JP3828905B2 - 局所麻酔薬を含有する細胞凝集抑制剤 - Google Patents
局所麻酔薬を含有する細胞凝集抑制剤 Download PDFInfo
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Description
末梢の知覚神経線維に作用して、求心性インパルスの伝導を遮断することにより、意識や反射機能を損なわないで目的とする部分の知覚を鈍麻、又は消失させる薬剤として局所麻酔薬が知られている。局所麻酔薬は、神経線維に作用して細胞の内側からナトリウムイオンの細胞内への流入を抑制し、脱分極阻止(膜の安定化)、活動電位の発生抑制によりインパルス(興奮)の発生、伝導を抑制する。その化学構造からアミド型とエステル型に大きく2つに分けられる。アミド型としてリドカイン(キシロカイン)、メピバカイン、ジブカイン、ブピバカイン、プロピトカイン及びそれらの薬理学的に許容される塩(例えば、塩酸塩)が挙げられ、また、エステル型としてコカイン、プロカイン、クロロプロカイン、テトラカイン及びそれらの薬理学的に許容される塩(例えば、塩酸塩)が挙げられる。これらの中でも他の局所麻酔薬に比べて安全域が広く、麻酔作用及び細胞凝集抑制作用の何れもが速く、その持続時間も長いことから、リドカイン及びその薬理的に許容しうる塩が特に好ましい。種々の濃度の塩酸リドカインの注射液や外用液、ゼリー、ビスカス、及びスプレー等が、キシロカイン(商品名)としてアストラゼネカ社等から販売されている。
本発明の細胞凝集抑制剤は、培養細胞の培養液又は増殖させた細胞を酵素処理等により回収した高密度の細胞懸濁液に添加することによって凝集作用の抑制された細胞懸濁物を調製することができる。培養細胞としては、例えば、血小板等の血液細胞や、繊維芽細胞、上皮細胞、神経細胞、臓器の実質細胞及び幹細胞等の種々の培養細胞に対して用いることができるが、好ましくは通常の培養条件下で紡錘形をなしている繊維芽細胞である。皮膚の繊維芽細胞は容易に得られ増殖させることができる。皮膚繊維芽細胞培養は、例えば、被験者から採取した皮膚の3mm×5mm程度の生検材料から開始する。培養を始める前に、生検材料は、抗生物質や抗真菌剤で繰り返し洗浄する。次に、表皮及び皮下の脂肪細胞含有組織を取り、その結果得られる培養物を実質的に非繊維芽細胞を含まないようにし、真皮の試料をメス又はハサミで細切する。検体片をピンセットで組織培養フラスコの乾燥表面上に1つずつ置き、5〜10分の間付着させ、次に少量の培地を付着した組織断片をはがさないように注意しながらゆっくり加える。24時間インキュベーションした後、フラスコに追加の培地を入れる。75cm2の組織培養フラスコを使用して培養を開始する時は、培地の初期の量は3〜4mlである。生検検体からの細胞株の樹立には通常2〜3週間かかり、増殖するために、適切な時期に初期の培養容器から細胞をはがすことができる。
本発明の細胞懸濁物は通常の外科的手法により被験者の体内、特に顔へ注入することができる。具体的には、注入希望部位に、所望により局所麻酔薬含有テープ(例えば、ペンレス(商品名))を貼って15分程度放置する。急ぎの場合はこの過程は省略可能である。次に、上述した方法により調製した細胞懸濁物を、例えば、27ゲージの注射針を付けたシリンジで皮下に注入する。注入量の目安としては、1ml(3×107個の細胞)はおよそ皮膚表面積の6〜10cm2に注入可能な量である。具体的には、片側下眼瞼全体で約0.3ml〜0.6ml使用する。
(非染色細胞数/全体の細胞数)×100
Claims (6)
- リドカイン、メピバカイン、ジブカイン、ブピバカイン、プロピトカイン、コカイン、プロカイン、クロロプロカイン、テトラカイン及びその薬理的に許容しうる塩からなる群より選択される何れかの局所麻酔薬を含有することを特徴とする哺乳動物の継代された皮膚繊維芽細胞懸濁物用凝集抑制剤。
- 前記局所麻酔薬が、塩酸リドカインである請求項1に記載の凝集抑制剤。
- 前記局所麻酔薬が、皮膚繊維芽細胞懸濁物中に0.1〜5重量%添加される請求項1又は2に記載の凝集抑制剤。
- ヒト被験者の皮下組織に注入して前記被験者の皮膚欠損を修復するための、自己の継代皮膚繊維芽細胞の懸濁物であって、請求項1〜3の何れかに記載の凝集抑制剤を含有することを特徴とする凝集作用の抑制された継代皮膚繊維芽細胞懸濁物。
- 被験者へ注入する際に、被験者の痛みが低減されることを特徴とする請求項4に記載の継代皮膚繊維芽細胞懸濁物。
- (a)被験者の皮膚の生検により得られた皮膚繊維芽細胞を、0.5〜20容量%の自己血清を含む培地中で継代培養し、脂肪細胞、ケラチノサイト、及び細胞外マトリクスを実質的に含まない皮膚繊維芽細胞を提供する工程、
(b)継代培養した繊維芽細胞をタンパク質分解酵素に暴露して繊維芽細胞を懸濁する工程、及び
(c)前記繊維芽細胞懸濁物に0.1〜5重量%の局所麻酔薬を添加する工程
を含み、前記局所麻酔剤がリドカイン、メピバカイン、ジブカイン、ブピバカイン、プロピトカイン、コカイン、プロカイン、クロロプロカイン、テトラカイン及びその薬理的に許容しうる塩からなる群より選択されることを特徴とする凝集作用の抑制された継代皮膚繊維芽細胞懸濁物の製造方法。
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