩緩衝溶液により約5分間2回洗浄。
象牙芽細胞分化促進ペプチド(配列番号96)が含有された象牙質知覚過敏症緩和のための歯磨き剤組成物に、蛍光染色試薬が0.1%含まれるように添加してよく混合した後、象牙細管が露出された切断歯を約1分間ブラッシングする。
反応させた切断歯を蒸留水により3回洗浄した後、ダイヤモンドソーにより切断歯の象牙細管が長く見えるよう縦に約0.5mm厚さで切った後、蛍光顕微鏡で浸透程度を観察(図3を参照)。
イ.人工唾液の調製
人工唾液の組成は下記表26の通りである。
※精製水に下記表2の各成分の最終濃度になるように添加して混合しており、リン酸カリウム(K2HPO4)は最後に添加した。
※人工唾液のpHは人の唾液に近い7.2付近で測定される。
抜歯した人の歯をダイヤモンドソーにより横に切断して、1mm厚さの象牙細管が露出された象牙質試験片を作製した。
※象牙質試験片は、象牙細管を完全に露出させるために、約32%リン酸(phosphoric acid)溶液に約5分間反応させた後、精製水により約5分間3回洗浄した。その後、象牙質試験片は、超音波洗浄機で約5分間6回洗浄して、象牙細管を完全に露出されるようにした。
その後、リン酸塩緩衝溶液により3回洗浄して保管する。
実施例3による象牙質知覚過敏症緩和のための歯磨き剤組成物を用いて、象牙細管試験片に約1分間ブラッシングして蒸留水により3回洗浄した後、人工唾液に約24時間反応させる。
比較例3−1で用意された精製水を用いて象牙細管試験片に約1分間ブラッシングして蒸留水により3回洗浄した後、人工唾液に約24時間反応させる。
比較例3−2で用意された歯磨き剤組成物を用いて象牙細管試験片に約1分間ブラッシングして蒸留水により3回洗浄した後、人工唾液に約24時間反応させる。
Claims (8)
- 下記一般式1のアミノ酸配列からなるペプチドを含む象牙質知覚過敏症緩和のための歯磨き剤組成物において、
KY−R1−R2−R3−R4−R5−R6−R7−R8(一般式1)
前記一般式1において、
R1は、アルギニン(R)、リジン(K)またはグルタミン(Q)であり、
R2は、アルギニン(R)またはグルタミン(Q)であり、
R3、R4およびR5は、それぞれアルギニン(R)またはリジン(K)であり、
R6は、アスパラギン(N)またはセリン(S)であり、
R7およびR8は、リジン(K)またはチロシン(Y)であり、
前記歯磨き剤組成物100重量部に対して、前記ペプチド0.0015重量部〜0.0025重量部、リン酸三カルシウム31.00重量部〜33.00重量部、ヒドロキシアパタイト0.045重量部〜0.055重量部および精製水20重量部〜22重量部を含み、
前記歯磨き剤組成物は、象牙質表面に薄い膜を形成し、前記リン酸三カルシウムから遊離されたリン酸−カルシウムイオンと結合して、象牙細管および象牙質表面に再石灰化を誘導することを特徴とする、象牙質知覚過敏症緩和のための歯磨き剤組成物。 - 前記ペプチドは、配列番号1〜96のいずれかのアミノ酸配列からなることを特徴とする、請求項1に記載の象牙質知覚過敏症緩和のための歯磨き剤組成物。
- 前記組成物100重量部に対して、湿潤剤33重量部〜37重量部、粘度調整剤4.4重量部〜5.4重量部、界面活性剤1.1重量部〜1.3重量部、および着香剤0.8重量部〜0.9重量部をさらに含むことを特徴とする、請求項1に記載の象牙質知覚過敏症緩和のための歯磨き剤組成物。
- 前記組成物100重量部に対してアミノカプロン酸0.15重量部〜0.25重量部、アラントイン1.9重量部〜2.1重量部を含むことを特徴とする、請求項1に記載の象牙質知覚過敏症緩和のための歯磨き剤組成物。
- 前記湿潤剤は、D−ソルビトール液、ピロリドンカルボン酸ナトリウム(sodium PCA Solution、PCA−Na)液、濃グリセリン、またはこれらの混合物であり、
前記粘度調整剤は、含水ケイ酸、キサンタンガム、CMC、またはこれらの混合物であり、
前記界面活性剤は、ココイルメチルタウリン酸ナトリウムであり、
前記着香剤は、緑茶エキス、カモミール抽出物、ローズマリーエキス、ミルラチンキ、ラタニアチンキ、カモミールチンキ、マスチックオイル40 HF−60662、プロポリス抽出液、グレープフルーツ種子抽出物、スピアミントB71228、ペパーミントオイル81689、またはこれらの混合物であることを特徴とする、請求項3に記載の象牙質知覚過敏症緩和のための歯磨き剤組成物。 - 前記湿潤剤は、前記D−ソルビトール液80重量%〜83重量%、前記PCA−Na液7重量%〜8重量%、前記濃グリセリン10重量%〜12重量%を含むことを特徴とする、請求項5に記載の象牙質知覚過敏症緩和のための歯磨き剤組成物。
- 前記粘度調整剤は、前記含水ケイ酸79重量%〜85重量%、前記キサンタンガム4重量%〜8重量%、前記CMC11重量%〜13重量%を含むことを特徴とする、請求項5に記載の象牙質知覚過敏症緩和のための歯磨き剤組成物。
- 請求項1に記載の歯磨き剤組成物を、これを必要とする個体の口腔に処理する段階を含む、象牙質知覚過敏症緩和方法。
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