JP6533213B2 - 抗ウイルス剤 - Google Patents
抗ウイルス剤 Download PDFInfo
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- JP6533213B2 JP6533213B2 JP2016505287A JP2016505287A JP6533213B2 JP 6533213 B2 JP6533213 B2 JP 6533213B2 JP 2016505287 A JP2016505287 A JP 2016505287A JP 2016505287 A JP2016505287 A JP 2016505287A JP 6533213 B2 JP6533213 B2 JP 6533213B2
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- antiviral
- polymer particles
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Description
で表される構造を繰り返し単位中に有する、粒径5μm以下のポリマー粒子を有効成分として含有する。式中の波線は、一般式(I)で示される構造が結合している、ポリマー構造中の他の構造部分を示す。電子吸引基とα位に存在するカルボキシル基とを有する一般式(I)の構造は、糖タンパク質と高い親和性を示す。一方、ウイルス粒子の表面、特にエンベロープ上には、ウイルス糖タンパク質が存在する。そのため、一般式(I)の構造を有するポリマー粒子は、ウイルス粒子の表面と高い親和性を示し、ウイルス粒子表面に特異的に接着してエンベロープを破壊し、ウイルス粒子の感染性を低下ないしは喪失させるものと考えられる。
で表される少なくとも1種のモノマーを、アミノ酸、アミノ酸誘導体、それらのオリゴマー及びポリマー、糖並びにポリソルベートからなる群より選択される少なくとも1種の共存下で重合させることにより製造することができる。このような構造を有するモノマーとしては、例えばアクリレート系モノマーが挙げられるが、アクリレート系モノマーは種々のものが市販されており、そのような市販品を好ましく用いることができる。また、その他のモノマーは、例えば市販のアクリレート系モノマーから化学合成分野における周知の常法を用いて容易に調製することができる。
アミノ酸系分子含有量=(アミノ酸系分子添加量)−(フィルター通過液中のアミノ酸系分子の量)
アミノ酸系分子含有率(%)=アミノ酸系分子含有量÷アミノ酸系分子添加量×100
特許文献8、9に記載されている方法に準じてアミノ酸又はデキストランを含有するナノサイズのポリマー粒子を製造した。具体的な手順は以下の通りである。
10 mLの0.001N HClに、100mgのアミノ酸を溶解して、その液性pHを要時1N塩酸を用いてpH=3に調整した。アミノ酸としては、グリシン、アラニン、及びアスパラギン酸を使用した。
(2) デキストラン合成系
10 mLの0.01N HClに100mgのデキストラン60Kを溶解し、その液性pHを要時1N塩酸を用いてpH=2に調整した。
供試ウイルスとして、魚病ウイルスの一種であるコイヘルペスウイルスCyprinid herpesvirus 3(CyHV-3)を用いた。供試ナノ粒子として、上記で合成した4種類のナノ粒子を用いた。
抗ウイルス効果の検証に先立ち、ナノ粒子のコイ細胞に対する毒性を確認するため、コイヒレ由来の細胞を3.5cmディッシュで単層になるように培養後、各種濃度(粒子濃度10mg/mLの液体を原液(1倍希釈)とし、10倍ずつ段階希釈)のナノ粒子を添加し、細胞の変化を顕微鏡下で観察して毒性の有無を判定した。その結果、4種類の粒子いずれも、10倍希釈(1mg/mL)の高濃度でもコイヒレ細胞に対する毒性は認められなかった。
CyHV-3の0.2%溶液をウイルス原液とし、10倍ずつ段階希釈した希釈系列を調製して抗ウイルス試験に用いた。10倍段階希釈の各ナノ粒子液とウイルス液を混合し、4℃で1時間インキュベートした後、コイヒレ由来の単層培養細胞(106 cells/well)に添加した。25℃で24時間インキュベート後、細胞内のDNAを抽出し、ウイルス遺伝子(ORF89遺伝子)のコピー数をリアルタイムPCRにより確認した。
24穴プレートの各ウエルからマイクロピペットを用いて、チップの先で細胞をこすって剥がした後、回収した細胞懸濁液を2mlのマイクロチューブに移した。Proteinase Kを0.2μl添加し、軽くボルテックス後、55℃で2時間インキュベートした。インキュベート後フェノール・クロロホルムを300μl加えて20℃で15,000rpm、5分間の遠心を行った後、マイクロピペットで上清200μlを回収した。同様の操作を3回繰り返した後、100%エタノールを500μl、3M酢酸ナトリウム20μlを加え−30℃で一晩保存した。一晩保存したサンプルを20℃で15,000rpm、1時間の遠心後、デカントで上清を廃棄した。沈殿に70%エタノール1,000μlを加え、20℃で15,000rpm、10分の遠心後、デカントで上清を廃棄してDNAの沈殿を乾燥させた後、Tris-EDTA buffer溶液30μlを加え、DNAを懸濁しDNAサンプルとした。
抽出したDNA量をQ5,000(TOMY)を用いて測定し、DNA濃度が約200ng/μlになるように蒸留水で希釈調整した。スクリーニングではPCRチューブにcDNA溶液1.0μl、TaqMan Gene Expression Master Mix(Applied Biosystems)5μl、Probe(CTTCCTCTGCTCGGCGAGCACG、配列番号1)0.5μl、プライマーKHV-86F(GACGCCGGAGACCTTGTG、配列番号2)及びKHV-163R(CGGGTTCTTATTTTTGTCCTTGTT、配列番号3)をそれぞれ0.9μl、精製水1.7μlを加え、Step One PlusTM リアルタイムPCRシステム(Applied Biosystems)にてリアルタイムPCR反応を行い、ウイルス濃度を求めた。PCR反応は、まず、熱変性95℃で10分間行った後、1サイクルの条件を、熱変性を95℃で30秒間、アニーリングおよび伸長反応を60℃で1分間とし、40サイクル行った。なお、スクリーニングは2本立てで行った。
小フラスコ(25cm2)10本のコイヒレ由来細胞にCyHV-3(moi=1)を接種し、25℃で10日間インキュベートした後、−80℃で凍結、融解を2回繰り返した。つづいて、50mlの遠沈管に懸濁液を移して5,000rpm、4℃で30分の遠心を行った後、上清を回収した。次に、上清を40,000rpm、4℃で1時間30分、超遠心(Beckman)を行って目的の試料を沈殿させた。上清を捨てペレットを無血清のDMEM培地500μlに再懸濁後、30%、40%、50%のスクロース重層の上に注意深く重層し、40,000rpm、4℃で1時間30分の超遠心を行った。その後、50%層より上に検出されたバンドを回収し、500mlのPBSに対して一晩透析を行ったものを供試ウイルス液とした。供試ウイルス液50μlとそれぞれ4種類のナノ粒子溶液5μlを混合し、24h反応させた後、透過型電子顕微鏡H-7100(日立)を用いて観察を行った。
Claims (8)
- グリシン、アラニン、アスパラギン酸及びデキストランからなる群より選択される少なくとも1種を含み、かつ、抗ウイルス活性成分を含まない、平均粒径1000nm未満のn-ブチル-2-シアノアクリレートポリマーの粒子を有効成分として含有する、抗ウイルス剤。
- 前記ウイルスがエンベロープを有するウイルスである請求項1記載の抗ウイルス剤。
- 前記ウイルスがインフルエンザウイルス及びヘルペスウイルスから選択される少なくとも1種である請求項2記載の抗ウイルス剤。
- ウイルス感染防止剤である請求項1ないし3のいずれか1項に記載の抗ウイルス剤。
- グリシン、アラニン、アスパラギン酸及びデキストランからなる群より選択される少なくとも1種を含み、かつ、抗ウイルス活性成分を含まない、平均粒径1000nm未満のn-ブチル-2-シアノアクリレートポリマーの粒子の有効量を、植物体、植物の種子、土壌、鉢土、苗箱、農業機具、又は園芸器具と接触させることを含む、植物ウイルス病害の防除方法。
- 繊維、不織布又は樹脂にポリマー粒子を付着又は練り込ませてなる、抗ウイルス用衛生用品材料であって、前記ポリマー粒子が、グリシン、アラニン、アスパラギン酸及びデキストランからなる群より選択される少なくとも1種を含み、かつ、抗ウイルス活性成分を含まない、平均粒径1000nm未満のn-ブチル-2-シアノアクリレートポリマーの粒子である、抗ウイルス用衛生用品材料。
- 請求項6記載の抗ウイルス用衛生用品材料を用いて製造された、抗ウイルス用の衛生用品であって、前記衛生用品は、マスク、白衣、防護服、手袋、ゴーグル、避妊用の器具、おむつ、シーツ、又は空気清浄器若しくはエアコンのフィルターである、抗ウイルス用の衛生用品。
- グリシン、アラニン、アスパラギン酸及びデキストランからなる群より選択される少なくとも1種を含み、かつ、抗ウイルス活性成分を含まない、平均粒径1000nm未満のn-ブチル-2-シアノアクリレートポリマーの粒子を含む、抗ウイルス用の衛生用品であって、前記衛生用品は、ルームミスト、ハンドソープ、うがい薬、スプレー、又は洗剤である、抗ウイルス用の衛生用品。
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