JP4916969B2 - ガス状のフッ化化合物充填脂微小球 - Google Patents
ガス状のフッ化化合物充填脂微小球 Download PDFInfo
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- JP4916969B2 JP4916969B2 JP2007190697A JP2007190697A JP4916969B2 JP 4916969 B2 JP4916969 B2 JP 4916969B2 JP 2007190697 A JP2007190697 A JP 2007190697A JP 2007190697 A JP2007190697 A JP 2007190697A JP 4916969 B2 JP4916969 B2 JP 4916969B2
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- gas
- liposomes
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- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- AQTQHPDCURKLKT-JKDPCDLQSA-N vincristine sulfate Chemical compound OS(O)(=O)=O.C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C=O)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 AQTQHPDCURKLKT-JKDPCDLQSA-N 0.000 description 1
- 229960002110 vincristine sulfate Drugs 0.000 description 1
- 229960004854 viral vaccine Drugs 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 239000011800 void material Substances 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 229920001285 xanthan gum Polymers 0.000 description 1
- 235000010493 xanthan gum Nutrition 0.000 description 1
- 239000000230 xanthan gum Substances 0.000 description 1
- 229940082509 xanthan gum Drugs 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
- 229910052727 yttrium Inorganic materials 0.000 description 1
- VWQVUPCCIRVNHF-UHFFFAOYSA-N yttrium atom Chemical compound [Y] VWQVUPCCIRVNHF-UHFFFAOYSA-N 0.000 description 1
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 description 1
- 239000002076 α-tocopherol Substances 0.000 description 1
- 235000004835 α-tocopherol Nutrition 0.000 description 1
- 150000008495 β-glucosides Chemical class 0.000 description 1
- 239000002132 β-lactam antibiotic Substances 0.000 description 1
- 229940124586 β-lactam antibiotics Drugs 0.000 description 1
Classifications
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- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1806—Suspensions, emulsions, colloids, dispersions
- A61K49/1815—Suspensions, emulsions, colloids, dispersions compo-inhalant, e.g. breath tests
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
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- A61K9/10—Dispersions; Emulsions
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- A61K9/1277—Processes for preparing; Proliposomes
- A61K9/1278—Post-loading, e.g. by ion or pH gradient
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
- A61M5/178—Syringes
- A61M5/31—Details
- A61M5/3145—Filters incorporated in syringes
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y10S977/906—Drug delivery
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Description
化が最も顕著に現れる。超音波がそのような界面に遭遇すると、音響インピーダンスが変化する結果として、音波がより強く反射してより強いシグナルが超音波画像に現れる。音の反射効率に影響を与える追加的要因は、その反射する界面の弾性である。この界面の弾性が高ければ高いほど、音の反射効率が高くなる。気泡の如き物質が高い弾性を示す界面を与える。このように、上に示した原理の結果として、研究者達は気泡または気体含有体を基とする超音波コントラスト剤の開発およびそれらの有効な製造方法を開発することに焦点を当てていた。
。特異的治療薬送達方法は、毒性のある副作用を最小限にし、必要とされる投薬量を低くし、そして患者にかかる費用を低くする働きをする可能性がある。
有用である。内部に液体が入っている従来技術のリポソーム(これらが適切なのは水溶性薬物をカプセル封じする場合のみである)とは異なり、本発明に従って製造する気体前駆体充填リポソームは、親油性薬物をカプセル封じするに特に有用である。更に、薬物の親油性誘導体、例えば二ハロゲン化メタロセンのアルキル化誘導体などを容易に脂質層の中に組み込むことができる。Kuo他、J.Am.Chem.Soc.1991、113、9027−9045。
本発明は、温度で活性化される気体前駆体充填リポソームを製造する方法および装置に向けたものである。内部が水溶液で満たされているリポソームを製造することに向けられた従来技術の方法とは異なり、本発明の方法は、内部に気体前駆体および/または最終的に気体が入っているリポソームを製造することに向けたものである。
例えば、気体前駆体であるパーフルオロブタンをリポソームの中に捕捉させることができ、そして温度を高めて3℃(パーフルオロブタンの沸点)を越えさせると、リポソームに捕捉されているフルオロブタンのガスが発生する。さらなる例として、乳化剤および安定化剤、例えばグリセロールまたはプロピレングリコールなどが入っている水懸濁液の中に気体前駆体であるフルオロブタンを懸濁させて市販ボルテクサー(vortexer)で渦流を起こさせることができる。この気体前駆体が液状であるに充分なほど低い温度で渦流を起こさせ、そして液体状態から気体状態に移行する相転移温度を過ぎるまでそのサンプルの温度を高くしながら渦流を継続する。このようにすることで、この微細乳化過程中に該前駆体は気体状態に変化する。適当な安定剤を存在させると、驚くべきことに、安定な気体充填リポソームが生じる。
PV=nRT
ここで、
P=気圧で表す圧力
V=リットルで表す体積
n=気体のモル数
T=度Kで表す温度
R=理想気体定数=22.4L気圧度−1モル−1。
体積(球)=4/3πr3
[式中、r=球の半径である]
で決定され得る。
Vgas=4/3π(rgas)3
に理想気体法則
RV=nRT
を代入すると、
Vgas=nRT/Pgas
になる、即ち
(A)n=4/3[πrgas 3]P/RT
量n=4/3[πrgas 3P/RT]*MWn、
これを液体の体積に戻して換算すると、
(B)Vliq=[4/3[πrgas 3]P/RT]*MWn/D]
[ここで、D=前駆体の密度]
になり、これを液滴の直径で解くと、
(C)直径/2=[3/4π[4/3*[πrgas 3]P/RT]MWn/D]1/3になり、これを換算すると、
直径=2[[rgas 3]P/RT[MWn/D]]1/3
になる。
濾過として使用することが可能になる。
Inxa=In(1−Xb)=ΔHfus/R(1/To−1/T)
[ここで、
Xa=溶媒のモル分率
Xb=溶質のモル分率
ΔHfus=溶媒の融解熱
To=溶媒の標準凝固点]
を用いて溶媒の凝固点降下を表すことができる。
Ma=溶媒の分子量、および
m=1000グラム当たりのモル数で表す溶質の重量モル濃度]。
ΔT=[MaRTo 2/1000ΔHfus]m
または ΔT=Kfm
[ここで、Kf=MaRTo 2/1000ΔHfus]
になる。
本発明の気体前駆体充填リポソームが入っている水懸濁液に渦流を起こさせる方法[この方法に関する変法には、振とうする前に任意にオートクレーブにかける変法、気体前駆体と脂質が入っている水懸濁液を任意に加熱する変法、この懸濁液が入っている容器の排気を任意に行う変法、任意に振とうするか或は自然発生的にこの気体前駆体リポソームを生じさせてこの気体前駆体充填リポソームの懸濁液を冷却する変法、そして気体前駆体と脂質が入っている水懸濁液を約0.22μmのフィルターに通して任意に押出すか、別法として、結果として生じるリポソームのインビボ投与中に約0.22μmのフィルターを用いたのと同様な濾過が生じるように濾過を実施してもよい変法が含まれる];
微細乳化方法[ここでは、本発明の気体前駆体充填リポソームが入っている水懸濁液を撹拌で乳化させそして加熱することで患者に投与する前の微小球を生じさせる]、および
加熱および/または撹拌で脂質懸濁液中の気体前駆体を生じさせる方法[ここでは、容器の中に入っている他の微小球を膨張させて追い出しそしてその容器の排気を行うことで空気を放出させることにより、より低い密度を有する気体充填微小球をその溶液の上部に浮遊させる]。
ポソーム類を該脂質の相転移温度より低い温度で生じさせると、そのゲル相はより堅いにも拘らず、生物学的系内で利用可能である。
、含まれるのは完全に泡である。
って脂質水溶液74を生じる。また、この乾燥脂質51を混合容器66の導入するに先立ってこれを水和させることにより、脂質を水溶液の状態で導入することも可能である。このリポソーム製造方法の好適な態様では、溶液74の初期仕込みを、この溶液が混合容器66の容量の一部のみを占めるように行う。更に、連続方法では、この脂質溶液74の体積が混合容器66容量の前以て決めたパーセントを越えないように、この脂質水溶液74の添加速度および生じる気体前駆体充填リポソームを取り出す速度を調節する。
に挿入してプランジャー106を引くことで前以て決めた量の泡を容器104の中に吸い込むことにより、抽出を達成することができる。以下で更に考察するように、針102の末端を泡73の中に位置させる場所選択を利用することでその抽出する気体前駆体充填リポソームの大きさを調節することができる。
ができる。このように、混合容器66内における抽出用管67の鉛直位置を設定することで、その抽出すべき気体前駆体充填リポソームの大きさを調節することができ、ここでは、この管の位置を高くすればするほど、その抽出される気体前駆体充填リポソームのサイズが大きくなる。更に、混合容器66内における抽出用管67の鉛直位置を周期的または連続的に調整することにより、継続を基礎にして、その抽出すべき気体前駆体充填リポソームの大きさを調節することができる。混合容器66内における抽出用管67の鉛直位置を正確に調整することを可能にする、抽出用管67に取り付けたねじ付きスリーブ72に合致するねじ付きカラー71であってもよいデバイス68を組み込むことにより、上記抽出を容易に行うことができるようにしてもよい。
或はほとんど残存していない気体前駆体充填リポソームが生じる。図8Aは、オートクレーブにかけた後であるが濾過を行う前のその生じた気体前駆体充填リポソームを示しており、このように、10μm以上の大きさを有する気体前駆体充填リポソームが多数生じていた。図8Bは、10μmの「NUCLEPORE」フィルターに通して濾過した後の気体前駆体充填リポソームを示しており、ここでは、約10μmの均一な大きさが生じていた。
オロ、プロパン−1,2−エポキシ、プロパン−2,2ジフルオロ、プロパン−2−アミノ、プロパン−2−クロロ、プロパン−ヘプタフルオロ−1−ニトロ、プロパン−ヘプタフルオロ−1−ニトロソ、プロパン−パーフルオロ、プロペン、プロピル−1,1,1,2,3,3−ヘキサフルオロ−2,3ジクロロ、プロピレン−1−クロロ、プロピレン−クロロ−(トランス)、プロピレン−2−クロロ、プロピレン−3−フルオロ、プロピレン−パーフルオロ、プロピン、プロピン−3,3,3−トリフルオロ、スチレン−3−フルオロ、六フッ化硫黄、硫黄(ジ)−デカフルオロ(S2F10)、トルエン−2,4−ジアミノ、トリフルオロアセトニトリル、トリフルオロメチルパーオキサイド、トリフルオロメチルスルフィド、六フッ化タングステン、ビニルアセチレン、ビニルエーテル、キセノン、窒素、空気および他の周囲ガス。
テイン、および/またはそれらの組み合わせなどの如き脂質が含まれる。このリポソーム類を単層または2層として生じさせてもよく、そしてこれらに被膜を持たせるか或は持たせなくてもよい。
リポソームに結合させるには一般に正に帯電した脂質を用いる必要があると今まで考えられていたからである。このリポソームの中にホスファチジン酸を5から10モル%入れると、該気体前駆体充填リポソームの安定性とサイズ分布が改良される。
でない。この気体前駆体を用いる結果として得られる気体充填微小球は空気より安定なことから、有効なコントラスト増強を与えるようにこれらを更に設計することができ、例えばこれらは、1種以上の被覆材または乳化剤で特に安定化しなくても、末梢静脈注入後、肺循環の中を通り抜ける。しかしながら、柔軟性のある安定化用材料のような被覆材または安定剤を1種以上用いるのが好適である。多糖類、ガングリオシド類およびポリマー類で安定化された気体微小球は、アルブミンおよび他の蛋白質で安定化されたものよりも有効である。脂肪族化合物を用いて製造したリポソームが好適である、と言うのは、このような化合物で安定化された微小球の方がずっと高い柔軟性を示すと共に圧力変化に対して安定であるからである。
25%のみにし、より好適にはその放出される気体の量を約10%のみにし、最も好適にはその放出される気体の量を約1%のみにする。
これらは送達中に安定であることに加えて、共振周波数の超音波、無線周波数のエネルギー(例えばマイクロ波)および/またはエコー源性をかけると破裂し得ることでこれらを選択したからである。
た、望まれるならば、この溶液に緩衝剤を入れてpHの範囲を約pH5から約pH7.4にしてもよい。適切な緩衝剤には、これらに限定するものでないが、酢酸塩、クエン酸塩、燐酸塩、重炭酸塩および燐酸塩緩衝食塩水、5%デキストロースおよび生理学的食塩水(標準食塩水)が含まれる。
のサイズを調整する好適な方法である。
ソームを投与することができる。
ル、パルミチン酸イソプロピル、鉱油、ミリスチルアルコール、オクチルドデカノール、オリーブ油、ピーナッツ油、桃仁油、ゴマ油、大豆油、スクアレン、オレイン酸ミリスチル、オレイン酸セチル、パルミチン酸ミリスチルに加えて、アルキル鎖脂肪酸(C=2−22)にエステル化した他の飽和および不飽和アルキル鎖アルコール類(C=2−22)が含まれる。
は移植してもよい。一般的には、超音波をより低い強度で始めて短期間行った後、このリポソームが超音波で可視化される(診断用超音波用途の場合)か或は破裂する(治療用超音波用途の場合)まで、強度、時間および/または共振周波数を高めて行く。
周波数の音を用いるのが好適である。非常に高い周波数を用いると、例えば10メガヘルツ以上の周波数を用いると、その音波エネルギーが流体および組織の中に浸透する深さが一般に制限される。皮膚および他の表面組織では音波エネルギーを外部からかけるのが好適であり得るが、深い所に存在する構造物の場合、間隙探針または筋肉内超音波カテーテルを通して音波エネルギーをかけるのが好適であり得る。
料に結合し得る基をその脂質層の中に組み込み、これを後で上記材料に結合させる。遺伝材料(DNA、RNA、1本鎖および2本鎖両方のアンチセンスおよびセンスオリゴヌクレオチド類)の場合、この組み込みは、乾燥脂質出発材料内に組み込み可能なカチオン脂質またはカチオンポリマーを用いることで容易に達成される。
チン(VLB)、硫酸ビンクリスチン、ブレオマイシン、硫酸ブレオマイシン、メトトレキセート、アドリアマイシンおよびアラビノシル、ヒドロキシ尿素、プロカルバジンおよびダカルバジンなど、有糸分裂阻害剤、例えばエトポシドおよびビンカアルカロイド類、放射性薬品、例えば放射線ヨウ素およびリン製品など、ホルモン類、例えばプロゲスチン類、エストロゲンおよびアンチエストロゲン類、駆虫薬、抗マラリア薬および抗結核薬、生物学剤、例えば免疫血清、抗毒素および抗ヘビ毒素、狂犬病予防品、細菌ワクチン、ウイルスワクチン、アミノグリコシド類、呼吸用製品、例えばキサンチン誘導体テオフィリンおよびアミノフィリンなど、乾燥甲状せん製剤、例えばヨウ素製品および抗甲状せん薬など、心臓血管用製品(キレート剤および水銀利尿薬および心臓グリコシド類を含む)、グルカゴン、血液製品、例えば非経口鉄、ヘミン、ヘマトポルフィリン類およびそれらの誘導体、生物学的応答修飾因子、例えばムラミルジペプチド、ムラミルトリペプチド、微生物細胞壁成分、リンフォカイン類(例えば細菌のエンドトキシン、例えばリポポリサッカライド、マクロファージ活性化因子など)、細菌のサブユニット(例えばミコバクテリア、コリネバクテリアなど)、合成ジペプチドであるN−アセチル−ムラミル−L−アラニル−D−イソグルタミン、抗カビ剤、例えばケトコナゾール、ニスタチン、グリセオフルビン、フルシトシン(5−FC)、ミコナゾール、アンホテリシンB、リシン、シクロスポリン類およびβ−ラクタム抗生物質(例えばスルファゼシン)など、ホルモン類、例えば成長ホルモン、メラノサイト刺激ホルモン、エストラジオール、二プロピオン酸ベクロメタゾン、ベタメタゾン、酢酸ベタメタゾンおよび燐酸ベタメタゾンナトリウム、燐酸ベタメタゾン二ナトリウム、燐酸ベタメタゾンナトリウム、酢酸コルチゾン、デキサメタゾン、酢酸デキサメタゾン、燐酸デキサメタゾンナトリウム、フルンソリド、ヒドロコルチゾン、酢酸ヒドロコルチゾン、シクロペンタンプロピオン酸ヒドロコルチゾン、燐酸ヒドロコルチゾンナトリウム、こはく酸ヒドロコルチゾンナトリウム、メチルプレドニゾロン、酢酸メチルプレドニゾロン、こはく酸メチルプレドニゾロンナトリウム、酢酸パラメタゾン、プレドニゾロン、酢酸プレドニゾロン、燐酸プレドニゾロンナトリウム、プレドニゾロンテルブテート(terbutate)、プレドニゾン、トリアムシノロン、トリアムシノロンアセトニド、二酢酸トリアムシノロン、トリアムシノロンヘキサアセトニドおよび酢酸フルドロコルチゾン、オキシトシン、バソプレシンおよびそれらの誘導体など、ビタミン類、例えばシアノコバラミンネイノイックアシッド(neinoic acid)、レチノイド類および誘導体、例えばパルミチン酸レチノールおよびα−トコフェロールなど、ペプチド類、例えばマンガンスーパーオキシドジムターゼなど、酵素類、例えばアルカリ性ホスファターゼなど、抗アレルギー薬、例えばアメレキサノックスなど、抗凝血薬、例えばフェンプロクモンおよびヘパリンなど、循環薬、例えばプロプラノロールなど、代謝増強薬、例えばグルタチオンなど、抗結核薬、例えばパラアミノサリチル酸、イソニアジド、硫酸カプレオマイシンシクロセリン、塩酸エタムブトールエチオナミド、ピラジナミド、リファムピンおよび硫酸ストレプトマイシンなど、抗ウイルス薬、例えばアシクロビル(acyclovir)、アマンタジンアジドチミジン(AZTまたはジドブジン)、リバビリンおよびビダラビン一水化物(アデニンアラビノシド、アラ−A)など、抗狭心薬、例えばジルチアゼム、ニフェジピン、ベラパミル、四硝酸エリトリチル、二硝酸イソソルビド、ニトログリセリン(三硝酸グリセリル)および四硝酸ペンタエリスリトールなど、抗凝血薬、例えばフェンプロクモンおよびヘパリンなど、抗生物質、例えばダプソン、クロラムフェニコール、ネオマイシン、セファクロール、セファドロキシル、セファレキシン、セフラジン、エリスロマイシン、クリンダマイシン、リンコマイシン、アモキシシリン、アンピシリン、バカムピシリン、カルベニシリン、ジクロキサシリン、シクラシリン、ピクロキサシリン、ヘタシリン、メチシリン、ナフシリン、オキサシリン、ペニシリン(ペニシリンG、ペニシリンV、チカルシリンリファムピンおよびテトラシクリンを含む)など、抗炎症薬、例えばジフニサール、イブプロフェン、インドメタシン、メクロフェナメート、メフェナム酸、ナプロキセン、オキシフェンブタゾン、フェニルブタゾン、ピロキシカム、スリンダック、トルメチン、アスピリンおよびサリチレート類など、抗原虫薬、例えばクロロキン、ヒドロキシクロロキン、メトロニダゾール、キニ
ンおよびアンチモン酸メグルミンなど、抗リューマチ薬、例えばペニシルアミンなど、麻酔薬、例えばアヘン安息香チンキなど、アヘン剤、例えばコデイン、ヘロイン、メタドン、モルフィネおよびアヘンなど、心臓グリコシド類、例えばデスラノシド、ジギトキシン、ジゴキシン、ジギタリンおよびジギタリスなど、神経筋遮断薬、例えばアトラクリウムベシレート、ガラミントリエチオジド、臭化ヘキサフルオレニウム、メトクリンヨージド、パンクロニウムブロマイド、スクシニルコリンクロライド(スキサメトニウムクロライド)、ツボクラリンクロライドおよびベクロニウムブロマイドなど、鎮静薬(催眠薬)、例えばアモバルビタール、アモバルビタールナトリウム、アプロバルビタール、ブタバルビタールナトリウム、クロラール水化物、エトクロルビノール、エチナメート、塩酸フルラゼパム、グルテチミド、塩酸メトトリメプラジン、メチプリロン、塩酸ミダゾラム、パラアルデヒド、ペントバルビタール、ペントバルビタールナトリウム、フェノバルビタールナトリウム、セコバルビタールナトリウム、タルブタール、テマゼパムおよびトリアゾラムなど、局所麻酔薬、例えば塩酸ブピバカイン、塩酸クロロプロカイン、塩酸エチドカイン、塩酸リドカイン、塩酸メピバカイン、塩酸プロカインおよび塩酸テトラカインなど、一般麻酔薬、例えばドロペリドール、エトミデート、ドロペリドールと組み合わせたクエン酸フェンタニール、塩酸ケタミン、メトヘキシタールナトリウムおよびチオペンタールナトリウムなど、および放射性粒子またはイオン、例えばストロンチウム、ヨウ化物、レニウムおよびイットリウムなどが含まれる。
例えば、単一のリポソームに2種以上の治療薬を含めてもよいか、或は異なる治療薬を含むリポソームを共投与してもよい。例として、黒色腫抗原に結合し得るモノクローナル抗体とIL−2の少なくとも一部をコード化するオリゴヌクレオチドを同時投与してもよい。本明細書で用いる如き用語「の少なくとも一部」は、その代表する遺伝子部分が有効に遺伝子発現をブロックする限り、このオリゴヌクレオチドが必ずしも全遺伝子に相当する必要はないことを意味する。
トレキセート)、5’−アシレートを用いたシトシンアラビノシド、ナイトロジェンマスタード(2,2’−ジクロロ−N−メチルジエチルアミン)、アミノメチルテトラシクリンを用いたナイトロジェンマスタード、コレステロールまたはエストラジオールまたはデヒドロエピアンドロステロンエステルを用いたナイトロジェンマスタードおよびアゾベンゼンを用いたナイトロジェンマスタードなどが含まれる。
4−ジメチルバレロニトリル)などが含まれる。
パーフルオロブタン(Pfaltz and Bauer、Waterbury、CT)を用いた気体前駆体含有リポソームの製造を下記の如く行った:ゴムストッパーが付いているガラス製ボトル(ボトルの容積=15.8mL)の中に、8:1:1の標準食塩水
:グリセロール:プロピレングリコール中に脂質が1mL当たり5mgの量で入っている溶液[脂質=87モル%のDPPC、8モル%のDPPE−PEG5,000、5モル%のジパルミトイルホスファチジン酸(全ての脂質をAvanti Polar Lipids、Alabaster、ALから入手)]を5mL入れた。Model Welch
2−Stage DirecTorr Pump(VWR Scientific、Cerritos、CA)真空ポンプを用い、上記ゴムストッパーに穴を開けて通した18ゲージの針で上記ボトルにホースを連結することにより、このボトルから空気を排除した。この気体を真空で除去した後、パーフルオロブタンが入っている缶に取り付けた配管に別の18ゲージ針を連結することによって、このストッパー付きボトルの中にパーフルオロブタンを入れた。このストッパー付きボトルから如何なる痕跡量の空気も除去されそして脂質溶液の上部空間がパーフルオロブタンで完全に満たされるように、上記過程を5回繰り返した。この18ゲージの針を1瞬または2瞬排気することで、上記ガラス製ボトルの内圧を周囲圧力と平衡状態にした後、この18ゲージの針を上記ストッパーから引き抜いた。このボトルをパーフルオロブタンで満たした後、ゴムバンドを用いてこのボトルをWig−L−Bug(商標)(Crescent Dental Mfg.Co.、Lyons、IL)のアームにしっかりと取り付けることで、このボトルを固定した。次に、このWig−L−Bug(商標)を用いて上記ボトルを60秒間振とうした。泡だった懸濁液が生じ、そしてこの泡層がその底に在る透明な溶液からいくらか分離するに数分間かかることが認められた。振とう後、この材料の体積は5ccから約12ccに上昇し、このことは、このリポソームがパーフルオロカーボン気体前駆体を約7cc捕捉したことを示唆していた。Accusizer(Model 770、Particle Sizing System、Santa Barbara、CA)を用いてこの材料の大きさを合わせ、そしてまた偏光顕微鏡(Nikon TMS、Nikon)を用い、150x倍率で検査した。このリポソームは、平均直径が約20から50ミクロンであるいくらか大きい球形構造物として現れた。次に、このリポソームの一部を、シリンジにより、孔サイズが8ミクロンのCostarフィルター(Syrfil 800938、Costar、Pleasanton、CA)に通して注入した。光顕微鏡およびAccusizer
Systemを用いて再びこのリポソームを検査した。このリポソームの平均サイズは約3ミクロンであり、そして体積重量平均(volume weighted mean)は約7ミクロンであった。このリポソームの99.9%以上が11ミクロン以下のサイズであった。上記実験は、気体前駆体ガスであるパーフルオロブタンを用いて振とうおよび濾過方法により非常に望ましい大きさを有するリポソームを製造することができることを示している。
で、アセプロマジンが20mg/mL)で鎮静化した後、Acoustic Imaging、モデルNo.7200臨床用超音波機を用いて走査し、7.5MHzの変換器を用い、カラードップラーでその腎臓を検査した。腎臓を検査すると同時にまた、2番目のAcoustic Imaging超音波機モデル番号5200を用いてこのラビットの心臓を走査し、7.5MHzの変換器を用い、その心臓の画像形成をグレースケールで行った。8ミクロンのフィルター(上を参照)を取り付けたシリンジを用いて、パーフルオロブタンで満たしたリポソームを耳の静脈を通して注入することでこれを投与した。この気体前駆体であるパーフルオロブタンが入っているリポソームを0.5cc(1kg当たり0.15cc)注入した後、腎臓の劇的および持続的増強を30分に渡って観察した。これは腎実質内の明るい色として表れ、これは、腎弓状動脈および微小循環内のシグナルが高まることを反映している。同時に行った心臓の画像形成は、心臓の可視化を妨害する影が最初の数分間表れることを示していた、即ち反射があまりにも強すぎる結果として、超音波ビームが完全に反射して吸収された。しかしながら、数分後、心室および血液プールに明るい持続した増強が観察され、これはまた50分以上持続した。また、グレースケール超音波機を用いて肝臓の画像も得た。これは心臓および血液プールが増強されるのと同時に実質および血液も増強されることを示していた。
気体前駆体を添加した後その内容物をMicrofluidicsミクロフルイダイザー(Microfluidics Inc.、Newton、MA)に6回通すことでこれに微細流動化を受けさせる以外は実施例1と同様にして気体前駆体充填脂質二重層を製造した。ストローク圧を10,000から20,000psiの範囲にした。実施例1に従う製造を継続することで、気体前駆体がカプセル封じされた気体充填脂質二重層が生じた。
DPPCを5モル%量のホスファチジン酸(Avanti Polar Lipids、Alabaster、AL)と組み合わせて用いること以外は実施例1に挙げたのと同様にして気体充填脂質二重層を製造した。気体充填脂質二重層が生じる結果として、下方の水系ビヒクル層の中に存在する脂質粒子の量が少なくなることで例示されるように、溶解度上昇が生じた。結果として起こる大きさ合わせにより、明らかに、DPPC単独に比較して全平均サイズが40um以下にまで低下した。
8:1:1(体積:体積:体積)の標準食塩水:プロピレングリコール:グリセロールから成るビヒクルの中にジパルミトイルホスファチジルコリンが82モル%、ジパルミトイルホスファチジン酸が10モル%およびジパルミトイルホスファチジルエタノールアミン−PEG5,000(Avanti Polar Lipids、Alabaster、AL)が8モル%入っている脂質調合物を用いる以外は実施例3で考察したのと同様に
してパーフルオロブタンがカプセル封じされた脂質二重層を形成させ、その結果として、泡と、主に如何なる粒子も含まれていない下方のビヒクル層が生じた。このビヒクルを変化させると、下方のビヒクル層の透明度が変化した。実施例3と同様にして調合物を製造することでパーフルオロブタンが入っている気体充填脂質二重層を生じさせた。濾過を行う前に、Particle Sizing SYstems Model 770オプティカルサイザー(optical sizer)(Particle Sizing Systems、Santa Barbara、CA)を用いて気体充填微小球の大きさを測定した。大きさ測定の結果、全粒子の99%が34μm以内であった。次に、その結果として得られる生成物を8μmのフィルターに通して濾過することで均一なサイズを有する微小球が得られた。この続いて行った微小球の大きさ測定の結果、全粒子の99.5%が10μm以内であった。この生成物を実施例1のインビボ実験で用いた。
ホスファチジン酸と気体前駆体を含有するリポソームおよび気体含有リポソームとDNAの結合。ジステアロイル−sn−グリセロホスフェート(DSPA)(Avanti Polar Lipids、Alabaster、AL)の7mM溶液を標準食塩水の中に懸濁させて50℃で渦流を起こさせた。この材料を室温に冷却した。この脂質溶液にpBR322プラスミドDNA(International Biotechnologies,Inc.、New Haven、CT)を40ミクログラム加えて穏やかに振とうした。Beckman TJ−6遠心分離器(Beckman、Fullerton、CA)を用いて上記溶液を10分間遠心分離にかけた。Hoefer TKO−100 DNA Fluorometer(Hoefer、San Francisco、CA)を用いてその上澄み液と沈澱物をDNA含有量に関して評価した。この方法では挿入用染料であるHoechst 33258(これはDNAに特異的である)を用いたことから、これで検出されるのは2本鎖DNAのみである。ホスファチジン酸を用いて製造した、負に帯電したリポソームまたは正味の負電荷を有する脂質が驚くべきことにDNAと結合することを見出した。DPPCで構成させた中性リポソームを対照として用いて上記実験を繰り返した。このDPPCリポソームを用いた場合、DNAは評価されるほどの量で検出されなかった。微小球中の脂質がDPPC:DEPPE−PEG500:DPPAが87:8:5モル%である混合物を用いて製造した気体充填リポソームを用いて上記実験を繰り返した。再び、このジパルミトイルホスファチジン酸を含む気体充填リポソームにDNAが結合した。
ミクロフルイダイザー(Microfluidics、Newton、MA)を−20℃の冷室に入れた。この冷室に、脂質溶液が25cc入っておりそしてヘッドスペースにパーフルオロブタンが35cc含まれているストッパー付きガラス製フラスコを入れた。この脂質溶液は、8:1:1の燐酸塩緩衝食塩水(pH7.4):グリセロール:プロピレングリコールの中にDPPC:DPPE+PEG5,000:DPPAが83:8:5のモル比で入っている溶液であった。この溶液は上記冷室内で凝固しなかったが、パーフルオロブタンは液体になった。
が約30nmから約50nmである限定されたサイズを有する小胞が生じた。室温に温めると、約10ミクロンの安定化微小球が生じた。
遠心分離管の中に1,2−ジパルミトイル−sn−グリセロ−3−ホスホコリン(MW:734.05、粉末、ロット番号160pc−183)(Avanti−Polar Lipids、Alabaster、AL)を重量測定して50mg入れ、そして5.0mLの食塩水溶液(0.9%のNaCl)または燐酸塩緩衝食塩水(塩化ナトリウムを0.8%、塩化カリウムを0.02%、二塩基性燐酸ナトリウムを0.115%および一塩基性燐酸カリウムを0.02%入れて、pHを7.4に調整)を用いて水和させる。この懸濁液に2−メチル−2−ブテンを165μLmL−1加える。次に、この水和させた懸濁液を、渦流機(Scientific Industries、Bohemia、NY)を用い装置設定を6.5にして10分間振とうする。全体積が12mLになることを確認する。この食塩水溶液は5.0mLから約4mLにまで低下すると予測される。
遠心分離管の中に1,2−ジパルミトイル−sn−グリセロ−3−ホスホコリン(MW
:734.05、粉末)(Avanti−Polar
Lipids、Alabaster、AL)を重量測定して50mg入れる。次に、この脂質を5.0mLの食塩水溶液(0.9%のNaCl)で水和させる。この懸濁液に2−メチル−2−ブテンを165μLmL−1加える。次に、装置設定を6.5にして上記脂質に10分間渦流を起こさせる。渦流を起こさせた後、この溶液全体を液体窒素内で凝固させる。次に、このサンプルを凍結乾燥機上に置いて凍結乾燥させる。このサンプルをこの凍結乾燥機上に18時間置く。この乾燥させた脂質を上記凍結乾燥機から取り出し、5mLの食塩水溶液の中で再び水和させた後、6.5に設定して渦流を10分間起こさせる。ピペットを用いてこの溶液の少量サンプルをスライドに乗せてこの溶液を顕微鏡下で見る。次に、この気体前駆体充填リポソームの大きさを測定する。このリポソームの最大サイズが約60μmでありそして検出される最小サイズが約20μmであることを確認する。その平均サイズ範囲は約30から約40μmである。
遠心分離管の中に1,2−ジパルミトイル−sn−グリセロ−3−ホスホコリン(MW:734.05、粉末)(Avanti−Polar Lipids、Alabaster、AL)を重量測定して50mg入れる。この懸濁液に2−メチル−2−ブテンを165μLmL−1加える。円錐形遠心分離管の回りに約2フィートのラテックス製配管(内径0.25インチ)をコイル様式で巻く。次に、このラテックス製配管を電気用テープで上記遠心分離管にしっかりと固定する。次に、このラテックス製配管を一定温度の循環浴(VWR Scientific Model 1131)に連結する。この浴の温度を60℃に設定し、そしてこの水の循環を高速に設定して上記配管の中を循環させる。上記脂質溶液の中に温度計を入れて42℃から50℃の範囲であることを確認する。
遠心分離管の中に1,2−ジパルミトイル−sn−グリセロ−3−ホスホコリン(MW:734.05、粉末)(Avanti−Polar Lipids、Alabaster、AL)を重量測定して50mg入れる。次に、この脂質に5.0mLの0.9%NaClを加えることでこれを水和させる。この懸濁液に2−メチル−2−ブテンを165μLmL−1加える。装置設定を6.5にして上記脂質水溶液に10分間渦流を起こさせる。渦流を起こさせた後、この溶液全体を液体窒素内で凝固させる。次に、この溶液全体を室温(25℃)の水浴の中に入れて解凍を起こさせる。次に、この凍結解凍手順を8回繰り返す。次に、装置を6.5に設定して上記水和懸濁液に渦流を10分間起こさせる。次に、例6に記述したのと同様に気体前駆体充填リポソームの検出を行う。
各々にDPPCが50mg入っている2個の遠心分離管を調製する。この遠心分離管の1つにラウリル硫酸ナトリウムを1モル%(Duponol Cロット番号2832を〜0.2mg)加え、そしてもう1つの管に10モル%(Duponol Cロット番号2832を2.0mg)入れる。両方の遠心分離管に0.9%NaClを5mL加える。両方の管に2−メチル−2−ブテンを165μLmL−1加える。この管の両方を液体窒素
内で凝固させた後、凍結乾燥を約16時間行う。両方のサンプルを凍結乾燥機から取り出して両方の管に食塩水を5mL加える。6.5の位置にして両方の管に渦流を10分間起こさせる。
脂質である1,2−ジパルミトイル−sn−グリセロ−3−ホスホコリン(Avanti−Polar Lipids、Alabaster、AL)を50mg重量測定して、5mLの0.9%NaClで水和させる。この懸濁液に2−メチル−2−ブテンを165μLmL−1加える。渦流を起こさせる代わりに、Heat Systems Sonicator Ultrasonic Processor XL(Heat Systems,Inc.、Farmingdale、NY)Model XL 2020を用いて、この水溶液を音波処理する。周波数が20KHzである上記音波処理器のノブを位置4にして、この音波処理器を連続波に設定する。マイクロチップを用いて音波処理を4℃の温度で10分間行う。音波処理後、温度が40℃にまで上昇し、そしてその溶液を光学顕微鏡下で見る。気体前駆体充填リポソームが生じたことの証拠を確認する。
この実施例では、脂質濃度が下限濃度より低くなると気体前駆体充填リポソームの生成が休止するか否かを確認する。10mgの1,2−ジパルミトイル−sn−グリセロ−3−ホスホコリン(Avanti−Polar Lipids、Alabaster、AL)を10mLの食塩水に加える。この懸濁液に2−メチル−2−ブテンを165μLmL−1加える。6.5の位置にして上記脂質/食塩水/気体前駆体溶液に渦流を10分間起こさせる。この溶液を光学顕微鏡下で見てサイズを測定する。このリポソームの最大サイズが約30μmから約45μmの範囲でありそして検出される最小サイズが約7μmであることを確認する。その平均サイズ範囲は約30から約45μmである。
濾過していない気体前駆体充填リポソームを50mLのシリンジの中に吸い込ませ、最短で150μm離れて位置している「NUCLEPORE」10μmフィルターと8μmフィルターから成るカスケードに通す(図3および4)。また、例えば、互いに直接的に隣接している10μmフィルターと8μmフィルターの積み重ねに通してこのサンプルを濾過してもよい。流量が2.0mL分−1になるように、気体前駆体充填リポソームを加圧下で上記フィルターに通す。次に、その結果として濾過された気体前駆体充填リポソームを気体前駆体充填脂質リポソームの収率に関して測定し、その結果、その収率は未濾過体積の80−90%から成る体積である。
Electronics Limited、Luton、Beds.、英国)を用いて、サイズ測定を実施する。図5および6で分かるように、この気体前駆体充填リポソームのサイズは、濾過前の気体前駆体充填リポソームのサイズに比較して、より均一に8−10μm付近に分布している。このように、濾過した後の気体前駆体充填リポソームの方がずっと均一なサイズを有することが分かるであろう。
50mLのFalcon遠心分離管(Becton−Dickinson、Lincoln Park、NJ)の中にDPPC(ジパルミトイルホスファチジルコリン)を250mgおよび0.9%NaClを10mL入れて周囲温度(約20℃)に維持する。この懸濁液に2−メチル−2−ブテンを165μLmL−1加える。次に、この懸濁液を1μmのNuclepore(Costar、Pleasanton、CA)ポリカーボネート膜に通して窒素圧下で押出す。その結果として得られる懸濁物の大きさを、Particle Sizing Systems(Santa Barbara、CA)Model 370レーザー光散乱サイザーを用いて測定する。全ての脂質粒子が平均外側直径で1μmまたはそれ以下である。
50mLのFalcon遠心分離管(Becton−Dickinson、Lincoln Park、NJ)にDSPC(ジステアロイルホスファチジルコリン)を100mgおよび0.9%NaClを10mL入れる。この懸濁液に2−メチル−2−ブテンを165μLmL−1加える。次に、この懸濁液を1μmの「NUCLEPORE」(Costar、Pleasanton、CA)ポリカーボネート膜に通して300−800p.s.i.の窒素圧下で押出す。その結果として得られる懸濁物の大きさを、Particle Sizing Systems(Santa Barbara、CA)Sub Micron Particle Sizer Model 370レーザー光散乱サイザ
ーを用いて測定する。全ての粒子が1μmまたはそれ以下のサイズであることを確認する。
Systems Model 370レーザー光散乱サイザーを用いて測定し、サイズが均一に1μm以下であることを確認する。
50mLのFalcon遠心分離管(Becton−Dickinson、Lincoln Park、ニュージャージー州)に、前以て1μmのフィルターに通して押出してオートクレーブに20分間かけた1,2−ジパルミトイルホスファチジルコリンが0.9%NaCl中に25mg/mLの量で入っている溶液を10mL入れる。この懸濁液に2−メチル−2−ブテンを165μLmL−1加える。この脂質懸濁液を室温(約20℃)と平衡状態にした後、VWR Genie−2(120ボルト、0.5アンペア、60ヘルツ)(Scientific Industries,Inc.、Bohemia、NY)を用いて、10分間か或は気体前駆体充填リポソームの全体積が元の脂質水溶液体積の少なくとも2倍または3倍になるまで、上記脂質に渦流を起こさせる。この管の底に存在する溶液に無水の粒子状脂質はほとんど全く含まれておらず、気体前駆体充填リポソームが入っている泡が多量に生じる。従って、前以てオートクレーブにかけても、この脂質懸濁液が気体前駆体充填リポソームを生じる能力は影響を受けない。オートクレーブにかけてもリポソームのサイズは変化せず、そしてこの脂質懸濁液が気体前駆体充填リポソームを形成する能力は低下しない。
50mLのFalcon遠心分離管(Becton−Dickinson、Lincoln Park、NJ)に、前以て1μmのフィルターに通して押出してオートクレーブに20分間かけた1,2−ジパルミトイルホスファチジルコリンが0.9%NaCl中に25mg/mLの量で入っている溶液を10mL入れる。この懸濁液にパーフルオロペンタン(PCR Research Chemicals、Gainesville、FL)を165μLmL−1加える。この脂質懸濁液を室温(約20℃)と平衡状態にした後、VWR Scientific Orbital振とう機(VWR Scientific、Cerritos、CA)の上に上記管を直立に位置させ、そして300r.p.m.で30分間振とうする。結果として起こる振とうテーブル上の撹拌により、気体前駆体充填リポソームが生じる。
パーフルオロペンタンを六フッ化硫黄、ヘキサフルオロプロピレン、ブロモクロロフルオロメタン、オクタフルオロプロパン、1,1ジクロロ,フルオロエタン、ヘキサフルオロエタン、ヘキサフルオロ−2−ブチン、パーフルオロペンタン、パーフルオロブタン、オクタフルオロ−2−ブテンまたはヘキサフルオロブタ−1,3−ジエンまたはオクタフルオロシクロペンテンで置き換えて上記実験を実施すると、これらは全部、気体前駆体充填リポソームを生じ得る。
50mLのFalcon遠心分離管(Becton−Dickinson、Lincoln Park、NJ)に、前以て1μmのフィルターに通して押出してオートクレーブに20分間かけた1,2−ジパルミトイルホスファチジルコリンが0.9%NaCl中に25mg/mLの量で入っている溶液を10mL入れる。この懸濁液に2−メチル−2−ブテンを165μLmL−1加える。この脂質懸濁液を室温(約20℃)と平衡状態にした後、この管を、空の家庭用1ガロン塗料用容器の内側に固定した後、渦巻き運動を利用した塗料用機械ミキサーの中に15分間入れる。渦巻き混合した後、上記遠心分離管を取り出し、そして気体前駆体充填リポソームが生じたことを確認する。
50mLのFalcon遠心分離管(Becton−Dickinson、Lincoln Park、NJ)に、前以て1μmのnucleporeフィルターに通して押出してオートクレーブに20分間かけた1,2−ジパルミトイルホスファチジルコリンが0.9%NaCl中に25mg/mLの量で入っている溶液を10mL入れる。この懸濁液に2−メチル−2−ブテンを165μLmL−1加える。この脂質懸濁液を室温(約20℃)と平衡状態にした後、この管を手で力強く10分間振とうする。撹拌を停止した後、気体前駆体充填リポソームが生じる。
例17に記述したのと同様にしてDPPCから気体前駆体充填リポソームを製造する。その結果として生じる未濾過リポソームを50mLのシリンジの中に吸い込ませ、最短で150μm離れて位置している「NUCLEPORE」(Costar、Pleasanton、CA)10μmフィルターに続く8μmから成るカスケードフィルターシステムに通す。加うるに、別のサンプルでは、2つのフィルターを互いに隣接させて重ねた10μmフィルターと8μmフィルターの濾過アセンブリを用いる。2.0mL/分の流量で濾過されるように、気体前駆体充填リポソームを加圧下で上記フィルターに通す。その濾過された気体前駆体充填リポソームの収率は未濾過体積の80−90%から成る体積である。 その結果として得られる気体前駆体充填リポソームのサイズ測定を4つの異なる方法で行うことにより、そのサイズ分布を測定する。Particle Sizing Systems(Santa Barbara、CA)Model 770 Optical Sizing装置、画像処理用ソフトウエア(Universal Imaging、West Chester、PA)に連動させたZeiss(Oberkochen、ドイツ)Axioplan光学顕微鏡、およびCoulter Counter(Coulter Electronics Limited、Luton、Beds.、英国)を用いて、サイズ測定を実施する。図8に示すように、この気体前駆体充填リポソームのサイズは、濾過前の気体前駆体充填リポソームのサイズに比較して、より均一に8−10μm付近に分布する。
用いる振とう機をCrescent「Wig−L−Bug」(商標)(Crescent Manufacturing Dental Co.、Lyons、IL)にする以外は実施例6と同じ手順を実施する。次に、上に記述した通常5分間から10分間の代わりに、60秒間、この調合物の撹拌を行う。気体で満たされた脂質球が生じる。
5mg/mLの脂質懸濁液にパーフルオロペンタン(沸点29.5℃、PCR Research Chemicals、Gainesville、FL)を100μL加え、そしてGenie IIミキサー(Scientific Industries,Inc.、Bohemia、NY)を用い、パワーを6.5に設定して渦流を室温で起こさせた。次に、Richmar(Richmar Industries、Inola、OK)1MHz治療用超音波装置を用いて高体温を実施し、温度計を用いて測定して温度が約42℃以上に上昇した。相転移温度に到達した時点で、気体微小球を確認した。診断用超音波(Acoustic Imaging、Phoenix、AZ)を用いて同時に走査を実施した。また、臨床用の診断超音波を用いることでも、この気体微小球由来の音響シグナルを可視化することができた。
例23と同じ実験を実施したが、ここでは、該懸濁液に渦流を起こさせ、そして予め左大腿部領域にC5A腫瘍細胞系を生じさせたHarlan−Sprague Dawleyラット(300グラム)に注入した。次に、この腫瘍領域の上にRichmar 1MHz治療用超音波装置を置き、そしてアドリアマイシンを埋め込んだ脂質懸濁液を静脈注射した。次に、この治療用超音波を連続波(100%衝撃周波)に設定してその腫瘍を加熱する。左大腿部領域にC5A腫瘍細胞系を生じさせた2番目のラットにアドリアマイシンのエマルジョンを同じ量で与えたが、しかしながら、この動物では超音波を用いなかった。この超音波を用いなかった対照に比較して、腫瘍が3週間以内に顕著に小さくなることを確認した。
52 水系媒体供給容器
55 気体前駆体
66 混合容器
72 フィルター要素
73 泡
75 機械振とう装置
76 抽出用容器
77 リポソーム
Claims (7)
- 容器中でゲル状態相の脂質から形成された超音波画像形成用コントラスト剤として用いるためのパーフルオロカーボンガス充填脂質微小球であって、
該脂質がジパルミトイルホスファチジン酸、ジパルミトイルホスファチジルコリンおよびポリエチレングリコールに共有結合したジパルミトイルエタノールアミンを含有する脂質混合物であり、
該パーフルオロカーボンがパーフルオロプロパンであり、
該脂質微小球の平均直径が1μm〜25μmであり、そして
該脂質微小球がパーフルオロカーボンガスの存在下で該脂質混合物を含む水溶液を振とうすることを含む調製方法により得られたものであり、該振とうが該脂質のゲル状態相から液晶状態への相転移温度を下回る温度で行われた、
ことを特徴とする前記脂質微小球。 - 脂質微小球の平均直径が1μm〜8μmである、請求項1記載の脂質微小球。
- 請求項1記載の脂質微小球であって、該パーフルオロカーボンガスが該パーフルオロカーボンガス充填脂質微小球の内部容積の少なくとも50%を構成する、脂質微小球。
- 請求項1記載の脂質微小球であって、ポリエチレングリコールに共有結合したジパルミトイルエタノールアミンのポリエチレングリコールが分子量5000を有する、脂質微小球。
- 請求項4記載の脂質微小球であって、脂質混合物が10モル%のジパルミトイルホスファチジン酸、82モル%のジパルミトイルホスファチジルコリンおよび8モル%のポリエチレングリコールに共有結合したジパルミトイルエタノールアミンを含む、脂質微小球。
- 請求項1〜5のいずれかに記載の脂質微小球を含んでなる食塩水溶液。
- 請求項6記載の食塩水溶液であって、pH5〜pH7.4に緩衝化されている、前記食塩水溶液。
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US08/076,239 US5469854A (en) | 1989-12-22 | 1993-06-11 | Methods of preparing gas-filled liposomes |
US15967493A | 1993-11-30 | 1993-11-30 | |
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US08/159,687 US5585112A (en) | 1989-12-22 | 1993-11-30 | Method of preparing gas and gaseous precursor-filled microspheres |
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- 1994-05-20 JP JP50183995A patent/JP4274387B2/ja not_active Expired - Lifetime
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- 1994-05-20 WO PCT/US1994/005792 patent/WO1994028780A2/en active IP Right Grant
- 1994-05-20 AT AT94919208T patent/ATE233574T1/de active
- 1994-05-20 PT PT94919208T patent/PT712293E/pt unknown
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Also Published As
Publication number | Publication date |
---|---|
DE69432219T2 (de) | 2003-12-04 |
EP0712293B1 (en) | 2003-03-05 |
CA2164845A1 (en) | 1994-12-22 |
NL300267I2 (nl) | 2007-10-01 |
DE69432219D1 (de) | 2003-04-10 |
AU683900B2 (en) | 1997-11-27 |
JP4274387B2 (ja) | 2009-06-03 |
DK0712293T3 (da) | 2003-06-30 |
EP0712293A4 (en) | 1998-05-06 |
CA2164845C (en) | 2008-04-29 |
US6071495A (en) | 2000-06-06 |
EP1252885A3 (en) | 2003-04-16 |
CN1125389A (zh) | 1996-06-26 |
JP2007314559A (ja) | 2007-12-06 |
LU91325I2 (fr) | 2007-05-16 |
ATE233574T1 (de) | 2003-03-15 |
EP1252885A2 (en) | 2002-10-30 |
PT712293E (pt) | 2003-07-31 |
DE122007000024I2 (de) | 2011-06-16 |
JPH08511526A (ja) | 1996-12-03 |
US5585112A (en) | 1996-12-17 |
CN1125654C (zh) | 2003-10-29 |
WO1994028780A3 (en) | 1995-02-02 |
WO1994028780A2 (en) | 1994-12-22 |
US6479034B1 (en) | 2002-11-12 |
EP0712293A1 (en) | 1996-05-22 |
ES2193161T3 (es) | 2003-11-01 |
DE122007000024I1 (de) | 2007-10-04 |
US5853752A (en) | 1998-12-29 |
NL300267I1 (nl) | 2007-05-01 |
AU7043194A (en) | 1995-01-03 |
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