JP2003535017A5 - - Google Patents

Description

（式中、ｈはプランク定数であり、ωは角周波数であり、ｄ０は、付着力が最大である距離であり、およびｒ１およびｒ^２ は、２つの相互作用している粒子の半径である）。したがって、乾燥粉体のためのＶＤＷ力の大きさおよび強度を最小にするｍたの一つの方法は、接触の粒子間面積を減少させることであると認識されるであろう。ｄ０の大きさが、接触のこの面積の反映であることは、重要である。２つの対向している物体間の接触の最小の面積は、粒子は完全な球である場合に起こるであろう。加えて、粒子が非常に多孔性であるならば、接触の面積は更に最小にされるであろう。したがって、本発明の有孔ミクロ構造は、粒子間接触、および対応するＶＤＷ力を低減するように作用する。ＶＤＷ力でのこの低減が主に、幾何粒径の増大よりむしろ本発明の粉体のユニークな粒子形態の結果であることに注目することは、重要である。この点について、本発明の特に好ましい態様が比較的低いＶＤＷ引力を示す平均または小さい微粒子（例えば平均の幾何直径＜１０μｍ）を有する粉体を与えることが認識される。
【００９８】
更に、上記に示されたように、粒子のどちらか、または両方ともが電気的にチャージされた場合に、粉体に影響を及ぼす静電力が起こる。この現象は、電荷の類似性または非類似性に依存して、粒子間の引力または反発で起こるであろう。最も単純な場合、電荷はクーロンの法則を用いて記述することができる。粒子間の静電力を調整し、または減少させる一つの方法は、粒子のどちらか、または両方が非電導性表面を有するかどうかである。従って、有孔ミクロ構造粉体が、比較的に非電導性である賦形剤、界面活性剤または活性剤を含むならば、粒子で発生する任意の電荷でも不規則に表面に分布される。その結果、高い電荷の保持が材料の比抵抗によって書き取る時から、非電導性成分を含む粉体の電荷半減期は比較的短い。抵抗性のまたは非導電性の成分は、効率的な電子供与体または受容体のどちらとしても機能しない材料である。
【００９９】
参照することによりここに取り込むＤｅｒｊａｇｕｉｎら（Ｍｕｌｌｅｒ、Ｖ．Ｍ．、Ｙｕｓｈｃｈｅｎｋｏ、ＶＳ．、およびＤｅｒｊａｇｕｉｎ、Ｂ．Ｖ．、Ｊ．Ｃｏｌｌｏｉｄ Ｉｎｔｅｒｆａｃｅ Ｓｃｉ．、１９８０、７７、１１５〜１１９）は、電子を受容し、または供与するそれらの機能のための分子群をランクするリスト与える。この点について、例示的な群は、次のようにランクを付けることができる：
【０１００】
供与体：−ＮＨ_２＞−ＯＨ＞−ＯＲ＞ＣＯＯＲ＞−ＣＨ_３＞−Ｃ_６Ｈ_５＞−ハロゲン＞−ＣＯＯＨ＞−ＣＯ＞−ＣＮ受容体：
【０１０１】
本発明は、比較的非伝導性の材料の使用により、開示された粉体で静電効果の低減を与える。上記のランキングを用いて、好ましい非伝導性の材料は、ハロゲン化されたおよび／又は水素化された成分を含む。それらが粒子荷電への抵抗性を有するため、リン脂質およびフッ素化発泡剤等の材料（それは、スプレードライした粉体で、ある程度まで保持できる）は好ましい。粒子中の残留発泡剤（例えばフルオロケミカル）は、比較的低いレベルであっても、典型的にはスプレードライおよびサイクロン分離の間に付与されるため、微粒子または有孔ミクロ構造の荷電を最小化するのを助長すると認識されるであろう。一般的な静電原理およびここでの教示に基づき、当業者は、過度の実験なしで開示された粉体の静電カを低減するために役立つ追加的な材料を確認することができるであろう。この点について、非常にチャージされた剤は、単純なｐＨ調整または反対にチャージされた化合物（例えばカチオン性脂質と結合した核酸）とのキレート化により、静電的的に修正することができる。更に、必要であるならば、通電および荷電技術を用いて、静電力を操作して、最小にすることもできる。
【０１０２】
上述した驚くべき利点に加え、本発明は更に、水素および液体結合の減衰または低減を与える。当業者に公知のように、水素結合、および液体ブリッジングの両方は、粉体によって吸収された水分から生ずる。一般に、より高い湿度は、親水性の表面に対して、より高い粒子間力を生ずる。これは、ラクトース等の比較的に親水性の化合物を使用する傾向がある、吸入療法のための先行技術処方における本質的な問題である。しかしながら、ここでの教示に従って、吸着水による付着力は、接触表面の疎水性を増大させることによって調整または低減することができる。当業者は、粒子疎水性における増加が、賦形剤の選択および／又は流動床を用いて使用されるもの等の、製造後のスプレードライコーティング技術の使用により達成することができると認識できる。従って、好ましい賦形剤は、リン脂質、脂肪酸石鹸およびコレステロール等の疎水性界面活性剤を含む。ここでの教示の点から、当業者が過度の実験なしで同様の望ましい性質を示す材料を同定できるであろうことが言える。
【０１０３】
それらが乾燥粉体または非水性の懸濁媒体との組み合わせのいずれとして使われるにせよ、微粒子または有孔ミクロ構造は「乾燥」状態で好ましく与えられるであろう。すなわち、微小粒子は、外界温度での貯蔵の間に粉体を化学的および物理的に安定したままとさせ、且つ簡単に分散可能であるままとさせる含水量を有するであろう。従って、微小粒子の含水量は、典型的には６質量％（重量％）未満であり、好ましくは３質量％（重量％）未満である。いくつかの例において、含水量は１質量％（重量％）程度に低い。もちろん、含水量は、少なくとも部分的には、処方によって規定され、使用されるプロセス条件、例えば、入口温度、フィード濃度、ポンプ速度、発泡剤タイプ、濃度およびポスト乾燥によって対照されると認識されるであろう。
【０１０４】
当業者によって知られているように、安息角または剪断力等の方法は、乾燥粉体の流れ性質を評価するために使用可能である。安息角は、フラットな表面上に粉体の円錐を注いだ際に、形成される角度として定義される。４５°〜２０°の範囲にある安息角を有する粉体が好ましく、適当な粉体流れを示す。より具体的には、３３°〜２０°の間の安息角を有する粉体は、比較的低い剪断力で流動し、吸入療法（例えばＤＰＩｓ）に用いられる製剤処方のにおいて特に有用である。剪断インデックスは、測定するために安息角より時間がかかるが、より信頼性があり、決定が容易であると考えられる。当業者は、ＡｍｉｄｏｎおよびＨｏｕｇｈｔｏｎによって概説された実験的手順（参照することによりここに取り込む、Ｇ．Ｅ．Ａｍｉｄｏｎ およびＭ．Ｅ．Ｈｏｕｇｈｔｏｎ、Ｐｈａｒｍ． Ｍａｎｕｆ．、２、２０、１９８５）が、本発明の目的のための剪断インデックスを評価するために使用可能であると認識するであろう。これも参照することによりここに取り込むＳ． ＫｏｃｏｖａおよびＮ． Ｐｉｐｅｌ、Ｊ． Ｐｈａｒｍ． Ｐｈａｒｍａｃｏｌ．、 ８、３３〜３５、１９７３に記述されているように、剪断インデックスは降伏ストレス、内部摩擦の有効角、引張強さ、および比凝集力等の粉体パラメータから評価される。本発明において、約０．９８未満の剪断インデックスを有する粉体が好ましい。より好ましくは、開示された組成物、方法およびシステムで用いられる粉体は、約１．１未満の剪断力インデックスを有するであろう。特に好ましい態様において、剪断インデックスは、約１．３未満、または約１．５未満でさえあろう。もちろん、異なる剪断力インデックスを有する粉体は、重要なサイトにおける活性または生物活性剤の効果的な沈着の結果で、使用可能である。
【０１０５】
粉体の流れ性質が、バルク密度測定と良好に相関することが示されたも、認識されるであろう。この点については従来の先行技術の考察（Ｃ．Ｆ．ハーウッド、Ｊ．Ｐｈａｒｍ． Ｓｃｉ．、 ６０、１６１〜１６３，１９７１）は、バルク密度の増大が、材料の剪断インデックスによって予測されるように、改善された流れ性質と相関すると考えた。逆に、本発明の有孔ミクロ構造のために、比較的低いバルク密度を有する粉体により優れた流れ性質が示されたことが、驚くべきことに判明した。すなわち、本発明の中空の多孔性の粉体は、実質的に孔が全くない粉体に対して、優れた流れ性質を示した。このため、特に良い流れ性質を示す０．５ｇ／ｃｍ^３ 未満のバルク密度を有する粉体を提供可能であることが判明した。より驚くべきことに、優れた流れ性質を示す０．３ｇ／ｃｍ^３ 未満、約０．１ｇ／ｃｍ^３未満、更には０．０５ｇ／ｃｍ^３未満の桁のバルク密度を有する有孔ミクロ構造粉体が提供可能であることが判明した。優れた流動性を有する低いバルク密度粉体を製造する能力は、本発明の新規且つ予想外の性質を更に引き立たせる。
【０１０６】
これらの低いバルク密度は、ＤＰＩｓとともに開示された粉体を用いるとき、特に有利である。具体的には、異常に低いバルク密度を有する粉体を与えることによって、本発明は、乾燥粉体吸入器具のために商業的に実行可能な最小の充填重量の低減を与える。すなわち、ＤＰＩｓのために最も多くの設計されるユニット用量容器は、固定された体積または重力技術を用いて満たされる。多くの先行技術とは逆に、本発明は、生物活性剤およびインシピエントまたはバルキング剤が吸入された粒子全体を構成する（ｍａｋｅ−ｕｐ）粉体を与える。非常に低いバルク密度を有する粒子を与えることによって、ユニット用量容器に満たすことができる最小限の粉体質量は低減され、それはキャリア粒子の必要性を除去する。すなわち、本発明の粒子の比較的低い密度は、キャリア粒子の使用なしで、比較的低用量の薬剤化合物の再現性ある投与を与える。更に、キャリア粒子の除去は、先行技術の大きいラクトース粒子がそれらのサイズのために喉および上部気道に衝撃を与える傾向があるため、喉沈着および任意の「ギャグ」効果を最小化するように作用する。
【０１０７】
有孔ミクロ構造粉体によって与えられる低減された引力（例えば、ファンデルワールス、静電、水素と液体ブリッジング、その他）および優れた流動性は、吸入療法（例えば、ＤＰＩｓ、ＭＤＩｓ、ネブライザー等の吸入器具における）のための製剤における、それら特に有用とすると認識されるであろう。優れた流動性とともに、ミクロ構造の有孔、多孔性のおよび／又は中空設計も、放出された際の粉体の結果として生じるエアロゾル性質において重要な役割を演ずる。この現象は、ＭＤＩまたはネブライザーの場合、またはＤＰＩの場合のように乾燥形での有孔ミクロ構造のデリバリーにおいて、ディスパージョンとしてエアロゾル化された微粒子または有孔ミクロ構造のためにも有効である。この点で、分散された微小粒子の有孔構造および比較的に高い表面積は、匹敵するサイズの非有孔粒子より容易に、より長い距離で吸入の間にガスの流れに沿って、それらが運ばれることを可能にする。
【０１０８】
より具体的には、それらの高いポロシティのため、粒子の密度は実質的に１．０ｇ／ｃｍ^３未満、典型的には０．５ｇ／ｃｍ^３未満、よりしばしば０．１ｇ／ｃｍ^３の桁、および０．０１ｇ／ｃｍ^３程度に低い。幾何粒径と異なり、有孔ミクロ構造の空気力学的粒径、ｄ_ａｅｒ は、実質的に粒子密度、ρ：ｄ_ａｅｒ＝ｄ_ｇｅｏρ（式中、ｄ_ｇｅｏは幾何直径である）に依存する。０．１ｇ／ｃｍ^３の粒子密度に対して、ｄ_ａｅｒは約３倍ｄ_ｇｅｏより小さくなり、肺の周辺（ｐｅｒｉｐｈｅｒａｌ）領域への増大された粒子沈着、および対応して喉におけるより少ない沈着をもたらす。この点について、有孔ミクロ構造の平均の空気力学的な直径は、好ましくは約５μｍ未満、より好ましくは約３μｍ未満、特に好ましい態様においては、約２μｍ未満である。このような粒子分布は、ＤＰＩ、ＭＯまたはネブライザーを用いて投与されるか否かに関係なく、生物活性剤の深い肺沈着を増大させように作用する。
【０１０９】
以降の例で示されるように、本発明のエアロゾル処方の粒径分布は、カスケードインパクション等の従来の技術によって、または飛行時間分析法によって、測定可能である。加えて、参照することによりここに取り込む提案された米国薬局方の方法（Ｐｈａｒｍａｃｏｐｅｉａｌ Ｐｒｅｖｉｅｗｓ、２２（１９９６）３０６５）に従って、吸入器具から放出された用量は決定された。これらおよび関連する技術は、肺に効率的に沈着する可能性があるそれらの微粒子に対応するエアロゾルの「微粒子分率」が計算されることを可能にする。ここで用いるように、語「微粒子分率」は、ＤＰＩ、ＭＤＩまたはネブライザーのマウスピースから、８段のアンデルセンカスケードインパクターのプレート２−７の上へ、作動につきデリバリーされる活性薬物の総量のパーセンテージを言う。このような測定に基づき、本発明の処方は、好ましくは有孔ミクロ構造の重量に基づき約２０質量％（重量％）（ｗ／ｗ）以上の微粒子分率を有するであろうし、より好ましくは、それらは約２５質量％（重量％）〜９０質量％（重量％）（ｗ／ｗ）、更に好ましくは約３０〜８０質量％（重量％）（ｗ／ｗ）の微粒子分率を示すであろう。本発明の選ばれた態様において、約３０質量％（重量％）、４０質量％（重量％）、５０質量％（重量％）、６０質量％（重量％）、７０質量％（重量％）、８０質量％（重量％）または９０質量％（重量％）さえ超える微粒子分率を好ましくは含む。
【０１１０】
更に、本発明の処方が先行技術製剤と比較した際に、誘導ポート上で、およびインパクターのプレート０および１上で、比較的低い沈着割合を示すことも見出された。これらのコンポーネントへの沈着は、ヒトにおける喉での沈着とリンクする。より具体的には、最も商業的に入手可能なＭＤＩｓおよびＤＰＩｓは、全用量の約４０〜７０質量％（重量％）（ｗ／ｗ）の喉沈着をシミュレーションした一方で、本発明の処方は、典型的には約２０質量％（重量％）（ｗ／ｗ）未満を沈着させる。したがって、本発明の好ましい態様は、約４０質量％（重量％）、３５質量％（重量％）、３０質量％（重量％）、２５質量％（重量％）、２０質量％（重量％）、１５質量％（重量％）または１０質量％（重量％）（ｗ／ｗ）未満の喉沈着さえシミュレーションした。当業者は、本発明によって与えられる喉沈着における有意な減少が、喉刺激作用等の関連する局所的副作用における対応する減少を生じることを認識するであろう。
【０１１１】
本発明により与えられる有利な沈着プロフィルに関して、ＭＤＩ推進剤が、典型的には喉の後方の方向へ、器具から懸濁粒子を高速度で押し出すことは周知である。先行技術の処方が、典型的には大径粒子および／又は凝集体の有意なパーセンテージを含むため、放出された用量の２／３程度に多い量以上が喉に衝撃を与える可能性がある。更に、従来の粉体製剤の好ましくないデリバリープロフィルはまた、ＤＰＩ器具で生ずるような、低い粒子速度の条件下で示される。一般的に、凝集し易い固体の、濃い微粒子をエアロゾル化する際に、この問題は固有である。それでも、上述したように、本発明の安定化ディスパージョンの新規且つ予想外の性質は、ＤＰＩ、ＭＤＩアトマイザーまたはネブライザー等の吸入器具からの投与に際して、驚くほど低い喉沈着しか生じない。
【０１１２】
如何なる特定の理論によっても束縛されることを欲っしないが、本発明によって与えられる低減された喉沈着は、粒子凝集における減少から、および取り込まれたミクロ構造の中空のおよび／又は多孔質形態から生ずるように思われる。すなわち、分散されたミクロ構造の中空および多孔質の性質は、中空／多孔質ウィフル（ｗｈｉｆｆｌｅ）ボールがベースボールより迅速に減速するように、推進剤流れ（またはＤＰＩｓの場合ガス流れ）中の粒子の速度を遅くする。従って、喉の後部に衝撃を与え、付着するよりはむしろ、比較的遅く移動する粒子は、患者により吸入され易い。更に、粒子の非常に多孔性の性質は、有孔ミクロ構造内の推進剤を迅速に去らせ、喉に衝撃を与える前に粒子密度を低下させる。したがって、投与された生物活性剤の実質的により高いパーセンテージが、それが能率的に吸収可能な肺の気道に沈着される。
【０１１３】
Ｅ．粉体形成
【０１１４】
上記の節から理解されるように、本発明のミクロ微粒子には、種々の成分を結合させ、または取り込むことができる。同様に、いくつかの技術は、所望の形態（例えば有孔または中空／多孔質コンフィグレーション）、分散性および密度を有する微粒子を与えるために使用可能である。他の方法の中で、本発明と適合性を有する微粒子は、スプレードライ、減圧乾燥、溶媒抽出、エマルション化、凍結乾燥、およびそれらの組み合わせを含む技術によって形成することができる。これらの技術の多くの基本的な概念が当該技術で周知であり、ここでの教示の点から、所望の粒子コンフィグレーションおよび／又は密度を与えるように、それらを適合させるために過度の実験を必要としないことが、更に認識されるであろう。
【０１１５】
いくつかの手順が一般的に本発明と適合性を有するが、特に好ましい態様は、典型的には、スプレードライによって形成された微粒子または有孔ミクロ構造を含む。周知であるように、スプレードライは液体のフィードを乾燥した微粒子の形に変換する一段階過程である。薬剤応用に関して、吸入を含む種々の投与経路のための粉体物質を与えるために、スプレードライが使われてきたと認識されるであろう。例えば、参照することによりここに取り込む吸入エアロゾルズ：「治療の物理学的および生物学的基礎」、Ａ． Ｊ． Ｈｉｃｌｅｙ編集、マーセルデッカー社、ニューヨーク、１９９６におけるＭ． Ｍ． ＳａｃｈｅｔｔｉおよびＭ ．Ｍ． Ｖａｎ Ｏｏｒｔを参照。
【０１１６】
一般的に、スプレードライは、高度に分散された液体と、液滴の蒸発および乾燥を与えるための充分な体積の熱い空気とをまとめることから成る。スプレードライまたはフィード（または供給原料）となるための製剤は、任意の水性溶液、経過ディスパージョン、スラリー、コロイドディスパージョンまたは選ばれたスプレードライ装置を用いてアトマイズできるペーストでありえる。好ましい態様において、供給原料は、エマルション、逆エマルション、ミクロエマルション、複エマルション、微粒子ディスパージョンまたはスラリー等のコロイド系を含む。典型的には、フィードは、溶媒を蒸発させて、乾燥した生成物をコレクターに運ぶ暖かい濾過された空気の流れにスプレーされる。費やされる空気は、次いで溶媒とともに排気される。当業者は、所望の生成物を与えるために、装置のいくつかの異なるタイプが使用できると認識するであろう。例えば、Ｂｕｃｈｉ社またはＮｉｒｏ社によって製造される商用のスプレードライ装置、所望のサイズ、形態および密度の粒子を効果的に製造するであろう。
【０１１７】
これらのスプレードライ装置、および特にそれらのアトマイザーは、専門化された応用、すなわち重複ノズル技術を用いる２つの溶液の同時のスプレー）のために改造、またはカスタマイズすることができることが、更に認識されるであろう。より具体的には、Ｗ／Ｏ型（ｗａｔｅｒ−ｉｎ−ｏｉｌ）エマルションは、１つノズルからアトマイズすることができ、およびマンニトール等の反被着剤を含む溶液は第二ノズルから共アトマイズすることができる。他の場合において、高圧液体クロマトグラフィ（ＨＰＬＣ）ポンプを用いるカスタム設計のノズルを通してフィード溶液を押し出すことは、好ましいであろう。所望の形態および／又は組成物を含むミクロ構造が製造されるならば、装置の選択は重要でなくて、ここでの教示を考慮して容易に当業者にとって明らかであろう。
【０１１８】
結果として生じるスプレードライされた粉体は典型的には、ほぼ球状の形、大きさにおいてほぼ均一、およびしばしば中空であるが、取り込まれる薬物およびスプレードライ条件に依存して、形状におけるいくらかの程度の不規則はあり得る。多くの例において、ディスパージョン安定性、および微粒子または有孔ミクロ構造の分散性は、膨張剤（または発泡剤）それらの製造で使用されるならば改善されるように思われる。特に好ましい態様は、分散または連続相として膨張剤を有するエマルションを含むことができる。膨張剤は、好ましくは、例えば商業的に入手可能なミクロ流動化装置を約３４，４７４ｋＰａ〜１０３，２４１ｋＰａ（５，０００〜１５，０００ｐｓｉ）の圧力で用いて、界面活性剤とともに分散される。これはプロセスは、エマルション、好ましくは取り込まれた界面活性剤によって安定され、典型的には水性連続相中に分散された水非混和性の膨張剤のサブミクロン液滴を含むもの、を形成する。これおよび他の技術を用いるこのようなエマルションの形成は、当該技術においてありふれており、且つ当業者に周知である。発泡剤は、好ましくは、スプレードライ過程で蒸発して、選ばれた態様においては、比較的に中空の、多孔性の空気力学的に軽いミクロスフィアを後に残すフッ素化合物（例えば、パーフルオロヘキサン、パーフルオロオクチルブロミド、パーフルオロデカリン、パーフルオロブチルエタン）である。以下で更に詳細に議論されるように、他の適当な液体の発泡剤は、非フッ素化油、クロロホルム、フレオン、エチルアセテート、アルコールおよび炭化水素を含む。窒素および二酸化炭素も、適当な発泡剤として考えられる。
【０１１９】
上述した化合物の他に、製造後のステップでの昇華により減圧下で除去できる無機および有機物質も、本発明と適合性を有する。これらの昇華性化合物は、スプレードライフィード溶液中にミクロ化された結晶として溶解または分散可能であり、炭酸アンモニウムおよびカンファーを含むことができる。本発明と適合性を有する他の化合物は、フィード溶液中に分散でき、またはその場で（ｉｎ−ｓｉｔｕ）製造できるリジッド化性の固体構造を含む。これらの構造は、次いで、製造後の溶媒抽出ステップを用いて、初期の粒子生成後に抽出される。例えば、ラテックス粒子は分散でき、その後他の壁形成性化合物とともに乾燥され、次いで適当な溶媒で抽出される。
【０１２０】
微粒子は好ましくは上記しれた発泡剤を用いて製造されるが、いくつかの例において、追加的な発泡剤は必須でなく、薬剤および／又は賦形剤および１以上の界面活性剤の水性分散液は、直接スプレードライされると認識されるであろう。このような場合に、その処方は中空の、比較的多孔性の微小粒子の形成をもたらすことができるプロセス条件（例えば、高温）に従うことができる。更に、薬物は、このような技術における使用に特に適当とするような特別な物理化学的性質（例えば高い結晶化度、高い溶融温度、界面活性、その他）を有することができる。
【０１２１】
発泡剤が使用されるとき、得られる微粒子のポロシティおよび分散性の程度は、少なくとも部分的には、発泡剤の性質、供給原料（例えば、エマルションとして）でのその濃度、およびスプレードライ条件に依存するように思われる。ポロシティ、およびディスパージョンにおける分散性を制御することに関して、従来は発泡剤として好ましくないとされていた化合物の使用が、特に好ましい特性を有する有孔ミクロ構造を与えることができることが、驚くべきことに見出された。より具体的には、本発明のこの新規で、予想外の面において、比較的高い沸点（すなわち約４０℃を超える）を有するフッ素化合物の使用が、特に多孔質である微粒子を製造するために使用可能なことが見出された。このような有孔ミクロ構造は、特に吸入療法に適している。この点について、約４０℃、５０℃、６０℃、７０℃、８０℃、９０℃または９５℃さえ超える沸点を有するフッ素化または部分的フッ素化発泡剤を用いることができる。特に好ましい発泡剤は、水の沸点、すなわち、１００℃を越える沸点を有する（例えばパーフルブロン（ｐｅｒｆｌｕｂｒｏｎ）、パーフルオロデカリン）。加えて、それらが０．３μｍ未満の平均の重みつけ（ｍｅａｎ−ｗｅｉｇｈｔｅｄ）粒径を有する安定なエマルションディディスパージョンの製造を容易にするため、比較的に低水溶性（＜１０^−６Ｍ）を有する発泡剤が好ましい。
【０１２２】
以前に記述されたように、これらの発泡剤は、好ましくは、スプレードライの前に、エマルション化された供給原料内に取り込まれる。本発明の目的のために、この供給原料がまた、好ましくは１以上の生物活性剤、１以上の界面活性剤または１以上の賦形剤をも含むであろう。もちろん、上述したされた成分の組み合わせも、本発明の範囲内にある。高沸点（＞１００℃）フッ素化発泡剤が本発明の１つの好ましい面を含むが、同様の沸点（＞１００℃）を有する非フッ素化発泡剤も、適合性を有する微粒子を与えるために使用可能であると認識されるであろう。本発明における使用に適当な例示的な非フッ素化発泡剤は、式：
【０１２３】
Ｒ^１−Ｘ−Ｒ^２またはＲ^１−Ｘ
（式中、Ｒ^１またはＲ^２は、水素、アルキル、アルケニル、アルキニル、芳香族、環式またはそれらの組み合わせであり、Ｘは炭素、イオウ、窒素、ハロゲン、リン、酸素、およびそれらの組み合わせ我を含む任意の基である）
を含む。
【０１２４】
如何なる形であれ本発明を制限しないが、水性フィード成分がスプレードライの間に蒸発するため、それが粒子表面で薄い外被を残すと仮定される。スプレーの初期の瞬間に形成される、得られた粒子壁または外被は、何百ものエマルション液滴（約２００〜３００ｎｍ）として、任意の高沸点発泡剤をトラップするように思われる。乾燥過程が続くに従い、微粒子の増大の内側の圧力は増大して、それにより取り込まれた発泡剤の少なくとも一部を蒸発させ、および比較的薄い外皮を介してそれを押し出す。これのベント、またはガス抜きは、見かけ上（ａｐｐａｒｅｎｔｌｙ）ミクロ構造における孔または他の欠陥の形成をもたらす。同時に、残っている微粒子の成分（おそらく、いくらかの発泡剤を含む）、粒子が固化するにつれて、内部から表面に移行する。この移行は、増大された内部粘性に起因する物質移動に、増大された抵抗性の結果、乾燥過程の間に、見かけ上遅くなる。一旦移行が生ずるならば、粒子が固化して、ボイド、孔、欠陥、中空、スペース、アパーチュア、パーフォレーション、またはホールを残す。孔または欠陥の数、それらのサイズ、および得られる壁厚さは、選ばれた発泡剤の処方および／又は性質（例えば沸点）、エマルション中のその濃度、全固体物濃度、およびスプレードライ条件に主に依存する。明細書を通して言及するように、この好ましい粒子形態が、少なくとも部分的には、改善された粉体分散性、懸濁安定性および空気力学に寄与するように思われる。
【０１２５】
これらの比較的高沸点の発泡剤の実質的な量が、結果として生じるスプレードライ生成物中で保持できることが、驚くべきことに判明した。すなわち、ここで記述されたスプレードライ微粒子は、残留発泡剤の１質量％（重量％）、３質量％（重量％）、５質量％（重量％）、１０質量％（重量％）、２０質量％（重量％）、３０質量％（重量％）または４０質量％（重量％）（ｗ／ｗ）の程度に多く含むことができる。このような場合に、この保持された発泡剤によって生ずる増大された粒子密度の結果、より高い製造収率が得られた。保持されたフッ素化発泡剤が、微粒子の表面特性を変え、それによりプロセシングの間の粒子凝集を最小にし、ディディスパージョン安定性を更に増大できることが、当業者によって認識されるであろう。粉体中の残留フッ素化発泡剤は、また、バリヤーを与えることによって、または製造の間に引力（例えば静電力）を低減することによって、粒子間の凝集力を低減することもできる。乾燥粉体吸入器とともに開示されたミクロ構造を用いるとき、この凝集力における低減は、乾燥粒子吸入器との組み合わせで開示されたミクロ構造を使用する際に、特に有利であることができる。
【０１２６】
更に、残留発泡剤の量は、プロセス条件（例えば出口温度）、発泡剤濃度または沸点により対照できる。出口温度が沸点またはより高いならば、発泡剤は粒子を逃れ、および製造収率は減少する。好ましい出口温度は、発泡剤沸点より、一般に２０、３０、４０、５０、６０、１０、８０、９０または１００℃低く操作されるであろう。より好ましくは、出口温度と沸点の間の温度差は、５０〜１５０℃の範囲にある。粒子ポロシティ、製造収率、静電性および分散性は、第一に、選ばれた活性剤および／又は賦形剤に適しているプロセス条件（例えば出口温度）の範囲を確認することによって最適化することができると、当業者によって認識されるであろう。好ましい発泡剤は、温度差が少なくとも２０℃で１５０℃までであるように、最高の出口温度を用いて選ぶことができる。いくつかの場合で、例えば、超臨界条件下で、または凍結乾燥技術を用いて微粒子を製造する際等に、温度差は、この範囲外側にある可能性がある。当業者は、発泡剤の好ましい濃度は、ここでの例で記述されるそれらと同様の技術を用いて、過度の実験なしで決定できることを更に認識するであろう。
【０１２７】
残留発泡剤が選ばれた態様において有利であり得るが、スプレードライした生成物から任意の発泡剤を実質的に除去することは、好ましいであろう。この点で、残留発泡剤は、減圧オーブン中で製造後の蒸発ステップで容易に除去することができる。更に、このような製造後の技術は、微粒子で有孔を与えるためにも使用することができる。例えば、孔は、生物活性剤と、減圧下で形成された微粒子から除去可能な賦形剤とをスプレードライすることによって形成することができる。
【０１２８】
いずれにせよ、供給原料での発泡剤の例示的な濃度は、２％〜５０％（ｖ／ｖ）の間、より好ましくは約１０％〜４５％（ｖ／ｖ）の間である。他の態様において、発泡剤濃度は好ましくは約５％、１０％、１５％、２０％、２５％または３０％（ｖ／ｖ）さえ越えることが好ましいであろう。それでも、他の供給原料エマルションは、３５％、４０％、４５％または５０％（ｖ／ｖ）の選ばれた化合物さえ含むことができる。
【０１２９】
好ましい態様において、フィードで用いられる発泡剤の濃度を確認する他の方法は、前駆体またはフィードエマルション中の発泡剤の濃度と、安定化界面活性剤（例えば、ホスファチジルコリンまたはＰＣ）のそれとの比として、それを与えることである。フルオロカーボン発泡剤（例えばパーフルオロオクチルブロミド）のために、および説明の目的のために、この比は、ＰＦＣ／ＰＣ比と呼ばれた。より一般的には、適合性を有する発泡剤および／又は界面活性剤が本発明の範囲の外にあることなく、例示的な化合物の代用にすることができると認識されるであろう。いずれにせよ、典型的なＰＦＣ／ＰＣ比は、約１〜約６０、より好ましくは、約１０〜約５０の範囲にある。好ましい態様のために、その比は一般に約５、１０、２０、２５、３０、４０または５０さえ超えるであろう。より高いＰＦＣ／ＰＣ比の使用は、一般に、より中空で多孔性の性質の構造を与えると認識すべきである。より具体的には、約４．８を超えるＰＦＣ／ＰＣ比を使用する方法は、ここで開示された乾燥粒子処方およびディスパージョンと適合性を有する構造を与える傾向があった。
【０１３０】
比較的高い沸点発泡剤は本発明の１つの好ましい面を含むが、他の発泡または膨張剤も、適合性を有するミクロ構造を与えるために使用可能と認識されるであろう。従って、発泡剤は、所望のミクロ構造を製造する目的でフィード溶液に組み込むことができる任意の揮発性物質をも含むことができる。発泡剤は、初期の乾燥過程の間で、または減圧乾燥または溶媒抽出等の製造後のステップで、除去することができる。適当な剤は：
【０１３１】
１．溶液を飽和させるために用いられる塩化メチレン、アセトン、エチルアセテートおよびアルコール等の水性溶液と混合できる、溶解された低沸点（１００℃未満）剤；
【０１３２】
２．高い圧力で用いられる、ＣＯ_２またはＮ_２等のガス、またはフレオン、ＣＦＣｓ、ＨＦＡｓ、ＰＦＣｓ、ＨＦＣｓ、ＨＦＢｓ、フルオロアルカン、および炭化水素等の液体；
【０１３３】
３．本発明の用途に適当な非混和性の低沸点（１００℃未満）液体のエマルションは、一般に式：
【０１３４】
Ｒ^１−Ｘ−Ｒ^２またはＲ^１−Ｘ
（式中、ＲｌまたはＲ^２は、水素、アルキル、アルケニル、アルキニル、芳香族、環式またはそれらの組み合わせであり、Ｘは、炭素、イオウ、窒素、ハロゲン、リン、酸素およびそれらの組み合わせを含む任意の基である）
である。
【０１３５】
４．アンモニウム塩、カンファー、その他等の、製造後のステップで、昇華によって減圧下に除去することができる溶解されるか、または分散された塩または有機物、
【０１３６】
５．製造後の溶媒抽出を用いる初期の粒子生成後に抽出することができる分散された固体、
を含む。
【０１３７】
より低沸点の膨張剤に関して、それらは、界面活性剤溶液の約１％〜４０％（ｖ／ｖ）の量で、典型的には供給原料に添加される。約１５％（ｖ／ｖ）の膨張剤は、本発明の方法で使用できるスプレードライ粉体を製造することが判明した。
【０１３８】
どの発泡剤が最終的に選ばれるにせよ、適合性を有する微粒子がＢｕｅｃｈｉミニスプレードライ装置（Ｂ−１９１型、スイス）等の商業的に入手可能な設備を用いて製造できることが判明した。当業者によって認識されるように、スプレードライ装置の入口温度と出口温度は、所望の粒子サイズを与え、取り込まれた生物活性剤の活性を維持するように、調節することができる。この点で、入口および出口温度は、処方成分および供給原料の組成物の溶融特性に従って調節される。入口温度は、従って、フィードの組成および所望の微粒子の特性に従って、６０℃〜１７０℃の間であることができ、出口温度は約４０℃〜１２０℃の間であることができる。好ましくは、これらの温度は、入口に対して９０℃〜１２０Ｄ℃、および出口に対して６０℃〜９０℃である。スプレードライ設備で使用される流速は、一般に毎分約３ｍｌ〜毎分約１５ｍｌであろう。アトマイザー空気流速は、毎分２５リットル〜毎分約６０リットルの値の間を変化する。商業上入手可能なスプレードライ装置は、当業者に周知であり、任意の特定のディスパージョンのための適当な設定は、以下に続く例への相応の参照とともに、実験的なテストを介して容易に決定することができる。
【０１３９】
ミクロ微粒子が好ましくは、エマルションの形のフッ素化発泡剤を用いて形成されるが、非フッ素化油が、ミクロ構造を低下させることなく、生物活性剤のローディング容量を増大させるために使用可能と認識されるであろう。この場合、非フッ素化油の選択は、活性または生物活性剤の溶解性、水溶性、沸点および引火点に基づく。生物活性剤は、油に溶解され、フィード溶液においてその後エマルション化されるであろう。好ましくは、油は選ばれた剤に関して、実質的な可溶化キャパシティ、より低い水溶性（＜１０^−３Ｍ）、水より高い沸点、および乾燥出口温度より高い引火点）を有する。可溶化キャパシティを増大させるための非フッ素化油への助溶媒（ｃｏ−ｓｏｌｖｅｎｔ）の、および界面活性剤の添加も、本発明の範囲内にある。
【０１４０】
特に好ましい態様において、水性組成物中で制限された溶解性を有する生物活性剤を可溶化するために、非フッ素化油を使用えきる。非フッ素化油の使用は、疎水性ペプチドおよびタンパク質のローディング容量を増大させるための特定の用途である。好ましくは、これらを可溶化するための油または油混合物は、１．３６〜１．４１の間の屈折率を有する（例えば酪酸エチル、炭酸ブチル、ジブチルエーテル）。加えて、温度および圧力等のプロセス条件は、選ばれた剤の溶解性を押し上げるために調節することができる。剤のローディング容量を最大にするための適当な油または油混合物およびプロセシング条件の選択は、ここでの教示を考慮して、当業者の範囲内であり、および過度の実験なしで達成可能と認識されるであろう。
【０１４１】
本発明の特に好ましい態様は、リン脂質等の界面活性剤と、少なくとも１つの生物活性剤を含む。他の態様は、それが更に、任意の選ばれた界面活性剤に加えて、例えば炭水化物（すなわちグルコース、ラクトースまたはデンプン）等の親水性部分を含むことができる賦形剤を更に含むことができるスプレードライ製剤を含む。この点について、種々のデンプンおよび誘導体化されたデンプンは、本発明における使用に適している。他の任意的な成分は、粘度調整剤、リン酸緩衝剤または他の従来の生体適合性の緩衝剤等の緩衝剤または酸または塩基等のｐＨ調整剤、および浸透圧剤（等張性、高モル濃度（ｍｏｌａｒｉｔｙ）または低モル濃度を与えるため）を含むことができる。適当な塩の例は、リン酸ナトリウム（一塩基性および二塩基性）、塩化ナトリウム、リン酸カルシウム、塩化カルシウムおよび他の生理的に許容できる塩を含む。
【０１４２】
如何なる成分が選ばれるにせよ、微粒子製造における第一段階は典型的には供給原料製造含む。好ましくは、選択された薬物は、濃厚溶液を製造するために水中に溶解される。薬物は、特に水不溶性剤の場合、エマルション中にで直接分散することもできる。または薬物は固体粒子ディスパージョンの形で取り込むことができる。用いられる活性または生物活性剤の濃度は、最終的な粉体で必要な剤の量、および使用されるデリバリー器具（例えばＭＤＩまたはＤＰＩのための微粉用量）の性能に依存する。必要に応じて、ポロキサマー１８８またはスパン（ｓｐａｎ）８０等の補助界面活性剤は、この付加的な溶液へ分散されてもよい。加えて、糖およびデンプン等の賦形剤も、加えることができる。
【０１４３】
選ばれた態様において、水中油型エマルションが、次いで別の容器で形成される。使用される油は、好ましくは、長鎖飽和リン脂質等の界面活性剤を用いてエマルション化されるフルオロカーボン（例えばパーフルオロオクチルブロミド、パーフルオロデカリン）である。例えば、１グラムのリン脂質は、１５０ｇの熱い蒸留水（例えば６０℃）中に、適当な高剪断の機械的ミクサー（例えばウルトラ−トゥラックス（Ｕｌｔｒａ−Ｔｕｒｒａｘ）のＴ−２５型ミクサー）を８０００ｒｐｍで５分間用いてされてにおいて均質化することができる。典型的には、５〜２５ｇのフルオロカーボンが、混合しつつ、分散された界面活性剤溶液に滴下される。水エマルション中の結果として生じるパーフルオロカーボンは、次いで、粒子サイズを低減するために高圧ホモジナイザを用いて処理される。例えば、そのエマルションは８２，７３７〜１０３，４２１ｋＰａ（１２，０００〜１８，０００ｐｓｉ）で、別個の５パスで処理することができ、且つ５０〜８０℃に維持することができる。
【０１４４】
生物活性剤溶液およびパーフルオロカーボンエマルションは、次いで、組み合わされ、スプレードライ装置に供給することができる。エマルションが好ましくは水性連続相を含むために、２つの製剤は典型的には混和性であろう。生物活性剤は本議論の目的のために別々に可溶化されるが、他の態様においては、活性または生物活性剤は、エマルション中に直接可溶化（または分散）することができると認識されるであろう。このような場合に、活性または生物活性なエマルションは、分離した薬物製剤を組み合わせることなく、単にスプレードライされる。
【０１４５】
いずれにせよ、入口および出口温度、フィード速度、微粒化圧力、乾燥空気の流速およびノズルのコンフィグレーション等の操作条件は、結果として生じる乾燥構造の必要な粒径、および製造収率を与えるために、製造者のガイドラインに従って調節することができる。例示的な設定は、次の通りである：６０℃〜１７０℃の間の空気入口温度；４０℃〜１２０℃の間の空気出口；毎分約３ｍｌ〜１５ｍｌの間のフィード速度；および３００Ｌ／分の吸引空気流量、および２５〜５０の間の微粒化空気流速。適当な装置およびプロセシング条件の選択は、ここでの教示を考慮した当業者の範囲内であって、過度の実験なしで達成することができる。いずれにせよ、これらおよび実質的に均等の方法の使用は、肺へのエアロゾル沈着に適当な粒径を有する、中空の多孔性の空気力学的に軽いミクロスフェアの形成を与える。中空および多孔質であるミクロ構造は、外観上、ほぼハチの巣状、または発泡体状である。特に好ましい態様において、有孔ミクロ構造は、中空、多孔質、スプレードライされたミクロスフェアを含む。
【０１４６】
スプレードライとともに、本発明において有用な有孔ミクロ構造は、凍結乾燥（ｌｙｏｐｈｉｌｉｚａｔｉｏｎ）によって形成することができる。当業者は、凍結乾燥が、それが凍結した後に、水が組成物から昇華されるフリーズドライプロセスであると認識するであろう。凍結乾燥プロセスに伴う特定の利点は、水溶液中で比較的不安定である生物学的製剤および他の薬剤が、高い温度（それにより、有害な熱効果を除去して）なしで乾燥することができ、次いで、安定性の問題が殆どない乾燥状態で貯蔵できるということである。本発明に関して、このような技術は、生理学的な活性を低下（ｃｏｍｐｒｏｍｉｓｅ）させることなく、微粒子または有孔ミクロ構造での、ペプチド、タンパク質、遺伝物質および他の天然および合成高分子とのないの取込みと、特に適合性を有する。凍結乾燥された微粒子を与える方法は当業者に公知であり、ここでの教示に従って適合性を有するミクロ構造を与えることは、明らかに、過度の実験を必要としない。微細な気泡状の構造を含む凍結乾燥されたケークは、５μｍまたは１０μｍ未満で平均直径を有する粒子を与えるために、当業者において公知の技術を用いて微粉化することができる。したがって、所望の特性を有するミクロ構造を与えるために凍結乾燥プロセスが使用できる範囲で、それらは本発明の範囲内にあると明白に考えられる。
【０１４７】
上記技術の他に、本発明の微粒子および有孔ミクロ構造は、また、壁形成剤を含むフィード溶液（エマルションまたは水性）が、減圧力下で迅速に加熱油（例えばパーフルブロンまたは他の高沸点ＦＣｓ）のリザーバに添加される方法を用いて形成することができる。フィード溶液の水および揮発性溶媒は、迅速に沸騰され、および蒸発される。このプロセスは、パフ化米またはポップコーンと同様に、壁形成剤から有孔構造を与えるために使用可能である。好ましくは、壁形成剤は加熱油中に不溶である。結果として生じる粒子は、次いでフィルタリング技術を用いて加熱油から分離され、その後、減圧下で乾燥することができる。
【０１４８】
加えて、本発明の粒子または有孔ミクロ構造は、二重エマルション方法を用いて形成することもできる。二重エマルション方法においては、薬物は第１に音波処理または均質化によって有機溶媒（例えば塩化メチレン）中に溶解されたポリマー中に分散される。この第１次のエマルションは、ポリビニルアルコール等のエマルション化剤を含む連続水相中で、複エマルションを形成することによって次いで安定化される。従来の技術および装置を用いる蒸発または抽出により、次いで有機溶媒が除去される。結果として生じる微小粒子は、次いで、本発明に従ってそれらを適当な懸濁媒体と組み合わせ、または使用される前に、洗浄され、濾過され、および乾燥される。
【０１４９】
Ｆ．投与
【０１５０】
ミクロ微粒子の製造のために如何なる方法が最終的に選ばれるにせよ、結果として生じる粉体は、粉体形で、または非水性の懸濁媒体を含むディスパージョンとしてそれらが効果的に用いられるような多数の有利な性質を有する。特に好ましい態様において、乾燥粉体またはディスパージョンの形のいずれでも、吸入療法を介して、生物活性な組成物が気道（すなわち肺および／又は鼻道）の粘膜の表面に投与されるであろう。このような投与は、ＭＤＩｓ、ＤＰＩｓ、ネブライザー、鼻ポンプ、アトマイザー、スプレーボトルを用いて、または滴の形での直接の点滴注入より行うことができる。しかしながら、吸入療法は本発明と極めて適合性があるが、投与の他の形および／又は経路も、有用であると認識されるであろう。
【０１５１】
この点について、本発明の粉体および安定化ディスパージョンは、体の任意の位置に対する化合物の局所的または全身の投与のためにも使用できる。したがって、好ましい態様において、（これらに制限されないが）局所的、筋肉内、経皮的、皮内、腹腔内、鼻、肺、バッカル、膣、直腸、耳に、または目への投与を含む多くの異なる経路を用いて製剤を投与できることが強調されるべきである。好ましい標的サイトは、例えば、胃腸管、尿生殖路または気道に見出すことができる。より一般的には、本発明の安定化ディスパージョンは、局所的に、または任意の体腔への投与によって剤をデリバリーするために使用できる。好ましい態様において、体腔は腹膜、副鼻洞空洞、直腸、尿道、胃、鼻腔、膣、耳道、口腔、頬側嚢および肋膜からなる群から選ばれる。当業者は、投与の選択された経路が主に生物活性剤の選択と被験体の所望の応答によって決定されると認識するであろう。
【０１５２】
開示された粉体または安定化ディスパージョンのデリバリーに関して、本発明他の面は、患者への１以上の生物活性剤、または生物学的製剤の投与のためのシステムに向けられる。上記したように、本発明と適合性を有する例示的な吸入器具は、アトマイザー、鼻ポンプ、ネブライザーまたはスプレーボトル、乾燥粉体吸入器、用量計量型吸入器またはネブライザーを含むことができる。好ましい態様においては、これらの吸入システムは、エアロゾルとして生物活性剤を所望の生理学的サイト（例えば粘膜の表面）にデリバリーするであろう。文脈上の拘束によって別に規定されない限り、本出願の目的のために、用語「エアロゾル化される」は、微細な固体または液体の粒子のガス状ディスパージョンを意味するように維持される。すなわち、エアロゾル、またはエアロゾル化された薬物は、例えば、乾燥粉体吸入器、用量計量型吸入器、アトマイザー、スプレーボトルまたはネブライザーによって発生させることができる。もちろん、以下で更に詳細に説明されるように、本発明の組成物は、直接に（例えば、従来の注射または針無しの注射により）、または液体用量点滴注入等の技術を用いて、デリバリーすることができる。従って、本発明の更なる面は、開示された粉体またはディスパージョンを含む針無しのインジェクター（例えば、加圧気体ガン）に向けられる。
【０１５３】
Ｆ（ｉ）．乾燥粉体吸入器
【０１５４】
吸入療法に関して、当業者は、本発明の粉体、特に有孔ミクロ構造を含むものが、特にＤＰＩｓにおいてで有用であると認識するであろう。従来のＤＰＩｓ、または乾燥粉体吸入器は、粉体状処方と、単独でまたはラクトースキャリア粒子とのブレンドで、薬物の所定の用量が吸入のための乾燥粉体の微細なミストまたはエアロゾルとしてデリバリーされる器具とを含む。有用なＤＰＩ薬物は、典型的には、それらが０．５〜２０μｍまでの間のサイズを有する個々の粒子に容易に分散するように処方される。粉体は、吸気によって、または加圧された空気等の外部のあるデリバリー力によって作動される。ＤＰＩ処方は、典型的には単一用量ユニットでパックされ、または用量の器具へのマニュアル移行によって、多数の用量を計測可能なリザーバシステムを使用する。
【０１５５】
ＤＰＩｓは、一般に、使用される用量デリバリーシステムに基づいて分類される。この点で、ＤＰＩｓの２つの主要なタイプは、ユニット用量デリバリー器具と、バルクリザーバデリバリーシステムとを含む。ここで用いるように、用語「リザーバ」は一般のセンスで使用され、文脈上の拘束によって別に規定されない限り、両方のコンフィグレーションを包含するように維持される。いずれにせよ、ユニット用量デリバリーシステムは、単一のユニットとして器具に与えられる粉体処方の用量を必要とする。このシステムにより、処方は、フォイル包装され、または水分侵入を防ぐためにブリスターストリップで与えることができる投与ウェルに予備充填される。他のユニット用量パッケージは、硬質ゼラチンカプセルを含む。ＤＰＩｓのために設計された大部分のユニット用量容器は、固定体積技術を用いて充填される。その結果、粉体流動性とバルク密度によって規定されるユニットパッケージ内に計量できる最小用量に、物理的限界（ここで密度）がある。
【０１５６】
上述したように、本発明の粉体は、先行技術キャリア製剤に伴う困難の多くを取り除く。すなわち、ＤＰＩ性能における改良は、ここで開示されたように、粒径、空気力学、形態および密度、湿度、および電荷を調節することによって得ることができる。この点で、本発明は、薬物、賦形剤または増量剤が、好ましくは有孔ミクロ構造と組み合わされ、または有孔ミクロ構造を含む処方を与える。上述したように、本発明に従う好ましい組成物は、典型的には０．１ｇ／ｃｍ^３ 未満、しばしば０．０５未満ｇ／ｃｍ^３ のバルク密度を有する粉体を産する。従来のＤＰＩより低い大きさのオーダーのバルク密度を有する粉体を提供することにより、選ばれた生物活性剤の遙かに低い用量がユニット用量容器に充填され、またはリザーバベースのＤＰＩｓを介して計量することが可能となる。効果的に小さい量を計量する能力は、ホルモン等の比較的効能ある生物活性剤のために重要である。更に、関連キャリア粒子なしで効果的に微粒子をデリバリーする能力は、生成物処方および充填を単純化し、および好ましくない副作用を低減する。
【０１５７】
本発明の粉体が特に単一の作動で生物活性剤の比較的高い服用量をデリバリーすることにおいて、効果的と認識されるであろう。先行技術と異なり、その粉体状処方は、効果的な充填およびデリバリーのために増量剤の使用を必要とせず、したがって、重量−重量ベースで生物活性剤のより高いレベルを含むことができる。重要なことに、開示された組成物は、単一の作動で約１０ｍｇ程度に多い生物活性剤をデリバリーするために使用できる。このような利点は、例えば、他の適合性を有する剤ほどには有力でない可能性がある受動免疫化のための免疫調節剤または抗体をデリバリーする際に、恐らく特に重要であろう。もちろん、この議論が特にＤＰＩｓの使用に向けられているが、この同じ利点は、ディスパージョン、およびＭＤＩｓ、鼻ポンプおよび針無しのインジェクター等の他の形の投与にも等しくに適用できる。
【０１５８】
前記の利点に加えて、本発明の好ましい態様は、それらを特にＤＰＩｓのために、効果的にする良好な空気力学の性質を示す。より具体的には、微小粒子の有孔構造と比較的高い表面は、吸入の間、匹敵するサイズの比較的に非有孔の粒子と比べて、より容易に、およびより長い距離で、ガスの流れでそれらが運ばれることを可能にする。それらの高いポロシティと低比重のため、ＤＰＩによる有孔ミクロ構造の投与は、鼻道および肺の周辺における粘膜の表面等の標的サイトでの増大された粒子沈着と、これに対応して、喉でのより少ない沈着を与える。このような粒子分布は、全身性投与のために好ましい、投与された剤の深い肺沈着を増大させるために使用することができる。更に、先行技術ＤＰＩ製剤に対する実質的な改良において、本発明の低い密度、高度に多孔性の粉体は、好ましくはキャリア粒子の必要性を除去する。
【０１５９】
Ｆ（ｉｉ）．安定化された分散性
【０１６０】
乾燥粉体コンフィグレーションでのそれらの使用とともに、本発明の粉体は、安定化ディスパージョンを与えるために懸濁媒体中の取り込むことができると認識されるであろう。好ましくは、安定化ディスパージョンは、非水性ディスパージョンを含む。数ある使用の中で、安定化ディスパージョンは、ＭＤＩｓ、アトマイザーまたはスプレーボトル、鼻ポンプ、針無しのインジェクター、ネブライザーまたは液体の用量点滴注入（ＬＤＩ技術）を用いて、患者の肺の気道への生物活性剤の効果的デリバリーを与える。
【０１６１】
当業者は、開示されたディスパージョンまたはディスパージョンの強化された安定性が、主に、懸濁粒子の間でのファンデルワールス引力を下げることによって、および懸濁媒体と粒子の間で密度における差違を低減することによって、達成されることを認識するであろう。ここでの教示に従って、懸濁安定性の増大は、それが次いで適合性を有する懸濁媒体中に分散される有孔ミクロ構造を設計することによって付与することができる。上述したように、有孔ミクロ構造は、流体懸濁媒体を微粒子の境界に自由に浸透させ、または灌流（ｐｅｒｆｕｓｅ）させる孔、ボイド、中空、欠損、または他の介在（ｉｎｔｅｒｓｔｉｔｉａｌ）スペースを含む。特に好ましい態様は、中空および多孔質の両方である、外観においてほぼハチの巣状または発泡体状の有孔ミクロ構造を含む。特に好ましい態様においては、有孔ミクロ構造は、中空、多孔性のスプレードライされたミクロスフェアを含む。もちろん、比較的無孔の、固体の微粒子を含む他の態様において、強化された安定性は、微粒子成分（例えば界面活性剤）の選択を介して付与することができる。
【０１６２】
有孔ミクロ構造が懸濁媒体（すなわちヒドロフルオロアルカン推進剤または液体のフルオロカーボン）中に配置される、懸濁媒体は粒子に浸透することができ、それにより、連続相および分散相が見分けがつかないホモ分散性を作る。画されたまたは「仮想」粒子（すなわち、ミクロ微粒子マトリックスによって境界線を描かれた（ｃｉｒｃｕｍｓｃｒｉｂｅｄ）体積を含む）それらが懸濁される媒体中のほぼ全体で作製されるため、粒子凝集（フロキュレーション）を駆動する力は最小にされる。加えて、画された粒子と連続相との間の密度における差違は、ミクロ構造を媒体で充填させることによって最小にされ、それにより粒子クリーミングまたは遠心沈降を効果的に遅くさせる。従って、本発明の微粒子と安定化ディスパージョンは、多くのエアロゾル化技術（例えばＭＤＩ、スプレーボトルを介する微粒化、鼻ポンプ、ネブライゼーション、その他等と、特に適合性を有する。更に、安定化ディスパージョンは、（これらに限定されないが）液体の用量点滴注入、針無しの注入、従来の注入および局所適用を含む投与の他の経路と適合性を有する。
【０１６３】
先行技術組成物と異なり、本発明の好ましいディスパージョンは、粒子の間で反発を増大させず、粒子の間でむしろ引力を減少させるように設計されている。この点で、非水性媒体中のフロキュレーションを駆動する主要な長所がファンデルワールス引力であると認識すべきである。上述したように、ＶＤＷ力は、量子力学起原であって、変動している双極子の間（すなわち誘起双極子−誘起双極子の相互作用）で、引力として視覚化することができる。ディスパージョン力は、極めて短距離で、および原子間の距離の６乗としてのスケールである。２つの巨視的な物体が互いに接近する時は、原子間のディスパージョン引力は総計される。結果として生じる力は、かなりより長距離で、相互に作用している物体の幾何学に依存する。
【０１６４】
より具体的には、２つの球状粒子に対して、ＶＤＷポテンシャル（ＶＡ）の大きさは、：
【０１６５】
【数２】

（式中、Ａ_ｅｆｆは、粒子および媒体の性質を説明する有効Ｈａｍａｋｅｒ定数であり、Ｈ_０ は粒子間の距離であり、Ｒ^１およびＲ^２は、球状の粒子１および２の半径である）
により近似することができる。有効Ｈａｍａｋｅｒ 定数は、分散粒子と懸濁媒体の分極率における差違に比例する：
【数３】

（式中、Ａ_ＳＭおよびＡ_ＰＡＲＴは、それぞれ、懸濁媒体および粒子のためのハメーカー（Ｈａｍａｋｅｒ）定数である）。懸濁粒子と分散媒が本質的に類似してくると、Ａ_ＳＭおよびＡ_ＰＡＲＴが大きさにおいてより近くなり、Ａ_ｅｆｆおよびＶ_Ａ は、より小さくなる。すなわち、懸濁媒体に伴うハメーカー定数と、分散粒子に伴うハメーカー定数の間で差違を低減することによって、有効ハメーカー定数（および対応するファンデルワールス引力）は、低減することができる。
【０１６６】
ハメーカー定数における差違を最小にする一つの方法は、上述したように連続相および分散相の両方が本質的に見分けがつかない、「ホモディスパージョン」を作製することである。有効ハメーカー定数を低減するために粒子の形態を利用する他に、構造的マトリックス（有孔ミクロ構造を画する）の成分は、懸濁媒体のそれに比較的近いハメーカー定数を示すように好ましくは選ばれるであろう。この点で、ディスパージョン成分の適合性を決定するため、および製剤の安定性に関して良好な指標を与えるために、懸濁媒体および微粒子成分のハメーカー定数の実際の値を使用することができる。代わりに、測定可能なハメーカー定数と符号するが、より容易により識別可能な特徴的な物理的な値を用いて、比較的に適合性を有する微粒子または有孔ミクロ構造成分と、懸濁媒体とを選ぶことができるであろう。
【０１６７】
この点で、多くの化合物の屈折率値が、対応するハメーカー定数に応じて変化する（ｓｃａｌｅ ｗｉｔｈ）傾向があることが判明した。したがって、容易に実質的にの屈折率値は、それに関して、懸濁媒体および粒子賦形剤の組み合わせが、比較的低い有効ハメーカー定数、およびこれに伴う安定性を有するディスパージョンを与えるであろうところの、かなり（ｆａｉｒｌｙ）良好な指標を与えるために使用可能である。化合物の屈折のインデックスが広く利用でき、または容易に誘導できるため、このような値の使用により、過度の実験をすることなく、本発明に従う安定化ディスパージョンの形成が可能となると認識されるであろう。説明のみの目的で、開示されたディスパージョンと適合性を有するいくつかの化合物の屈折のインデックスを、直ぐ下の表Ｉに与える：
【０１６８】
【表１】

【０１６９】
上述した適合性を有するディスパージョン成分と調和して、当業者は、複数の成分が約０．５未満の屈折率差を有するディスパージョンの形成が好ましいことを認識するであろう。すなわち。懸濁媒体の屈折率は、好ましくは粒子または有孔ミクロ構造に伴う屈折率のうちの約０．５の範囲内にある。懸濁媒体および粒子の屈折率は直接測定することができ、または個々それぞれの相における主成分の屈折率を用いて近似できることが、更に認識されるであろう。微粒子または有孔ミクロ構造に対して、主要な成分は、重量パーセントベースで決定できる。懸濁媒体に対して、主要な成分は、典型的には体積パーセントベースで導かれる。本発明の選ばれた態様において、屈折率差の値は、好ましくは約０．４５未満、約０．４、約０．３５または約０．３未満でさえあるであろう。低い屈折率差が、より高いディスパージョン安定性を暗示するならば、特に好ましい態様は、約０．２８未満、約０．２５、約０．２、約０．１５または約０．１未満の指数差を含む。当業者は、本開示を与えられたならば、過度の実験なしで、どの賦形剤が特に適合性を有するかについて決定できることが言える。好ましい賦形剤の最終的な選択は、また、生体適合性、規則の事情、製造の容易さ、コストを含む他の要素よって影響される。
【０１７０】
上述したように、粒子と連続相の間の密度差の最小化は、懸濁媒体が粒子体積の大部分を構成するように、有孔および／又は中空ミクロ構造を用いることによって達成することができる。ここで用いるように、用語「粒子体積」は、それらが固体（すなわち粒子境界によって定義される体積）であるならば、取り込まれた中空／多孔質粒子によって置き換えられるであろう懸濁媒体の体積に対応する。説明の目的のため、および上述したように、これらの流体充填された微粒子の体積は、「仮想的な微粒子」と言うことができる。好ましくは、生物活性剤／賦形剤シェルまたはマトリックスの平均体積（すなわち、有孔ミクロ構造によって実際に置き換えられる媒体の体積）は、平均の粒子体積の８０％未満（または仮想微粒子の８０％未満）を含む。より好ましくは、ミクロ微粒子マトリックスの体積は、平均の粒子体積の約５０％、４０％、３０％または２０％未満さえ含む。より好ましくは、シェル／マトリックスの平均体積は、平均の粒子体積の約１０％、５％、３％または１％未満を含む。当業者は、このようなマトリックスまたはシェル体積が、典型的には、そこに見出される懸濁媒体によって圧倒的に規定される仮想粒子密度に、殆ど寄与しないと認識するであろう。
【０１７１】
このようなミクロ構造の使用は、仮想粒子の見掛けの密度が、引力的なファンデルワールス力を実質的に除去している懸濁媒体のそれに近似させると、更に認識されるであろう。更に上述したように、ミクロ微微粒子マトリックスの成分が、懸濁媒体の密度に近似するために、可能な与えられた他の考慮をできる限り多くするように、選ぶことが好ましい。したがって、本発明好ましい態様において、仮想粒子と懸濁媒体は、約０．６ｇ／ｃｍ^３ 未満の密度差を有するであろう。すなわち、仮想粒子の平均の密度（マトリックス境界によって画されるように）は、懸濁媒体の約０．６ｇ／ｃｍ^３ の範囲内にある。より好ましくは、仮想粒子の平均の密度は、選ばれた懸濁媒体の０．５、０．４．０．３または０．２ｇ／ｃｍ^３ の範囲内にあるであろう。より好ましい態様において、密度差は、約０．１、０．０５．０．０１未満、または０．００５ｇ／ｃｍ^３ 未満の範囲内にあるであろう。
【０１７２】
前記の利点に加えて、開示された微粒子の使用は、懸濁物中の遙かに高い体積の粒子分率を含むディスパージョンを形成させる。密充填（ｃｌｏｓｅ ｐａｃｋｉｎｇ）にアプローチする体積分率での先行技術ディスパージョンの処方が、一般に、ディスパージョン粘弾性挙動の劇的な増大を生ずることが認識されるべきである。このタイプのレオロジー的挙動は、ＭＤＩまたはネブライザー応用に適当でない。当業者は、粒子の体積分率が粒子の見かけ体積（すなわち粒子体積）と、そのシステムの総容積への比として定義できることを認識するであろう。各システムは、最大体積分率または充填分率を有する。例えば、単一の立方配置での粒子が０．５２の最大充填分率に到達し、他方、面心立方／六方晶の密充填コンフィグレーションにおけるそれらが約０．７４の最大充填分率に到達する。非球状粒子または多分散システムに対して、誘導された値は異なる。したがって、最大充填分率は、しばしば与えられたシステムのための経験的なパラメータであると思われる。
【０１７３】
ここで、本発明の好ましい微粒子が、密充填に近似する高い体積分率でさえ、好ましくない粘弾性な挙動を示さないことが、驚くべきことに判明した。逆に、それらは、固体粒子を含む同様な懸濁物と比較して、殆どまたは全く降伏応力を有さない自由流れの、低粘度懸濁物のままである。開示された懸濁物の低い粘度は、少なくとも大部分は、流体充填された中空、多孔質粒子の間の比較的低いファンデルワールス引力のためであると思われる。従って、選ばれた態様において、開示されたディスパージョンの体積分率は、約０．３を超える。他の態様は、密充填条件に近似するより高い値で、０．３〜０．８のオーダー、または０．５〜約０．８のオーダーのパッキング値を有していてもよい。更に、体積分率が密充填に近似するとき、粒子沈降が当然に減少する傾向があるため、比較的濃縮された濃いディスパージョンの形成は、処方の安定性を更に増大させことができる。
【０１７４】
本発明の方法および組成物は比較的に濃縮された懸濁物を形成するために使用できるが、安定化因子は、遙かに低いパッキング体積で等しく良好に機能し、且つこのようなディスパージョンは、本開示の範囲内にると考えれれる。この点について、低い体積分率を含むディスパージョンが、先行技術のテクニックを用いて安定させることは極めて難しいと認識されるであろう。逆に、ここで記述したように、生物活性剤を含む微粒子を取り込んでいるディスパージョンは、低い体積分率でさえ特に安定である。したがって、本発明は、安定化ディスパージョン、特に０．３未満の体積分率で形成され、使用されるべき呼吸のためのディスパージョンを当てる。好ましいいくつかの態様において、体積分率は約０．０００１〜０．３、より好ましくは、約０．００１〜０．０１である。更に、他の好ましい態様は、約０．０１〜約０．１まで体積分率を有する安定化ディスパージョンを含む。
【０１７５】
本発明の有孔ミクロ構造は、微粉化された生物活性剤の希薄ディスパージョンを安定させるためにも使用できる。このような態様において、有孔ミクロ構造は懸濁物中の粒子の体積分率を増大させるめに添加することができ、それによりクリーミングまたは沈降に対する懸濁安定性を増大させる。更に、これらの態様において、取り込まれたミクロ構造は、また、微粉化された薬物粒子の近いアプローチ（凝集）を防ぐように作用することができる。このような態様において取り込まれた有孔ミクロ構造は、必ずしも生物活性剤を含まなくてもよいと認識すべきである。むしろ、それらは界面活性剤を含む、種々の賦形剤のみで形成することができる。
【０１７６】
当業者は、本発明の安定化懸濁物またはディスパージョンが、それが容器またはリザーバに次いで配置できる選ばれた懸濁媒体中の、ミクロ構造の分散によって製造できると更に認識するであろう。この点について、本発明の安定化製剤は、最終的な所望のディスパージョン濃度を製造するために、充分な量で成分を単に混合することによって作製できる。ミクロ構造は、機械的エネルギーなしで容易に分散されるが、分散（例えば、音波処理を用いて）を助長するための機械的エネルギーの印加も考えられる。代わりに、複数の成分は単純な振盪または他のタイプの攪拌によって混合することができる。そのプロセスは、好ましくは、懸濁安定性に対する水分の悪影響を除くために、無水条件下で行われる。一旦形成されたならば、そのディスパージョンは、フロキュレーションまたは沈降に対して低減された感受性を有する。
【０１７７】
本明細書を通して示したように、本発明のディスパージョンは好ましくは安定される。広義に、用語「安定化ディスパージョン」は、生物活性剤の効果的デリバリーを与えるために必要とされる程度に、凝集、フロキュレーションまたはクリーミングに対して抵抗性を有する任意のディスパージョンを意味するように維持される。更に、開示されたディスパージョンおよび粉体が室温で安定であって、それらの活性を効果的に維持するために冷蔵または凍結を必要としないことは、本発明の重要な利点である。貯蔵寿命を引き伸ばす他に、注目に値する温度安定性は、出荷（ｓｈｉｐｐｉｎｇ）および投与を著しく単純化する。
【０１７８】
当業者は、与えられたディスパージョンの安定性を評価するために使用可能ないくつかの方法があると認識するであろうが、本発明の目的のための好ましい方法は、動的光沈降法を用いるクリーミングまたは沈降時間の決定を含む。好ましい方法は、懸濁粒子を遠心力に供し、時間の関数として懸濁物の吸光度を測定することを含む。吸光度における迅速な減少は、貧弱な安定性を有する懸濁物を確認する。当業者が過度の実験なしで、その手順を特定のディスパージョンに適応可能なことが言える。
【０１７９】
本発明の目的のために、クリーミング時間は、懸濁された薬物微粒子が懸濁媒体の体積の１／２までクリーム化するのための時間として定義される。同様に、沈降時間は、微粒子が液体媒体の体積の１／２に沈降物するために必要とされる時間として定義することができる。上述した光沈降技術の他に、製剤のクリーミング時間を決定する比較的簡単な方法は、シールされたガラスバイアル中に微粒子のディスパージョンを与えることである。バイアルは攪拌または振盪されて比較的均一なディスパージョンを与え、それは次いで、わきに置かれ、適当な機器を用いて、または目視査によって観察される。懸濁粒子が懸濁媒体の体積の１／２までクリーム化するため（すなわち、懸濁媒体の上半分まで上昇する）、または体積の１／２まで沈降する（すなわち、媒体の１／２の底に落ちつく）ために必要な時間が、次いで記録される。１分より長いクリーミング時間を有するディスパージョン処方が好ましく、適当な安定性を示す。より好ましくは、安定化されたディスパージョンは、１、２、５、１０、１５、２０または３０分より長いクリーミング時間を含む。特に好ましい態様において、安定化ディスパージョンは、約１、１．５、２、２．５または３時間を超えるクリーミング時間を示す。沈降時間のための実質的に均等の期間は、適合性を有するディスパージョンを示す。
【０１８０】
他の成分を本発明の安定化ディスパージョンに含むことができると理解されるであろう。例えば、浸透圧剤、安定剤、キレート化剤、緩衝剤、粘度調節剤、塩および糖を、最大寿命および投与の容易さを微調整するために、安定化ディスパージョンに添加することができる。このような成分は、直接懸濁媒体に添加することができ、または有孔ミクロ構造と組み合わせ、またはその中に取り込むことができる。滅菌性、等張性および生体適合性等の考慮は、開示された組成物に従来の添加剤の使用を支配する可能性がある。このような剤の使用は当業者にとって理解されるであろうし、特定の量、比および剤のタイプは、過度の実験なしで経験的に決定することができる。
【０１８１】
Ｆ（ｉｉｉ）．計量型用量吸入器
【０１８２】
上述したように、安定化ディスパージョンは、好ましくは、用量計量型吸入器等のエアロゾル適用を介して、患者の鼻または肺の気道に投与することができる。このような安定化製剤の使用は、優れた用量再現性、および上述したように標的サイトで改善された沈着を与える。ＭＤＩｓは当該技術において周知であり、過度の実験なしでクレームされたディスパージョンの投与のために容易に使用することができた。呼吸活性化型ＭＤＩｓ、並びに既に開発され、または将来開発されるであろう他のタイプの改善を含むものも、安定化ディスパージョンおよび本発明で両立でき、およびそれらの範囲内と考えられる。
【０１８３】
ＭＤＩカニスターは、一般に、プラスチックまたはプラスチック−コートされたガラスボトル、または好ましくは、所望により陽極酸化され、ラッカーコートされ、および／又はプラスチックコートされていてもよい金属（例えばアルミニウム）等の推進剤の蒸気圧に耐えることができる容器またはリザーバを含み、その容器が計量型バルブで閉じられている。計量型バルブは、作動につき処方の計量された量をデリバリーするように設計されている。バルブは、そのバルブを介する推進剤のリークを防ぐためのガスケットを取り込む。ガスケットは、例えば、低密度ポリエチレン、クロロブチル、白および黒のブタジエン−アクリロニトリルゴム、ブチルゴム、およびネオプレン等の任意の適当なエラストマー性材料を含むことができる。適当なバルブは、例えばフランスのバロア社（Ｖａｌｏｉｓ）（例えば、ＤＦＩＯ、ＤＦ３０、ＤＦ３１／５０ＡＣＴ、ＤＦ６０）、ベスパク社（Ｂｅｓｐａｋ ｐｌｃ、ＬＴＫ）（例えばＢＫ３００、ＢＫ３５６）、および３Ｍ−ネオテクニックＬＴＫ（例えば、スプレーマイザー（Ｓｐｒａｙｍｉｓｅｒ））等のエアロゾル産業界で周知の製造者から商業的に入手可能である。
【０１８４】
個々の充填されたカニスターは、患者の肺または口または鼻の空洞への薬物の投与のための用量計量型吸入器を形成するために、使用の前に、便利に適当なチャネリング器具または作動装置にはめ込まれる。適当なチャネリング器具は、それにより、例えば、患者の鼻または口（例えばマウスピース作動装置）に対して、充填されたカニスターから計量バルブを介して薬物がデリバリーできる、例えばバルブ作動装置および円柱形または円錐形状のパスを含む。計量型用量吸入器は、例えば、作動につき１０〜５０００マイクログラムの範囲で生物活性剤等の、作動につき固定されたユニット薬用量をデリバリーするように設計されている。典型的には、単一充填型のカニスターは、数十または数百のショットまたは用量を与える。
【０１８５】
ＭＤＩｓに関して、推進剤として作用するために適当な蒸気圧を有する任意の生体適合性の懸濁媒体をも使用できることは、本発明の利点である。特に好ましい懸濁媒体は、用量計量型吸入器との使用に適合性を有する。すなわち、それらは計量型バルブの作動および関連する圧力の放出の際に、エアロゾルを形成できるであろう。一般的に、選ばれた懸濁媒体は、生体適合性（すなわち、比較的に非毒性）で、生物活性剤を含む懸濁された有孔ミクロ構造に関して非反応性であるべきである。好ましくは、懸濁媒体は、有孔ミクロスフェア中に取り込まれる任意の成分に対して実質的な溶媒の働きをしない。本発明の選ばれた態様は、フルオロカーボン（他のハロゲンで置換されたものを含む）、ヒドロフルオロアルカン、パーフルオロカーボン、炭化水素、アルコール、エーテルまたはそれらの組み合わせからなる群から選ばれる懸濁媒体を含む。懸濁媒体は、特定の特性を付与されるために選ばれる種々の化合物の混合物を含むことができると認識されるであろう。
【０１８６】
本発明のＭＤＩ懸濁媒体に用いられる特に適当な推進剤は、室温での圧力下の液化性でき、吸入または局所的使用において安全であり、毒物学的に無害で、副作用のないような推進剤ガスである。この点について、適合性を有する推進剤は、用量計量型吸入器の作動の際に、エアロゾルを能率的に形成するために充分な蒸気圧を有する、任意の炭化水素、フルオロカーボン、水素含有フルオロカーボンまたはそれらの混合物を含むことができる。それらの推進剤（典型的にはヒドロフルオロアルカンまたはＨＦＡｓと呼ばれる）は、特に適合性を有する。例えば、適当な推進剤は、短鎖炭化水素、 ＣＨ_２ＣｌＦ、ＣＣｌ_２Ｆ_２ＣＨＣｌＦ、ＣＦ_３ＣＨＣｌＦ、ＣＨＦ_２ＣＣｌＦ_２、ＣＨＣｌＦＣＨＦ_２、ＣＦ_３ＣＨ_２Ｃｌ、およびＣＣｌＦ_２ＣＨ_３等の Ｃ_１〜Ｃ_４水素含有クロロフルオロカーボン；ＣＨＦ_２ＣＨＦ_２、ＣＦ_３ＣＨ_２Ｆ、ＣＨＦ_２ＣＨ_３ およびＣＦ_３ＣＨＦＣＦ_３等のＣ_１〜Ｃ_４水素含有フルオロカーボン（例えばＨＦＡｓ）；およびＣＦ_３ＣＦ_３およびＣＦ_３ＣＦ_２ＣＦ_３ 等のパーフルオロカーボンを含む。好ましくは、単一のパーフルオロカーボンまたは水素含有フルオロカーボンが推進剤として使用される。推進剤として特に好ましいものは、１，ｌ，１，２−テトラフルオロエタン（ＣＨ_３ＣＨ_２Ｆ）（ＨＦＡ−１３４ａ）、および１，１，１，２，３，３，３−ヘプタフルオロ−ｎ−プロパン（ＣＦ_２ＣＨＦＣＦ_３）（ＨＦＡ−２２７）、パーフルオロエタン、モノクロロジフルオロメタン、１，１−ジフルオロエタン、およびそれらの組み合わせである。その処方は、成層圏のオゾンを枯渇させる成分を含まないことが好ましい。ＣＣｌ_３Ｆ、ＣＣ_１２Ｆ_２とＣＦ_３ＣＣｌ_３等のクロロフルオロカーボンを実質的に含まなくことが特に好ましい。
【０１８７】
本発明の好ましい態様は生態学的に優しい懸濁媒体を含むが、従来のクロロフルオロカーボンおよび置換されたフッ素化合物も、ここでの教示に従って懸濁媒体として使用できる。この点で、環境的な懸念が伴う可能性があるものの、ＦＣ−１１（ＣＣｌ_３Ｆ）、ＦＣ−１１Ｂ１（ＣＢｒＣｌ_２Ｆ）、ＦＣ−１１６２（ＣＢｒ_２ＣｌＦ）、ＦＣ１２Ｂ２（ＣＦ_２Ｂｒ_２）、ＦＣ２１（ＣＨＣｌ_２Ｆ）、ＦＣ−２１Ｂ１（ＣＨＢｒＣｌＦ）、ＦＣ−２１Ｂ２（ＣＨＢｒ_２Ｆ）、 ＦＣ−３１Ｂ１（ＣＨ_２ＢｒＦ）、ＦＣ１１３Ａ（ＣＣｌ_３ＣＦ_３）、 ＦＣ−１２２（ＣＣｌＦ_２ＣＨＣｌ_２）、ＦＣ−１２３（ＣＦ_３ＣＨＣｌ_２）、ＦＣ−１３２（ＣＨＣｌＦＣＨＣｌＦ）、ＦＣ−１３３（ＣＨＣｌＦＣＨＦ_２）、ＦＣ−１４１（ＣＨ_２ＣｌＣＨＣｌＦ）、ＦＣ−１４１Ｂ（ＣＣｌ_２ＦＣＨ_３）、 ＦＣ−１４２（ＣＨＦ_２ＣＨ_２Ｃｌ）、ＦＣ−１５１（ＣＨ_２ＦＣＨ_２Ｃｌ）、 ＦＣ−１５２（ＣＨ_２ＦＣＨ_２Ｆ）、ＦＣ−１１１２（ＣＣｌＦ＝ＣＣｌＦ）、ＦＧ−１１２１（ＣＨＣｌ＝ＣＦＣｌ）およびＦＣ１１３１（ＣＨＣｌ＝ＣＨＦ）の全ては、ここでの教示と適合性を有する。従って、これらの化合物の各々は、単独または他の化合物（すなわち揮発性がより小さいフルオロカーボン）と組み合わせて、本発明に従って呼吸性のディスパージョンを安定させるために使用できる。
【０１８８】
前記の態様とともに、本発明の安定化ディスパージョンは、必要とする際の患者の肺の気道に投与できるエアロゾル化された薬物を与えるように、ネブライザーとともに用いることもできる。ネブライザーは当該技術において周知であり、過度の実験なしでクレームされたディスパージョンの投与のために容易に使用することができた。呼吸活性化ネブライザー、並びに既に開発された、または開発されるであろう改良の他のタイプを含むものも、安定化ディスパージョンと適合性を有し、本発明の範囲内と考えられる。
【０１８９】
ネブライザーは、バルク液体を呼吸可能なガス中に懸濁された小さい液滴に変換するエアロゾルによって動く。ここで、（好ましくは、肺の気道に）投与されるべきエアロゾル化された薬物は、生物活性剤を含む有孔ミクロ構造と組み合わされた懸濁媒体の小さい液滴を含む。このような態様において、本発明の安定化ディスパージョンは、ネブライザーと操作可能に結合される流体リザーバ中に、典型的には配置される。与えられる製剤の特定の体積、リザーバを充填する手段、その他は主に、個々のネブライザーの選択に依存し、充分に当業者の範囲内にある。もちろん、本発明は、単一用量ネブライザーおよび多数回投与ネブライザーと、完全に適合性を有する。
【０１９０】
本発明は、好ましくはフッ素化合物（すなわちフルオロケミカル、フルオロカーボン、またはパーフルオロカーボン）を含む懸濁媒体を有する安定化ディスパージョンを与えることによって、これらおよび他の困難を克服する。本発明の特に好ましい態様は、室温での液体であるフルオロケミカルを含む。上述したように、このような化合物の使用は、連続相として、または懸濁媒体としてのいずれでも、先行技術の液体吸入製剤に勝るいくつかの利点を与える。この点について、多くのフルオロケミカルは肺における安全性と生体適合性の証明された歴史を有することは、充分に確立されている。更に、水溶液に対比して、フルオロケミカルは肺の投与の後の気体交換に否定的に衝撃を与えない。逆に、それらが実際に気体交換を改善できること、およびそれらのユニークな湿潤性特性により、粒子のエアロゾル化された流れを、肺の中に深く運んで、それにより所望の薬剤化合物の全身性デリバリーを改善する。加えて、フルオロケミカルの比較的に非反応性の性質は、取り込まれた生物活性剤の任意の分解（タンパク質分解または加水分解による）を遅らせるように作用する。
【０１９１】
いずれにせよ、ネブライザー媒介されたエアロゾル適用は、液滴の増大された表面積を与え、いくつかの場合には、微粉化され、またはエアロゾル化された薬物の輸送を与えるために、典型的には、エネルギーの入力を必要とする。エアロゾル適用の１つの共通の方法は、ノズルから流体の流れを押し出して放出させ、それにより液滴が形成される。霧状にされた投与に関して、肺中へ深く輸送されるために充分に小さくなるように、追加的なエネルギーが液滴に一般的に付与される。従って、高速ガス流れまたは圧電結晶によって与えられるような、追加的なエネルギーが必要である。ネブライザーの２つのポピュラーなタイプ、ジェットネブライザおよび超音波ネブライザーは、アトマイゼーションの間に、追加的なエネルギーを流体に印加する前記の方法に依存する。
【０１９２】
ネブライゼーションを介する体循環への生物活性剤の肺デリバリーに関して、最近の研究は、計量型溶液とも呼ばれる移動式の手で持てる（ｈａｎｄ−ｈｅｌｄ）超音波ネブライザーの使用に集中した。単一ボラス（ｂｏｌｕｓ）ネブライザーとして知られるこれらの器具は、一回または二回の呼吸における深い肺デリバリーに対して効率的な粒径を有する水溶液中の薬剤の単一ボラスをエアロゾル化する。これらの器具は、３つの広いカテゴリーに分類される。第１のカテゴリーは、Ｍｕｅｔｔｅｒｌｅｉｎ ら（Ｊ． Ａｅｒｏｓｏｌ Ｍｅｄ． １９８８；１：２３１）によって記述されたもの等の純粋の圧電気の単一ボラスネブライザーを含む。他のカテゴリーにおいて、所望のエアロゾル雲は、米国特許第３，８１２，８５４号に記述されたもの等のミクロチャネル押出単一ボラスネブライザーによって生成することができる。最後に、第３のカテゴリーは、環状の加圧単一ボラスのネブライザーを記述しているロバートソンら（ＷＯ９２／１１０５０号）によって例証された器具を含む。前記の参照の各々は、それらの全部でここで取り込む。大部分の器具は手で作動されるが、存在するいくつかの器具は、呼吸で作動される。器具が患者の回路を介する呼吸感じるとき、エアロゾルを放出することによって呼吸作動の器具は動く。呼吸作動のネブライザーは、患者のための吸入ガスを含む、エアロゾルを空気流れの中に放出するためのベンチレータ回路とインラインに配置されてもよい。
【０１９３】
どのタイプのネブライザーが使用されるかに関係なく、または生体適合性の非水性化合物が懸濁媒体として使用できることは、本発明の利点である。好ましくは、それらはそれらへのにエネルギーの印加によりエアロゾルを形成することができる。一般的に、選ばれた懸濁媒体は、生体適合性であるべきであり（すなわち比較的に非毒性）、および生物活性剤を含む懸濁された有孔ミクロ構造に対して非反応性であるべきである。好ましい態様は、懸濁媒体が、フルオロケミカル、フルオロカーボン（他のハロゲンで置換されたものを含む）、パーフルオロカーボン、フルオロカーボン／炭化水素ジブロック、炭化水素、アルコール、エーテルまたはそれらの組み合わせから成る群から選ばれた懸濁媒体を含む。懸濁媒体は、特定の特性を付与するために選ばれた種々の化合物の混合物を含むことができると認識されるであろう。
【０１９４】
ここでの教示に従って、懸濁媒体は、炭化水素、フルオロカーボンまたは炭化水素／フルオロカーボンジブロックを含む多くの異なる化合物の任意の一つを含むことができる。一般に、考えられる炭化水素または高度にフッ素化されたパーフルオロ化された化合物は、線状または分岐または環式、飽和または不飽和の化合物であることができる。これらのフルオロケミカルと炭化水素の従来の構造上の誘導体も、本発明の範囲内にあると考えられる。これらの全くまたは部分的にフッ素化された化合物を含む選ばれた態様は、１以上のヘテロ−原子および／又は臭素または塩素の原子を含むことができる。好ましくは、これらのフルオロケミカルは、２〜１６個の炭素原子を含み、（これらに制限されないが）線状、環式または多環パーフルオロアルカン、ビス（パーフルオロアルキル）アルケン、パーフルオロエーテル、パーフルオロアミン、パーフルオロアルキルブロミドおよびパーフルオロアルキルクロリド（例えばジクロロオクタン）を含む。懸濁媒体に用いられる特に好ましいフッ素化合物は、パーフルオロオクチルブロミドＣ_８Ｆ_１７Ｂｒ（ＰＦＯＢまたはパーフルブロン）、ジクロロフルオロオクタンＣ_８Ｆ_１６Ｃｌ_２、およびヒドロフルオロアルカン、パーフルオロオクチルエタンＣ_８Ｆ_１７Ｃ_２Ｈ_５（ＰＦＯＥ）を含むことができる。他の態様に関して、パーフルオロヘキサンまたはパーフルオロペンタンの懸濁媒体としての使用は、特に好ましい。
【０１９５】
より一般的には、本発明における使用が考えられる例示的なフルオロケミカルは、ハロゲン化されたフルオロケミカル（すなわち、Ｃ_ｎＨ_２ｎ＋１Ｘ、ＸＣ_ｎＦ_２ｎＸ、（式中、ｎ＝２〜１０、Ｘ＝Ｂｒ、ＣｌまたはＩ））、および特に、１−ブロモ−Ｆ−ブタン、ｎ−Ｃ_４Ｆ_９Ｂｒ、 １−ブロモ−Ｆ−ヘキサン（ｎ−Ｃ_６Ｆ_１３Ｂｒ）、１−ブロモ−Ｆ−ヘプタン（ｎ−Ｃ_７Ｆ_１５Ｂｒ）、 １，４−ジブロモ−Ｆ−ブタンおよび１，６−ジブロモ−Ｆ−ヘキサンを含む。他の有用な臭素化されたフルオロケミカルは、Ｌｏｎｇへの米国特許第３，９７５，５１２号に開示され、これを参照することによりここに取り込む。パーフルオロオクチルクロリド（ｎ−Ｃ_８Ｆ_１７Ｃｌ）、１，８−ジクロロ−Ｆ−オクタン（ｎ−ＣｌＣ_８Ｈ_１６Ｃｌ）、１，６−ジクロロ−Ｆ−ヘキサン（ｎ−ＣｌＣ_６Ｆ_１２Ｃｌ）、 および１、４−ジクロロ−Ｆ−ブタン（ｎ−ＣｌＣ_４Ｆ８Ｃ_１）等のクロリド置換基を有する特定のフルオロケミカルも好ましい。
【０１９６】
エステル、チオエーテルおよびとアミン等の他のリンケージを含むんで、ハロゲン化フルオロケミカル、フルオロカーボン、フルオロカーボン−炭化水素化合物も、本発明における懸濁媒体としての使用に適している。例えば、一般式 Ｃ_ｎＦ_２ｎ＋１ＯＣ_ｍＦ_２ｍ＋１、またはＣ_ｎＦ_２ｎ＋１ＣＨ＝ＣＨＣ_ｍＦ_２ｍ＋１、（式中、ｎ およびｍは同一または異なり、ｎおよびｍは約２〜約１２の整数である）を有する化合物（例えば、Ｃ_４Ｆ_９ＣＨ＝ＣＨＣ_４Ｆ_９（Ｆ−４４Ｅ）、ｉ−Ｃ_３Ｆ_９ＣＨ＝ＣＨＣ_６Ｆ１３（Ｆ−ｉ３６Ｅ）、 およびＣ_６Ｆ１３ＣＨ＝ＣＨＣ_６Ｆ１３（Ｆ−６６Ｅ）として）は、ここでの教示と適合性を有する。有用なフルオロケミカル炭化水素ジブロックおよびトリブロック化合物は、一般式Ｃ_ｎＦ_２ｎ＋１−Ｃ_ｍＦ_２ｍ＋１、またはＣ_ｎＦ_２ｎ＋１−Ｃ_ｍＦ_２ｍ−１（式中、ｎ＝２〜１２；ｍ＝２〜１６）、またはＣ_ｐＦ_２ｐ＋１−Ｃ_ｎＦ_２ｎ−Ｃ_ｍＦ_２ｍ＋１ （式中、ｐ＝２〜１２；ｍ＝１〜１２；ｎ＝２〜１２）を有するものを含む。このタイプの好ましい化合物は、Ｃ_８Ｆ_１７Ｃ_２Ｈ_５、Ｃ_６Ｆ_１３Ｃ_１０Ｈ_２１、Ｃ_８Ｆ_１７Ｃ_８Ｈ_１７、Ｃ_６Ｆ_１３ＣＨ＝ＣＨＣ_６Ｈ_１３、 およびＣ_８Ｆ_１７ＣＨ＝ＣＨＣ_１０Ｈ_２１を含む。置換されたエーテルまたはポリエーテル（すなわち、ＸＣ_ｎＦ_２ｎＯＣ_ｍＦ_２ｍＸ、ＸＣＦＯＣ_ｎＦ_２ｎＯＣＦ_２Ｘ（式中ｎ、およびｍ＝１〜４、Ｘ＝Ｂｒ、ＣｌまたはＩ）、およびフルオロケミカル−炭化水素エーテルジブロックまたはトリブロック（すなわち、Ｃ_ｎＦ_２ｎ＋１−Ｏ−Ｃ_ｍＦ_２ｍ＋１Ｈ（式中、ｎ＝２〜１０；ｍ＝２〜１６）、Ｃ_ｐＦ_２ｐ＋１−Ｏ−Ｃ_ｍＦ_２ｍ−Ｏ−Ｃ_ｐＦ_２ｐ＋１ （式中、ｐ＝２〜１２；ｍ＝１〜１２；ｎ＝２〜１２）も、Ｃ_ｎＦ_２ｎ＋１−Ｏ−Ｃ_ｎＦ_２ｎ−Ｏ−Ｃ_ｍＦ_２ｍ＋１ （式中、ｎ、ｍおよびｐは、１〜１２である）と同様に使用できる。更に、応用に応じて、パーフルオロアルキル化エーテルまたはポリエーテルは、クレームされたディスパージョンと適合性を有することができる。
【０１９７】
Ｃ_１０Ｆ_１８等の多環および環式フルオロケミカル（例えばＦ−デカリンまたはパーフルオロデカリン）、パーフルオロパーヒドロフェナントレン、パーフルオロテトラメチルシクロヘキサン（ＡＰ−１４４）およびパーフルオロ−ブチルデカリンも、本発明の範囲内にある。追加的な有用なフルオロケミカルは、Ｆ−トリプロピルアミン（「ＦＴＰＡ」）およびＦ−トリブチルアミン（「ＦＴＢＡ」）、Ｆ−４−メチルオクタヒドロキノリジン（「ＦＭＯＱ」）、Ｆ−Ｎ−メチルデカヒドロイソキノリン（「ＦＭＩＱ」）、Ｆ−Ｎ−メチルデカヒドロキノリン（「ＦＨＱ」）、Ｆ−Ｎ−シクロヘキシルピロリジン（「ＦＣＨＰ」）およびＦ−２−ブチルテトラヒドロフラン（「ＦＣ−７５」または「ＦＣ−７７」）等のパーフルオロ化アミンを含む。更に他の有用なフッ素化合物は、パーフルオロフェナントレン、パーフルオロメチルデカリン、パーフルオロジメチルエチルシクロヘキサン、パーフルオロジメチルデカリン、パーフルオロジエチルデカリン、パーフルオロメチルアダマンタン、パーフルオロジメチルアダマンタンを含む。パーフルオロオクチルハイドライド等の非フッ素置換基を有する他の考えられるフルオロケミカル、および異なる数の炭素原子を有する類似化合物も、有用である。当業者は、他の種々に修飾されたフルオロケミカルが、本出願で用いるようなフルオロケミカルの広い定義内に包含され、本発明における使用に適当していることを更に認識するであろう。従って、本発明の安定化ディスパージョンを形成するために、前述の化合物の各々が単独で、または他の化合物との組み合わせで使用できる。
【０１９８】
懸濁媒体として有用な可能性がある追加的な例証的なフルオロカーボンまたはフッ素化合物のクラスは、（これらに制限されないが）フルオロヘプタン、フルオロシクロヘプタン、フルオロメチルシクロヘプタン、フルオロヘキサン、フルオロシクロヘキサン、フルオロペンタン、フルオロシクロペンタン、フルオロメチルシクロペンタン、フルオロジメチルシクロペンタン、フルオロメチルシクロブタン、フルオロジメチルシクロブタン、フルオロトリメチルシクロブタン、フルオロブタン、フルオロシクロブタン、フルオロプロパン、フルオロエーテル、フルオロポリエーテル、およびとフルオロトリエチルアミンを含む。このような化合物は、一般に環境的に健全で、生物学的に非反応性である。
【０１９９】
エネルギーの印加の際にエアロゾルを製造できる任意の液体もネブライザーとともに使用可能である、選ばれた懸濁媒体は、約５０６，６２５Ｐａ（５気圧）未満、より好ましくは約２０２，６５０Ｐａ（２気圧）未満の蒸気圧を好ましくは有するであろう。特に明記しない限り、ここで記述される全ての蒸気圧は、２５℃で測定される。他の態様において、好ましい懸濁媒体化合物は、約６６６．６Ｐａ〜約１０１，３２５Ｐａ（約５Ｔｏｒｒ〜約７６０Ｔｏｒｒ）のオーダーの蒸気圧を有し、より好ましい化合物は、約１０６７Ｐａ〜約７９９９３Ｐａ（約８Ｔｏｒｒ〜約６００Ｔｏｒｒ）のオーダーの蒸気圧を有し、とりわけ好ましい化合物は約１３３３Ｐａ〜約４６６３Ｐａ（約１０Ｔｏｒｒ〜約３５０Ｔｏｒｒ）のオーダーの蒸気圧を有する。このような懸濁媒体は、効果的な換気療法を与えるために圧縮空気ネブライザー、超音波ネブライザーまたは機械的アトマイザーとともに使用可能である。分散された生物活性剤の安定性または生体利用性を更に強化するた選ばれた特定の物理的特性を有する懸濁媒体を与えるために、より揮発性の化合物を、より低い蒸気圧の成分に混合することができる。
【０２００】
ネブライザーに向けられる本発明の他の態様は、周囲の条件（すなわち１気圧）下で、選ばれた温度で沸騰する懸濁媒体を含む。例えば、好ましい態様は、０℃より上、５℃より上、１０℃より上、１５℃より上、または２０℃より上で沸騰する懸濁媒体化合物を含むであろう。他の態様において、懸濁媒体化合物は、２５℃以上で、または３０℃より上で沸騰することができる。更に、他の態様において、選ばれた懸濁媒体化合物は、ヒト体温（すなわち３７℃）以上で、４５℃、５５℃、６５℃、７５℃、８５℃、または１００℃より上で沸騰することができる。
【０２０１】
Ｆ（ｖ）．直接投与
【０２０２】
ＭＤＩおよびネブライザーとともに、本発明の安定化ディスパージョンは、種々の経路を用いて種々の標的サイトに、生物活性剤を投与するために使用可能と認識されるであろう。例えば、開示された組成物は、液体の用量点滴注入（ＬＤＩ）技術とともに、直接肺にデリバリーすることができる。代わりに、安定化ディスパージョンは、鼻ポンプ、スプレーボトルまたはアトマイザーを用いて鼻道における粘膜の表面に効果的にデリバリーすることができた。まだ他の態様において、開示されたディスパージョンは、従来の注入を用いて、または圧縮ガスを用いる針無しのインジェクターにより、標的サイトに（例えば筋肉内に、または皮膚内に）投与することができた。後者は、針無しの接種の場合に特に好ましい。更に他の態様は、眼または耳等、またはより好ましくは、泌尿生殖器管または胃腸管におけるそれら等の粘膜表面の標的サイトへのディスパージョンの局所的デリバリーに向けられる。このような技術は、取り込まれた生物活性剤の透過を強化するために更にイオン泳動を使用することができる。いずれにせよ、安定化ディスパージョンは取り込まれた剤の活性を保存しつつ、優れた用量再現性を与える。
【０２０３】
当業者は、前記のデリバリー技術と適合性を有する懸濁媒体が、ネブライザーとの使用に対して上述したものと同様であることを認識するであろう。すなわち、安定化ディスパージョンは、フルオロケミカル、フルオロカーボン（他のハロゲンで置換されたものを含む）、パーフルオロカーボン、フルオロカーボン／炭化水素ジブロック、炭化水素、アルコール、エーテルまたはそれらの組み合わせからなる群から選ばれた懸濁媒体を好ましくは含む。特に好ましくは、本出願の目的のために、このようデリバリー技術のための適合性を有する懸濁媒体は、ネブライザーでの使用とともに受器で列挙されたものと同等である。特に好ましい態様において、選ばれた懸濁媒体は、周囲の条件下の液体であるフルオロケミカルを含む。
【０２０４】
更に、液体の用量点滴注入が、安定化ディスパージョンの肺への直接の投与を含むと認識すべきである。この点について、生物活性な化合物の直接の肺投与は、特に、肺の疾患部分の貧弱な血管循環が静脈内薬物デリバリーの有効性を低減するような、疾患の治療において特に効果的である。ＬＤＩに関して、安定化ディスパージョンは、部分的な液体換気または全体の（ｐｌｕｍｏｎａｒｙ）液体換気とともに好ましくは使用される。更に、本発明は、投与より前の、投与の間の、またはその後の薬剤微粒ディスパージョン内への生理学的に許容可能なガス（例えば一酸化窒素または酸素）の治療上有益な量を導入することを更に含むことができる。
【０２０５】
ＬＤＩのために、本発明のディスパージョンは、肺デリバリー導管を用いて、肺に投与することができる。当業者は、ここで用いるように、用語「肺デリバリー導管」が、肺内への液体の点滴注入または投与を与える任意の器具または装置またはそれらの部分を含むように、広義に解釈されると認識するであろう。この点で、肺デリバリー導管またはデリバリー導管は、それを必要とする際の患者の少なくとも肺気道の部分に、開示されたディスパージョンの投与または点滴注入を与える任意のボア、ルーメン、カテーテル、チューブ、導管、シリンジ、作動装置、マウスピース、気管内チューブまたは気管支鏡を意味するように維持される。デリバリー導管は、液体換気機またはガス換気機を伴ってもよく、または伴わなくてもよいと認識されるであろう。特に好ましい態様において、デリバリー導管は、気管内チューブまたは気管支鏡を含む。
【０２０６】
本発明の安定化ディスパージョンは患者の肺の機能的残気量（ｆｕｎｃｔｉｏｎａｌ ｒｅｓｉｄｕａｌ ｃａｐａｃｉｔｙ）まで投与することができるが、選ばれた態様は、遙かに小さい体積（例えば１ミリリットル以下のオーダー）の肺の投与を含むと認識されるであろう。例えば、治療されるべき疾患に依存して、投与される体積は、１、３、５、１０、２０、５０、１００、２００または５００ミリリットルのオーダーであることができる。好ましい態様において、液体の体積は、０．２５または０．５パーセントＦＲＣ未満である。特に好ましい態様のために、液体の体積は、０．１パーセントＦＲＣ以下である。安定化ディスパージョンの比較的少ない体積の投与に関して、懸濁媒体（特にフルオロケミカル）の湿潤性およびスプレッド特性が肺における活性剤の一様な分布を容易にすると認識されるであろう。しかしながら、他の態様において、０．５を超える、０．７５または０．９パーセントＦＲＣより大きい体積のディスパージョンを投与することは、好ましい可能性がある。もちろん、少なくともいくつかのフルオロケミカルに伴う非凡な湿潤およびスプレッド特性は、鼻道等の他の粘膜の表面への投与に関して、それらを特に適合性にさせる。
【０２０７】
ここで開示される粉体および安定化とディスパージョンに関して、当業者は、それらが滅菌され、プレパッケージされたキット形で、医師または他の健康管理の専門家に有利に供給することができると認識するであろう。より具体的には、すぐ患者に投与できる安定粉体、または予め形成されたディスパージョンとして、処方を供給することができる。逆に、別個の、すぐ混合できる複数の成分として、それらを提供することもできる。すぐ使用できる形で提供された際には、粉体またはディスパージョンは、一回使用の容器またはリザーバで、並びに多数回使用の容器またはリザーバでパッケージすることができる。いずれにせよ、容器またはリザーバは、こで記述されるように、選ばれた吸入または投与器具と組み合わせることができ、且つ用いることができる。個々の成分として（例えば、粉体状ミクロスフェアとして、およびニートな懸濁媒体として）提供された際には、次いで、いつでも使用前に指示されたように容器内の内容物を単に組み合わせることによって、安定化された製剤を形成できる。加えて、ユーザーが必要に応じてそれらを次いで投与することができるように、このようなキットは、すぐに混合できる多くプレ包装された投与ユニットを含むことができる。
【０２０８】
Ｇ．例
【０２０９】
上記の記述は、以下の例を参照することにより、より充分に理解されるであろう。このような例は、しかしながら、本発明を実施する好ましい方法の単なる代表的なもののみであって、いかなる方法でも、本発明の範囲を制限することと読まれ、または解釈されるべきではない。
【０２１０】
Ｉ．スプレードライによるＨＡペプチドの中空多孔質粒子の製造
【０２１１】
吸引：１００％、入口温度：８５℃、出口温度：５１℃；フィードポンプ：１０％：Ｎ_２ 流：８００Ｌ／ｈｒの条件下で、Ｂ−１９１のミニスプレードライヤー（Ｂｕｅｃｈｉ、Ｆｌａｗｉｌ、スイス）を用いるスプレードライ技術によって、中空多孔性のＨＡ１１０−１２０ペプチド（インフルエンザウィルス粒子の赤血球凝集素のアミノ酸残基１１０〜１２０）粒子（「プルモ・スフィア」（ＰｕｌｍｏＳｈｅｒｅｓ；商標））を製造した。そのフィードは、スプレードライの直前に２つの製剤（ＡおよびＢ）を混合することによって製造した。粒子収集を助長するためにサイクロン出口ポートに、１５０メッシュのステンレス鋼スクリーンを置いた。
【０２１２】
製剤Ａ：ｌ８ｍｇのＨＡ１１０〜１２０のペプチド（カイロン（Ｃｈｉｒｏｎ）社、エメリービル（Ｅｍｅｒｙｖｉｌｌｅ）、カリフォルニア州）と、ｌｍｇのヒドロキシエチル澱粉（味の素、日本）とを溶解するために、５ｇの脱イオン水を用いた。
【０２１３】
製剤Ｂ：リン脂質によって安定化された水中フルオロカーボン−エマルションを、以下のようにして製造した。そのリン脂質（０．３ｇのＥＰＣ−１００−３（リポイドＫＧ、Ｌｕｄｗｉｇｓｈａｆｅｎ、ドイツ）を、３３ｇの熱い脱イオン水（Ｔ＝５０〜６０℃）中でウルトラ−トゥラックスミクサー（Ｔ−２５型）を８０００ ｒｐｍで２〜５分間（Ｔ＝６０〜７０℃）用いて均質にした。８グラムのパーフルブロン（パーフルオロオクチルブロミド：アトケム（Ａｔｏｃｈｅｍ）、パリ、フランス）を、混合の間、滴下で加えた。フルオロカーボンを加えた後、エマルションを少なくとも４分間混合した。得られた粗製エマルションを、次いで、１２４，１０６ｋＰａ（１８，０００ｐｓｉ）で５パスで高圧ホモジナイザ（Ａｖｅｓｔｉｎ、オタワ、カナダ）に通した。
【０２１４】
製剤Ｂの８分の１を分離し、製剤Ａに添加した。得られたＨＡペプチド／パーフルブロンのエマルションフィード溶液を、上述した条件下でスプレードライヤーに供給した。粉体をサイクロンで集め、篩分けスクリーンを、パーフルブロンを用いて収集ジャー内に洗浄した。パーフルブロン中のＨＡ懸濁液を、その後−６０℃で凍結させ、凍結乾燥した。易流動性（ｆｒｅｅ ｆｌｏｗｉｎｇ）の白い粉体を得た。
【０２１５】
ＩＩ．ＨＡペプチドを含む中空多孔質微粒子のインビトロ活性
【０２１６】
プルモ・スフィア（ＨＡ−Ｐｕｌ）でのＨＡペプチドの、抗原を現出している細胞を活性化する機能性を、ニートなＨＡペプチドと比較した。例ｌからのＨＡペプチドのプルモ・スフィアを、５ｍｇ／ｍｌ（処方の重量／体積）の濃度で、滅菌ＰＢＳでインキュベートした。得られたＨＡ−Ｐｕｌ−ＰＢＳ溶液のシリアル希釈を、完全ＲＰＭＩ−ｌ０％ＦＣＳ中のＭｌ２抗原を現出している細胞（１×１０^４／ウェル）およびＨＡ特異的ＴｃＨ（２×１０^４／ウェル）を含むマイクロウェルに添加した。ＴｃＨセルラインは、ＩＬ−２プロモータ（ＩＬ−２／−ｇａｌ）によって制御されるリポーター遺伝子を有する。
【０２１７】
３７℃で１２時間のインキュベーションの後、マイクロウェルプレートを遠心分離し、パラホルムアルデヒド−グルタルアルデヒドで細胞を４℃、５分間で固定して、ＰＢＳで洗浄し、Ｘ−ｇａｌ基質を一晩中加えた。ウェルあたり５００個の細胞につき活性ＴｃＨの数を、光学顕微鏡を用いてカウントした。ウェルあたりの活性ＴｃＨの総数は、細胞の総数に青い細胞のパーセンテージを掛けることによって見積もった。図１に示す結果は、処方中の活性ペプチドの存在を示す。標準の活性化曲線（ＨＡ食塩水（ｓａｌｉｎｅ））による比較は、活性ペプチドの濃度が約５％質量／質量（重量／重量）であることを示し、それは逆相−ＨＰＬＣ測定と一致した。
【０２１８】
ＩＩＩ ．ＨＡペプチドプルモスフィアの作用のメカニズム
【０２１９】
Ｔ細胞エピトープＨＡ１１０−１２０（ＨＡ−Ｐｕｌ）を含むプルモ・スフィアミクロ粒子の内面化（ｉｎｔｅｒｎａｌｉｚａｔｉｏｎ）およびプロセシングの必要性を検討した。パーフルブロン中に懸濁されたＨＡ−Ｐｕｌ（５００ｎＭ／ウェル ＨＡ１１０化１２０のペプチド）を風乾（ａｉｒ−ｄｒｉｅｄ）し、および非固定、またはパラホルムアルデヒド固定のＭ１２抗原現出細胞（ＡＰＣ）細胞と、完全ＲＰＭＩ−１０％ＦＣＳ中の特異的ＴｃＨ細胞の存在下でインキュベートし、および同様の濃度でＰＢＳ中に懸濁されたＨＡ−Ｐｕｌ、およびニートなＨＡペプチドと比較した。それが細胞内プロセシングを必要とする（ｄｏｅｓ ｔｈａｔ ｒｅｑｕｉｒｅ）ことから、ショ糖−精製したＡ／ＰＲ／８／３４（Ｈ１Ｎ１）ウィルス（１５ｇ／ｍｌ）をポジティブ対照として使用した。ネガティブ対照は、ＮＰ１４７−１５５ペプチドと、処方されていないＮＰペプチドと、無関係な無関係なウィルスとからなっていた。例ＩＩにおいて記述した細胞の数および成長条件が、続いた。細胞を固定し、Ｘ−ｇａｌ基質にさらした。結果を、活性ＴｃＨｌの％として表した。
【０２２０】
図２は、固定した、および非固定のＡＰＣが、ニートなＨＡペプチドとＨＡＰｕｌを現出できたことを示す。対照的に、生存ＡＰＣのみが、ウィルス性状況からＨＡペプチドを現出することができた。更に、処方された、およびニートなＮＰペプチド、並びにＢ／Ｌｅｅウィルスは、特異的ＴｃＨを活性化しなかった。結果は、ＨＡ−Ｐｕｌの内面化およびプロセシングが、ＴｃＨの活性化の必要条件でないことを示す。むしろ、ＨＡ−ペプチドは、容易にプルモ・スフィアから放出され、Ｍ１２ ＡＰＣ上でＭＨＣクラスＩＩ分子（Ｉ−Ｅ^ｄ ）に結合して、ＴＣＲのエンゲージメントおよびＴｃＨの活性化を生じる。このプロセシングステップを、ＰＢＳまたはパーフルブロン中でデリバリーされたＨＡ−Ｐｕｌ、並びにニートなＨＡペプチドに対して観察した。更に、これらの結果は、プルモ・スフィア中で処方され、パーフルブロン中で安定化されたＨＡ １１０−１２０のペプチドが、その免疫原性を保持することを証明する。
【０２２１】
ＩＶ．スプレードライによる蛍光ラベルした中空多孔質ＨＡペプチド粒子の製造
【０２２２】
吸引：１００％、入口温度：８５℃、出口温度：５１℃；フィードポンプ：１０％：Ｎ_２ 流：８００Ｌ／ｈｒの条件下で、Ｂ−１９１のミニスプレードライヤー（Ｂｕｅｃｈｉ、Ｆｌａｗｉｌ、スイス）を用いるスプレードライ技術によって、中空多孔性のＨＡ−フルオロセイン（ｆｌｕｏｒｏｓｃｅｉｎ）１１０−１２０ペプチド／テキサスレッドＤＨＰＥ粒子を製造した。そのフィードは、スプレードライの直前に２つの製剤（ＡおよびＢ）を混合することによって製造した。粒子収集を助長するためにサイクロン出口ポートに、１５０メッシュのステンレス鋼スクリーンを置いた。
【０２２３】
製剤Ａ：２０ｍｇのＨＡ−フルオロセイン １１０〜１２０のペプチド（カイロン社、エメリービル（Ｅｍｅｒｙｖｉｌｌｅ）、カリフォルニア州）と、ｌｍｇのヒドロキシエチル澱粉（味の素、日本）とを溶解するために、５ｇの脱イオン水を用いた。
【０２２４】
製剤Ｂ：リン脂質によって安定化された水中フルオロカーボン−エマルションを、以下のようにして製造した。そのリン脂質（０．３ｇのＥＰＣ−１００−３（リポイドＫＧ、Ｌｕｄｗｉｇｓｈａｆｅｎ、ドイツ）、および０．３ｍｇの蛍光色素、テキサスレッドＤＨＰＥ（モレキュラープローブ、Ｅｕｇｅｎｅ、ＯＲ、３ｍｇ）を、先ずクロロホルムに溶解した。クロロホルムを、次いでＢｕｃｈｉ ＲｏｔｏＶａｐ を用いて除去した。そのＥｌ００−３／テキサスレッドＤＨＰＥ薄膜を、次いで３３ｇの熱い脱イオン水（Ｔ＝６０〜７０℃）中に分散させた。その界面活性剤を、更に、でウルトラ−トゥラックスミクサー（Ｔ−２５型）を１０，０００ｒｐｍ で約２分間（Ｔ＝５０〜６０℃）用いてプロセシングした。８グラムのパーフルブロン（アトケム、パリ、フランス）を、混合の間、滴下で加えた。フルオロカーボンを加えた後、エマルションを少なくとも４分間混合した。得られた粗製エマルションを、次いで、１２４，１０６ｋＰａ（１８，０００ｐｓｉ）で５パスで高圧ホモジナイザ（Ａｖｅｓｔｉｎ、オタワ、カナダ）に通した。
【０２２５】
製剤Ｂの８分の１を分離し、製剤Ａに添加した。得られたＨＡペプチド／テキサスレッドＤＨＰＥ／パーフルブロンのエマルションフィード溶液を、上述した条件下でスプレードライヤーに供給した。粉体をサイクロンで集め、篩分けスクリーンを、パーフルブロンを用いて収集ジャー内に洗浄した。パーフルブロン中のＨＡ懸濁液を、その後−６０℃で凍結させ、凍結乾燥した。易流動性の蛍光フクシア色の粉体を得た。
【０２２６】
Ｖ．蛍光ラベルしたＨＡプルモスフィアの生体利用性
【０２２７】
例ＩＶのように製造したフルオロセイン−ＨＡペプチド（２０％質量／質量（重量／重量％））を、パーフルブロン中に懸濁した。メトファン（Ｍｅｔｏｆａｎ）麻酔したマウスを、７０ｇのペプチド用量に対応する、パーフルブロン中のｆ−ＨＡＰｕｌの７０ ｌ体積で、鼻内に（ｉ．ｎ．）接種した。血液サンプルを目ブリージングによってヘパリン処理チューブ内に集め、血漿を分離し、ペプチドの濃度を蛍光分析で測定した。対照として、滅菌食塩水（全ての群に対してｎ＝４）の７０ ｌ中の７０ｇのｆ−ＨＡペプチドの静脈内（ｉ．ｖ．）接種を使用した。
【０２２８】
図３は、時間に対するｆ−ＨＡペプチドの血清濃度を示す。ｉ．ｎ．−デリバリーされたｆ−ＨＡペプチドの絶対生体利用性は約５％であり、Ｔ_ｍａｘ は２０分で生じた。ｉ．ｖ．投与に対する連続的な対数的減少と、ｉ．ｎ．投与の場合の一過性の増大と、後に続く指数関数的な減少で、薬物動態的なプロフィルは２つの投与経路の間で異なっていた。６時間までの総クリアランスで、ｆ−ＨＡの除去は、尿（図示しない）を介して生ずる。
【０２２９】
この例は、Ｐｕｌ中で処方されたＴ細胞エピトープ（約１．４ｋＤａの分子量）のｉ．ｎ．投与が全身性デリバリーと適合性を有することを示す。
【０２３０】
ＶＩ．スプレードライによるヒトＩｇＧ中空の多孔質微粒子の製造
【０２３１】
吸引：１００％、入口温度：８５℃、出口温度：６１℃；フィードポンプ：１０％：Ｎ_２ 流：８００Ｌ／ｈｒの条件下で、Ｂ−１９１のミニスプレードライヤー（Ｂｕｅｃｈｉ、Ｆｌａｗｉｌ、スイス）を用いるスプレードライ技術によって、中空多孔性のヒトＩｇＧ粒子を製造した。そのフィードは、スプレードライの直前に２つの製剤（ＡおよびＢ）を混合することによって製造した。
【０２３２】
製剤Ａ：５５ｍｇのヒトＩｇＧ（シグマケミカル、セントルイス、ミズーリ州）と、３．２ｍｇのヒドロキシエチル澱粉（味の素、日本）とを溶解するために、２ｍｇの正常食塩水（バクスター、シカゴ、イリノイ州）を用いた。
【０２３３】
製剤Ｂ：リン脂質によって安定化された水中フルオロカーボン−エマルションを、以下のようにして製造した。そのリン脂質、０．４１５ｇのＥＰＣ−１００−３（リポイドＫＧ、Ｌｕｄｗｉｇｓｈａｆｅｎ、ドイツ）を、４０．３ｇの熱い脱イオン水（Ｔ＝５０〜６０℃）中でウルトラ−トゥラックスミクサー（Ｔ−２５型） を８０００ｒｐｍで２〜５分間（Ｔ＝６０〜７０℃）用いて均質にした。５．２ｇのパーフルブロン（アトケム、パリ、フランス）を、混合の間、滴下で加えた。フルオロカーボンを加えた後、エマルションを少なくとも４分間混合した。得られた粗製エマルションを、次いで、１２４，１０６ｋＰａ（１８，０００ｐｓｉ）で５パスで高圧ホモジナイザ（Ａｖｅｓｔｉｎ、オタワ、カナダ）に通した。
【０２３４】
製剤Ｂの８分の１を分離し、製剤Ａに添加した。得られたＩｇＧ／パーフルブロンのエマルションフィード溶液を、上述した条件下でスプレードライヤーに供給した。粉体をサイクロンで集め、篩分けスクリーンを、パーフルブロンを用いて収集ジャー内に洗浄した。パーフルブロン中のＩｇＧ懸濁液を、その後−６０℃で凍結させ、凍結乾燥した。易流動性の白い粉体を得た。中空多孔性のＩｇＧ粒子は、飛行時間型の分析法（Ａｅｒｏｓｉｚｅｒ、Ａｍｈｅｒｓｔ Ｐｒｏｃｅｓｓ Ｉｎｓｔｒｕｍｅｎｔｓ、Ａｍｈｅｒｓｔ、マサチューセッツ州） によって決定された２．３７３±１．８８μｍの体積−重み平均空気力学的直径を有していた。
【０２３５】
ＶＩＩ． ポリクローナル性ヒトＩｇＧプルモ・スフィアのインビトロ活性
【０２３６】
例ＩＶからのポリクローナル性ヒトＩｇＧプルモ・スフィア（ｈＩｇＧ−Ｐｕｌ）の処方を、捕捉ｈＩｇＧ酵素免疫吸着測定法（ＥＬＩＳＡ）を用いて活性について確認した。パーフルブロン中の５ｍｇ／ｍＬのｈＩｇＧ−ＰｕＬ懸濁液を製造し、ウェル中へピペットで移し、風乾した。ＰＢＳを乾燥したｈＩｇＧ−Ｐｕｌに添加して、一晩中をインキュベートさせた。水和されたｈＩｇＧ−Ｐｕｌ溶液を希釈して、コーティング緩衝剤（ｄｉｌ．１：１０００、シグマ・イムノケミカル）中のマウス抗ヒトｋ鎖モノクローナル抗体でコートした酵素免疫吸着測定法プレート移し、その後、１５％のヤギ血清を含むＰＢＳで室温、２時間でブロックした。ウェルを洗浄し、ヤギ抗ヒトＩｇＧアルカリホスファターゼのコンジュゲート（ＰＢＳ−１５％ヤギ血清、０．０５％Ｔｗｅｅｎ中、１：１０００）を用い、その後ｐＮＰＰ基質を添加することによりアッセイを行った。４０５ｎｍに設定した自動プレートリーダーを用いて、光学濃度（ＯＤ）を読んだ。食塩水（標準）中のｈＩｇＧ、およびブランクのプルモ・スフィアと混合したｈＩｇＧを、アッセイ上での脂質の効果を除外するための対照として使用した。ブランクのプルモ・スフィアは、リン脂質とデンプンのみから成っていた。
【０２３７】
図４は、ｈＩｇＧ−Ｐｕｌ、ｈＩｇＧおよびｈＩｇＩＧ＋ブランクのプルモ・スフィアのためのキャリブレーション曲線を示す。ｈＩｇＧ−Ｐｕｌ処方は、約２０質量％（重量％）のｈＩｇＧを含むと決定された。加えて、ｈＩｇＧ−Ｐｕｌは、ｋ軽鎖および重鎖エピトープの発現を保持した。
【０２３８】
ＶＩＩＩ．プルモスフィア処方からのＨＡ１１０−１２０ペプチドおよびヒトＩｇＧの溶解動態
【０２３９】
プルモ・スフィアからの抗原およびｈＩｇＧ放出の動態を、０．ｌｕｍ直径フィルタを備えた溶解チャンバと、適合した２４ウェル平底セル培養プレートを用いて測定した。例ｌおよびＶＩからのプルモ・スフィア粉体の約３ｍｇを、溶解チャンバの下部コンパートメントに配置して、同時に滅菌ＰＢＳにさらした（１．３ｍ１／ウェル）。呼吸パターンをシミュレーションするために、プレートを３７℃で水平の振盪機（３０ＲＰＭ）上に配置した。２５μｌサンプルを、上部コンパートメントから集め、ｈＩｇＧの場合の捕捉ＥＬＩＳＡ（図５Ａ）、またはフルオロセインでラベルしたＨＡペプチド処方（ｆ−ＨＡ）の場合のバイオアッセイ（図５Ｂ）で分析した。ｆ−ＨＡに対する結果を、蛍光分析（図示せず）によって独立に確認した。ｈＩｇＧおよびＨＡペプチドプルモ・スフィアの溶解動態を、それらのそれぞれの水性対照と比較した。
【０２４０】
図５Ａおよび５Ｂに示す結果は、パーセント放出として表した。迅速な拡散制御の放出は、ＨＡペプチド処方に対して観察され、水性対照との間の差違は無かった。完全溶解は、２時間以内に生じた。対照的に、より遅い浸食制御動態は、ｈＩｇＧに対して観察された。完全溶解は、水のＩｇＧ対照に対する１時間と比較して、６時間より長い時間を必要とした。ここで記述した結果は、プルモ・スフィアからの溶解動態が、少なくとも部分的に、処方された化合物の分子量（それぞれ、１．４ｋＤａおよび１５０ｋＤａ）に依存することを証明する。それはまた、親水性および疎水性における差違は、同様の効果を有する可能性があることが認識されるであろう。
【０２４１】
ＩＸ． ｈＩｏＧプルモスフィアの生体利用性
【０２４２】
ケタミン／キシラジンで麻酔されたマウスに気管内で（２０ ｌのパーフルブロン中の２０ｇのｈＩｇＧ）、またはメトファンで麻酔されたマウスへ鼻ルートで（７０μｌのパーフルブロン中の７０ｇｈＩｇＧ）を介して、例ＶＩからのヒトＩｇＩＧプルモスフィア（ｈＩｇＧ−Ｐｕｌを）を投与した。滅菌ＰＢＳ中のｈＩｇＧの同一の体積を、対照群において静脈内投与した。種々の時間的間隔でマウスから血を抜き取り、およびｈＩｇＧの血清濃度を全ての群（ｎ＝３）で、捕捉酵素免疫吸着測定法によって評価した。ｉ．ｖ．対照と比較した血清濃度−時間曲線（ＡＵＣ）の下で面積から、絶対生体利用性を決定した。ＡＵＣ値は、台形法則を用いて計算した。
【０２４３】
血漿ｈＩｇＧ濃度曲線を、図６Ａ（ｉ．ｔ．）、および図６ＧＢ（ｉ．ｎ．）に示す。ｈＩｇＧ−Ｐｕｌの気管内デリバリーに対する絶対生体利用性は２７％であり、鼻腔内接種に対しては１．５％であった。両方の場合とも、Ｔ_ｍａｘは、約２日で生じた。ウエスタンブロッティングは、気道を介するデリバリー後の循環するｈＩｇＧに対する分子量が、ニートな材料から区別できないことを示した。そのｈＩｇＧは、１４日を越える循環において持続することが観察された。
【０２４４】
Ｘ．気管の経路を介してデリバリーされたｈＩｇＧプルモスフィアへの抗体応答
【０２４５】
パーフルブロン中に懸濁された、例ＶＩからのｈＩｇＧ−プルモ・スフィア（ｈＩｇＧ−Ｐｕｌ）で処理されたマウスにおける気管内投与（ｈＩｇＧの２０ｇ用量）を介する血液および気管支・肺胞洗浄（ＢＡＬ）中の液の応答。マウスを、以下の対照でも処理した：食塩水中の２０μｇのｈＩｇＧ、ｉ．ｔ．；１食塩水中の１００μｇのｈＩｇＧ、ｉ．ｖ．およびｉ．ｔ．；完全フロイントアジュバント（ＣＦＡ）中の１００μｇ、皮下、および食塩水、ｉ．ｔ．。個々の群を、三重実験（ｔｒｉｐｌｉｃａｔｅ）で行った。血液およびＢＡＬを、免疫化の２週後に集めた。
【０２４６】
抗原ｈＩｇＧ−マウスＩｇＧのタイターを、ｈＩｇＧまたは０．１％のＢＳＡでコートした酵素免疫吸着測定法プレートを用いて測定した。ウェルを、ＰＢＳ−１５％ヤギ血清でブロックして、血清またはＢＡＬの種々の希釈を用いて２時間インキュベートした。洗浄の後、抗マウスＩｇＧアルカリホスファターゼのコンジュゲート、およびこれに続くＰＮＰＰ基質の添加により、アッセイを行った。およびが続いた。プレートの光学濃度（ＯＤ）を、自動プレートリーダーを用いて５５０ｎｍで分析し、および結果を血清ＩｇＧの場合（図７Ａ）終点希釈タイターとして、またはＢＡＬ ＩｇＧ（図７Ｂ）に対する平均ＯＤとして表した。
【０２４７】
これらの結果は、食塩水中でｈＩｇＧを受けた用量／経路を調和させた群と比較して、気管内経路を介してｈＩｇＧ−Ｐｕｌで処理しるマウスにおける、増大した全身性および局所的な液性の応答を示す。更に、気管内または静脈内経路を介して、食塩水中のｈＩｇＧのより高い用量を受けたマウスと比較して、応答が強化された。血清抗体のタイターは、ＣＦＡ中のｈＩｇＧでｓ．ｃ．免疫したマウスで測定されたものと同様であった。興味あることに、液性の応答は全身の生体利用性（データ示さず）と相関せず、局所免疫の関与を暗示した。
【０２４８】
ＸＩ．気管の経路を介してデリバリーされたｈＩｇＧプルモスフィアへのＴ細胞応答
【０２４９】
パーフルブロン中に懸濁された、例ＶＩからのｈＩｇＧ−プルモ・スフィア（ｈＩｇＧ−Ｐｕｌ）により気管経路で免疫されたマウスの脾臓において誘導されたＴ細胞免疫のレベル。脾臓を単細胞懸濁に分離し、赤血球を除去するために低張緩衝剤で処理した。脾細胞を、４×１０６細胞／ｍｌで完全ＲＰＭＩ−１０％ＦＣＳ中に再懸濁し、２４−ウェルの平底プレート（１ｍｌ／ウェル）中で、６ｇ／ｍｌのｈＩｇＧの存在下でインキュベートした。７２時間のインキュベーションの後、上澄みを集め、酵素免疫吸着測定法（バイオソース（Ｂｉｏｓｏｕｒｃｅ）インターナショナル、Ｃａｍａｒｉｌｌｏ 、カリフォルニア州）によって、ＩＬ−２、ＩＦＮ−およびＩＬ−４の濃度を決定した。
【０２５０】
結果（図８）は、各群での個々のマウスの中のサイトカイン濃度の平均値として表され、ｈＩｇＧ食塩水対照と比較して、ｈＩｇＧ−Ｐｕｌで免疫したマウスにおける全３個のサイトカインの強化された産生を示した。ｈＩｇＧ−Ｐｕｌ処理した群に対する脾臓Ｔ細胞によるサイトカインの産生は、食塩水中のｈＩｇＧのｉ．ｖ．群に対して観察されたそれに匹敵した。これらの結果は、肺で活性化された記憶Ｔ細胞の全身性移行を強く示唆する。
【０２５１】
ＸＩ．鼻経路を介してデリバリーされたｈＩｇＧプルモスフィアへの抗体応答
【０２５２】
パーフルブロン中に懸濁され、または食塩水に溶解された例ＶＩからのプルモ・スフィア（ｈＩｇＧ−Ｐｕｌ）として処方されたいずれかの鼻腔内滴下（２０ｇ）を介してｈＩｇＧを受けたマウスの液性の応答を確認した。免疫化後の種々の時間的間隔で血清を得て、ｈＩｇＧに対する特異的マウスＩｇＧのタイターを、例Ｘで記述した酵素免疫吸着測定法の手順を用いた測定した。それらの結果（図９）は、平均の終点タイター（ｎ＝３）として表され、食塩水と比較して、ｈＩｇＧ−Ｐｕｌで処理したマウスにおける開始の動態がより速く、大きさが高く、免疫応答の被検体間（ｉｎｔｅｒｓｕｂｊｅｃｔ）再現性がより低いことを示した。
【０２５３】
Ｘ ＩＩＩ ．腹膜経路を介してデリバリーされたｈＩｇＧプルモ・スフィアへの抗体応答
【０２５４】
パーフルブロン中に懸濁された例ＶＩからのｈＩｇＧ−プルモ・スフィア（ｈＩｇＧ−Ｐｕｌ）で、腹膜（ｉ．ｐ．）経路（ｈＩｇＧの１００ｇ用量）を介して処理したマウスの体液性応答。マウスを、以下の対照でｌ００μｇのｈＩｇＧでもｉ．ｐ．処理した：食塩水中、多重膜ジパルミトイルホスファチジルコリン（ＤＰＰＣ）リポソーム食塩溶液（＋ｍｌ ｌｉｐ）中、単層（ｕｎｉｌａｍｅｌｌａｒ）ＤＰＰＣリポソーム食塩溶液（＋ｕｌ ｌｉｐ）中、およびブランクのプルモ・スフィア食塩溶液（＋ｅｍｐｔｙ Ｐｕｌ）中。ｈＩｇＧが全くないブランクのプルモ・スフィア溶液の追加的な対照群をも、テストした。ｍｌ ｌｉｐ（＞ｌ０μｌ）およびｕｌ ｌｉｐ（９０ｎｍ）の粒子メジアン径を、レーザ光散乱技術を用いて決定した。各群を、三重実験で行った。例Ｘと同じ酵素免疫吸着測定法技術を用いて、血清中のＩｇＧ体液免疫応答を７日および１４日で測定した。
【０２５５】
結果は終点タイターの平均として表され、ｈＩｇＧ−Ｐｕｌで接種された動物に対する抗体価の一貫した増大を示した。特に図１０Ａおよびｌ０Ｂは、それぞれ７日および１４日での終点タイターを示す。空Ｐｕｈに加えられたＩｇＧは、食塩水中のｈＩｇＧに類似するタイターを誘導した。更に、ｈＩｇＧへのいずれのＤＰＰＣリポソーム製剤の添加も、ｈＩｇＧ−Ｐｕｌで観察された増大した免疫を回復しなかった。従って、これらの結果は：（１）ｈＩｇＧ−Ｐｕｌの強化された免疫は、経路に依存する現象でない（例ＸおよびＸＩＩを参照）；（２）ｈＩｇＧ−Ｐｕｌの処方は、ｈＩｇＧの強化された免疫原性のための先行必要条件である；および（３）ＤＰＰＣまたはＰｕｌの他の成分は、独立したアジュバント効果を有しない、ことを証明する。更に、これらの結果は、ｈＩｇＧ−Ｐｕｌによって誘導された強化された免疫性の原因となるデリバリーの経路、並びに他の因子の重要性を解明する。
【０２５６】
ＸＩＶ．スプレードライによるインフルエンザウィルスＡ／ＷＳＮ／３２（Ｈ１Ｎ１）の中空多孔質粒子の製造
【０２５７】
吸引：１００％、入口温度：８５℃、出口温度：６１℃；フィードポンプ：１０％：Ｎ_２ 流：８００Ｌ／ｈｒの条件下で、Ｂ−１９１のミニスプレードライヤー（Ｂｕｅｃｈｉ、Ｆｌａｗｉｌ、スイス）を用いるスプレードライ技術により、８個の構造タンパク質複合体と８個の負荷電されたＲＮＡセグメントとを含む比較的複雑なエンベロープウィルスを含む、中空多孔性のインフルエンザウィルスＡ／ＷＳＮ／３２（Ｈ１Ｎ１）をミクロ粒子内に良好に取り込んだ。そのフィードは、スプレードライの直前に２つの製剤（ＡおよびＢ）を混合することによって製造した。処方前に、ウィルスは生存しており、ショ糖勾配遠心分離によって精製した。
【０２５８】
製剤Ａ：ｌｍｇのヒドロキシエチル澱粉（味の素、日本）を秤量し、食塩水中０．６ｇのインフルエンザウィルスを含むチューブへ移した。
【０２５９】
製剤Ｂ：リン脂質によって安定化された水中フルオロカーボン−エマルションを、以下のようにして製造した。そのリン脂質、０．１１１ｇのＥＰＣ−１００−３（リポイドＫＧ、Ｌｕｄｗｉｇｓｈａｆｅｎ、ドイツ）を、２０ｇの熱い脱イオン水（Ｔ＝５０〜６０℃）中でウルトラ−トゥラックスミクサー（Ｔ−２５型）を８０００ｒｐｍで２〜５分間（Ｔ＝６０〜７０℃）用いて均質にした。４．４ｇのパーフルブロン（アトケム、パリ、フランス）を、混合の間、滴下で加えた。フルオロカーボンを加えた後、エマルションを少なくとも４分間混合した。得られた粗製エマルションを、次いで、１２４，１０６ｋＰａ（１８，０００ｐｓｉ）で５パスで高圧ホモジナイザ（Ａｖｅｓｔｉｎ、オタワ、カナダ）に通した。
【０２６０】
製剤Ｂの体積で８分の１を分離し、製剤Ａに添加した。得られたインフルエンザウィルス／パーフルブロンのエマルションフィード溶液を、上述した条件下でスプレードライヤーに供給した。粉体をサイクロンで集め、篩分けスクリーンを、パーフルブロンを用いて収集ジャー内に洗浄した。パーフルブロン中のＨＡ懸濁液を、その後−６０℃で凍結させ、凍結乾燥した。易流動性の白い粉体を得た。
【０２６１】
ＸＶ．インフルエンザウィルスＡ／ＷＳＮ／３２（Ｈ１Ｎ１）プルモ・スフィアのインビトロ活性
【０２６２】
スプレードライした粒子への生存ウィルス性抗原の取込みを、以下の技術を用いて確認した：例ＸＩＶからのインフルエンザウィルスＡ／ＷＳＮ／３２（Ｈ１Ｎ１）プルモ・スフィア（ＷＳＮ−Ｐｕｌ）を、４０℃、６時間で５ｍｇ／ｍｌの濃度で滅菌ＰＢＳに溶解した。水和されたＷＳＮ−Ｐｕｌを、次いで９６−ウェルプレートにおいて３７℃、１時間で種々の希釈の非固定の、およびパラホルムアルデヒドで固体したＭ１２抗原を現出している細胞（ＡＰＣ）とともにインキュベートした。抗原標識（ｐｕｌｓｉｎｇ）の後、それらのＡＰＣを洗浄し、ＴｃＨとともに４時間インキュベートした。ホルムアルデヒド−グルタルアルデヒド固定した細胞をＸ−ｇａｌ基質でインキュベートし、および陽性の細胞をカウントした。
【０２６３】
結果を、パーセント活性化ＴｃＨとして表した（図１ｌＡ）。ショ糖精製した生存ＷＳＮウィルスの種々の濃度を、対照（図１ｌＢ）として使用した。ＷＳＮ−Ｐｕｌ処方は、約５質量％（重量％）のインフルエンザウィルスを含むと決定された。非固定のＡＰＣのもののみがウィルスを活性化することができ、抗原が分解しなかったことを示した。感染ウィルスの滴定が、ＭＤＣＫ（メダイン ダービー腎臓癌細胞）アッセイによって決定され（図１１Ｃ）、および全体のウィルスの約１％が、なお許容細胞中で感染および複製できることを示した。一緒に、これらの結果は、プルモ・スフィア粉体への比較的大きいインフルエンザウィルス抗原の成功した取込みを示す。
【０２６４】
ＸＶＩ．鼻経路を介してデリバリーされたインフルエンザウィルスＡＩＷＳＮＩ３２（Ｈ１Ｎ１）プルモスフィアへの抗体応答
【０２６５】
５ｇのウィルスおよび生存ウィルスの２×１０^３ＴＣＩＤ_５０（生存ウィルスの量に対応する全体の抗原ロードの１％）を含むインフルエンザウィルスＡ／ＷＳＮ／３２（Ｈ１Ｎ１）プルモ・スフィア（ＷＳＮ−Ｐｕｌ）処方によるＢＡＬＢ／ｃマウスの鼻内接種の後、ＷＳＮウィルスに対するウィルス特異的ＩｇＧ抗体応答の誘導を測定した。対照マウスを、２×１０^３ ＴＣＩＤ_５０生存ウィルス（全ウィルスのの０．０５ｇに対応）、またはＵＶ−死滅させたＷＳＮウィルス（５ｇ）で免疫化した。ｈＩｇＧで処理されたマウスからの血清を、ネガティブ対照として使用した。抗体応答を、以下の酵素免疫吸着測定法技術を用いて血清中で測定した：ウェルを、コーティング緩衝剤中で、ショ糖精製したＷＳＮウィルスでコートし、非哺乳類のタンパク質（ＳｅｒａＢｌｏｋ）でブロックし、血清サンプルのシリアル希釈とともにインキュベートした。サンプルを洗浄し、およびビオチンコンジュゲートのラット抗マウスｍＡｂ、続くストレプトアビジン−アルカリフォスファターゼおよびｐＮＰＰ基質によって、アッセイを行った。結果を、終点タイター逆数の幾何平均として表した。接種群につき、マウスの数は３であった。
【０２６６】
図１２に示された結果は、７日および１４日での食塩水中のＷＳＮ−Ｐｕｌまたは生存ＷＳＮウィルス（ＷＳＮ／Ｉｏ）で免疫化されたマウスにおける高タイターのＩＧ抗体の誘導を示す。対照的に、特異的ＩｇＧの小さいタイターのみが、塩水中で死滅したウィルスで免疫されたマウスにおいて検出された。
【０２６７】
ＸＶＩＩ．鼻経路を介してデリバリーされたインフルエンザウィルスＡ／ＷＳＮ／３２（Ｈ１Ｎ１）プルモ・スフィアへのＴ細胞応答
【０２６８】
Ｔ細胞応答を、上述したように免疫されたマウス（例ＸＶＩ）からのリンパ球のウィルスおよびエピトープ−特異的なサイトカイン産生に関して定義した。５ｇのウィルスおよび生存ウィルスの２×１０^３ＴＣＩＤ_５０（生存ウィルスの量に対応する全体の抗原ロードの１％）を含むインフルエンザウィルスＡ／ＷＳＮ／３２（Ｈ１Ｎ１）プルモ・スフィア（ＷＳＮ−Ｐｕｌ）処方によるＢＡＬＢ／ｃマウスの鼻内接種の後、Ｔ細胞応答の誘導を測定した。対照マウスを、２×１０^３ＴＣＩＤ_５０生存ウィルス（全ウィルスのの０．０５ｇに対応）、またはＵＶ−死滅させたＷＳＮウィルス（５ｇ）で免疫化した。検査した抗原は、ショ糖精製したＷＳＮウィルス、ＨＡ１１０−１２０のペプチド、およびＮＰ１４７−１５５ペプチドであった。未処理の食塩水の群を、対照として含せた。
【０２６９】
免疫化の１０日後に、フィコール勾配遠心によって末梢血液単核細胞（ＰＳＭＣ）を血液から単離した。応答細胞の種々の数を、完全ＲＰＭＩ−１０％ＦＣＳ中、３×１０^５ 細胞／ウェルでニトロセルロース／抗−ＩＦＮまたは抗ＩＬ−４（ＰｈａｒＭｉｎｇｅｎ）ＥＬＩＳＰＯＴプレート（ミリポア）でインキュベートした。刺激細胞（マイトマイシン処理された脾細胞、５×ｌ０^５ ／ウェル）、抗原、およびヒトｒＩＬ−２（２０ Ｕ／ｍｌ）を添加し、それらのプレートを４８時間共インキュベートした。細胞を次いでＰＢＳ−０．０５％Ｔｗｅｅｎで洗浄し、抗サイトカイン抗体（ＰｈａｒＭｉｎｇｅｎ）は一晩中インキュベートし、およびＨＲＰ−ストレプトアビジンコンジュゲート、これに続く不溶基質（ベクターラボラトリー）を用いてアッセイを行った。アッセイを水で停止させ、ウェルを風乾し、およびスポットを立体顕微鏡を用いてカウントした。
【０２７０】
結果を、バックグランド信号を差し引いた後の、ＩＦＮ−またはＩＬ−４／１０^６ＰＢＭＣ を産生する特異的な細胞の頻度として表した。バックグランドは、再現的に６／１０^６ 未満であった。ＰＢＭＣは、各群のマウスからプールした。図ｌ３Ａ、１３Ｂおよび１３Ｃにおける結果は、ＷＳＮ−ＰｕｌおよびＷＳＮウィルスによるワクチン接種が、一般に、ＩＦＮ−およびＩＬ−４を産生するＨＡ−、ＮＰ−およびＷＳＮ−特異的なＴ細胞を誘導したことを示す。対照的に、死滅したウィルスによる免疫化は、主に、ＩＬ−４を産生するＴ細胞を誘導した。更に、死滅したウィルスによる免疫化は、ＮＰ１４７−１５５のペプチドに対して特異的な、ＩＬ−４を産生するＴｃ２細胞の強化された部分個体群を誘導した。これらのデータは、生存対照および処方されたウィルス（すなわち、生存および死滅したウィルスを含む）によって誘発された細胞応答が、典型的な従来のワクチンに対応する死滅したウィルス対照によって誘発された応答より更に効果的であったことを示す。
【０２７１】
ＸＶ ＩＩＩ ．鼻経路．を介してデリバリーされたインフルエンザウィルスＡ／ＷＳＮ／３２（Ｈ１Ｎ１）プルモスフィアで免疫されたマウスの感染性攻撃に対する防御
【０２７２】
例ＸＶＩＩに記述したように免疫したマウスを、免疫化の３週間後に、鼻経路を介してデリバリーされた１．２×１０^６ のインフルエンザウィルスで攻撃した。ウィルス放散および体重変化の点での防御は、攻撃の４日後で定義した。結果を、図１４Ａおよびと１４Ｂに示す。
【０２７３】
鼻の洗浄液中のウィルスタイター測定を、生存ウィルスをＭＤＣＫアッセイで滴定することによって決定した。結果は、インフルエンザウィルスＡ／ＷＳＮ／３２（Ｈ１ＩＮ１）プルモ・スフィア（ＷＳＮ−Ｐｕｌ）または対照生存ＷＳＮウィルスで前もって免疫されたマウスにおいて、感染性ウィルスが無いことを示した（図１４Ａ）。ＵＶ死滅ＷＳＮウィルスで免疫されたマウス、または非投薬マウスは、鼻の洗浄液中でインフルエンザウィルスの顕著なタイターを示した。加えて、ＷＳＮ−ＰｕｌまたはＷＳＮウィルス（生存ウィルスの低用量）で免疫されたマウスは、攻撃の後にそれらの体重を保持した（図１４Ｂ）。非免疫化マウス、およびＵＶ死滅ＷＳＮウィルスで免疫されたマウスは、体重の顕著な低減、および続く死亡を示した（７日で、各群での２／３）。これらの結果は、ＷＳＮ−Ｐｕｌが粘膜のデリバリーの際に効果的なワクチン接種効率を与えることを証明した。
【０２７４】
ＸＩＸ．スプレードライによるＴＡ７ レトロウィルスの中空多孔質粒子の製造
【０２７５】
吸引：１００％、入口温度：８５℃、出口温度：６１℃；フィードポンプ：１０％：Ｎ_２ 流：８００Ｌ／ｈｒの条件下で、Ｂ−１９１のミニスプレードライヤー（Ｂｕｅｃｈｉ、Ｆｌａｗｉｌ、スイス）を用いるスプレードライ技術によって、ＴＡ７レトロウィルス粒子を製造した。そのフィードは、スプレードライの直前に２つの製剤（ＡおよびＢ）を混合することによって製造した。
【０２７６】
製剤Ａ：ｌｍｇのＴＡ７レトロウィルスを溶解するために、２ｇの脱イオン水を用いた。
【０２７７】
製剤Ｂ：リン脂質によって安定化された水中フルオロカーボン−エマルションを、以下のようにして製造した。そのリン脂質（０．３ｇのＥＰＣ−１００−３（リポイドＫＧ、Ｌｕｄｗｉｇｓｈａｆｅｎ、ドイツ）を、１６．５ｇの熱い脱イオン水（Ｔ＝５０〜６０℃）中でウルトラ−トゥラックスミクサー（Ｔ−２５型）を８０００ｒｐｍで２〜５分間（Ｔ＝６０〜７０℃）用いて均質にした。８．０ｇのパーフルブロン（アトケム、パリ、フランス）を、混合の間、滴下で加えた。フルオロカーボンを加えた後、エマルションを少なくとも４分間混合した。得られた粗製エマルションを、次いで、１２４，１０６ｋＰａ（１８，０００ｐｓｉ）で５パスで高圧ホモジナイザ（Ａｖｅｓｔｉｎ、オタワ、カナダ）に通した。
【０２７８】
製剤Ｂの８分の１（体積で）を分離し、製剤Ａに添加した。得られたＴＡ７レトロウィルス／パーフルブロンのエマルションフィード溶液を、上述した条件下でスプレードライヤーに供給した。粉体をサイクロンで集め、篩分けスクリーンを、パーフルブロンを用いて収集ジャー内に洗浄した。パーフルブロン中のＴＡ７レトロウィルス懸濁液を、その後−６０℃で凍結させ、凍結乾燥した。易流動性の白い粉体を得た。
【０２７９】
例ＸＩＸ．ＴＡ７レトロウィルスのスプレードライ粒子のインビトロ活性
例ＸＩＸで製造したスプレードライ粒子に取込みした後、ＴＡ７レトロウィルスの活性を検査した。スプレードライしたＴＡ７レトロウィルス粒子を食塩水に溶解し、ヒーラ（Ｈｅｌａ）細胞に１時間で加えた。接種の２４時間後、次いで、β−ｇａｌを用いてそれらの細胞をトランスジェニック発現について分析した。ニートおよびスプレードライしたＴＡ７レトロウィルス粒子の間で、差違は観察されなかった。これらの結果は、比較的に大きく且つ複雑なＴＡ７レトロウィルスが、見かけ上の活性のロスなしにスプレードライ粒子に効果的に取り込み可能であることを示す。
【０２８０】
ＸＸＩ．スプレードライによるウシ・ガンマグロブリンの中空の多孔質粒子の 製造
吸引：１００％、入口温度：８５℃、出口温度：６１℃；フィードポンプ：１０％：Ｎ_２ 流：８００Ｌ／ｈｒの条件下で、Ｂ−１９１のミニスプレードライヤー（Ｂｕｅｃｈｉ、Ｆｌａｗｉｌ、スイス）を用いるスプレードライ技術によって、ＴＡ７レトロウィルス粒子を製造した。そのフィードは、スプレードライの直前に２つの製剤（ＡおよびＢ）を混合することによって製造した。
【０２８１】
製剤Ａ：０．６ｇのＢＧＧ（Ｃａｌｂｉｏｃｈｅｍ、サンディエゴ、カリフォルニア州）、０．４２ｇのラクトース（シグマケミカル、セントルイス、ミズーリ州）、および２５ｍｇのプルロニックＦ−６８、ＮＦグレード（ＢＡＳＦ、Ｐａｒｓｉｐｐａｎｙ、ニューヨ−ク州）を溶解するために、０．２％の食塩溶液の２１ｇを使用した。
【０２８２】
製剤Ｂ：リン脂質によって安定化された水中フルオロカーボン−エマルションを、以下のようにして製造した。そのリン脂質（０．３ｇのＥＰＣ−１００−３（リポイドＫＧ、Ｌｕｄｗｉｇｓｈａｆｅｎ、ドイツ）を、３３ｇの熱い脱イオン水（Ｔ＝５０〜６０℃）中でウルトラ−トゥラックスミクサー（Ｔ−２５型）を８０００ｒｐｍで２〜５分間（Ｔ＝６０〜７０℃）用いて均質にした。３５ｇのＦ−デカリン（エアープロダクツ、Ａｌｌｅｎｔｏｗｎ、ペンシルベニア州）を、混合の間、滴下で加えた。フルオロカーボンを加えた後、エマルションを少なくとも４分間混合した。得られた粗製エマルションを、次いで、１２４，１０６ｋＰａ（１８，０００ｐｓｉ）で５パスで高圧ホモジナイザ（Ａｖｅｓｔｉｎ、オタワ、カナダ）に通した。
【０２８３】
製剤ＡおよびＢを組み合わせ、上述した条件下でスプレードライヤーに供給した。易流動性の白粉体を、サイクロン分離器で集めた。中空多孔質粒子は、飛行時間型の分析法（Ａｅｒｏｓｉｚｅｒ、Ａｍｈｅｒｓｔ Ｐｒｏｃｅｓｓ Ｉｎｓｔｒｕｍｅｎｔｓ、Ａｍｈｅｒｓｔ、マサチューセッツ州）によって決定された１．２７±１．４２μｍの体積−重みつけ平均空気力学的直径を有していた。
【０２８４】
ＸＸＩＩ ウシ・ガンマグロブリンＭＤＩ処方に対するアンデルセンカスケー ドインパクター結果
例ＸＸＩに従って製造した、ＢＧＧの多孔質粒子で処方された用量計量型吸入器（ＭＤＩ）の吸入性を、アンデルセンカスケードインパクターを用いて評価した。８３ｍｇの中空多孔質ＢＧＧ粒子を１０ｍｌのアルミニウム缶内に秤量し、４０℃、３〜４時間の窒素流れ下の減圧オーブンで乾燥した。その缶をＤＦ３１／５０ａｃｔ５０ ｌバルブ（米国バロア、グリニッジ、コネチカット州）を用いて圧着シールし、ステムによる超過圧により９．６４ｇのＨＦＡ−ｌ３４ａ（デュポン、ウィルミントン、デラウェア州）推進剤で充填した。
【０２８５】
装置の作動により、６１％の微粒子分率と６８μｇの微粉用量が観察された（図１５）。本例は、ＢＧＧ等の比較的大きい生物活性剤が処方でき、且つＭＤＩから効果的にデリバリーできることを説明している。
【０２８６】
当業者は、本発明がその精神または中心的な属性から逸脱することなく他の特定の形で具体化できると更に認識するであろう。本発明の前述した記述がその例示的な態様のみを開示するという点で、他の変形も本発明の範囲内にあると考えられることが理解されるべきである。したがって、本発明は、ここで詳述した特定の態様に、限られない。むしろ、本発明の範囲および内容の指標として、添付の請求項が参照されるべきである。
【図面の簡単な説明】
【図１】
図１は、本発明に従うミクロ構造における処方に従う機能性ＨＡペプチド（インフルエンザウィルスの赤血球凝集素の残基１１０〜１２０）のレベルの図示表現である。
【図２】
図２は、ミクロ構造中に処方された抗原が、Ｔ細胞を活性化するために細胞内プロセシングを必要としないという事実を説明する。
【図３】
図３は、鼻投与されたミクロ微粒子および静脈内注射を用いてデリバリーされたＨＡペプチドの血漿濃度を比較する。
【図４】
図４は、本出願で記述されたようなミクロ微粒子内で処方されたヒトＩｇＧに対するキャリブレーション曲線を選ばれた対照とともに表す。
【図５Ａ】
図５Ａは、ＩｇＧ処方されたミクロ微粒子とＨＡペプチド処方されたミクロ微粒子のための放出動態を図で説明する。
【図５Ｂ】
図５Ｂは、ＩｇＧ処方されたミクロ微粒子とＨＡペプチド処方されたミクロ微粒子のための放出動態を図で説明する。
【図６Ａ】
図６Ａは、処方されたミクロ微粒子を用いて気管内および鼻投与に従う血漿中のＩｇＧの持続性に示す。
【図６Ｂ】
図６Ｂは、処方されたミクロ微粒子を用いて気管内および鼻投与に従う血漿中のＩｇＧの持続性に示す。
【図７Ａ】
図７Ａは、本発明に従って処方されたミクロ微粒子として気管内投与されたＩｇＧに対する全身および局所的な抗体応答を示す。
【図７Ｂ】
図７Ｂは、本発明に従って処方されたミクロ微粒子として気管内投与されたＩｇＧに対する全身および局所的な抗体応答を示す。
【図８】
図８は、ＩｇＧミクロ微粒子のマウスへの気管内部投与の後のＴ細胞応答を示すサイトカインのレベルを図で説明する。
【図９】
図９は、鼻腔内に投与されたＩｇＧミクロ微粒子に対するネズミの抗体応答を示す。
【図１０Ａ】
図１０Ａは、ＩｇＧミクロ微粒子の腹腔内注射のそれぞれ７日および１４日後の、ネズミの抗体価を示す。
【図１０Ｂ】
図１０Ｂは、ＩｇＧミクロ微粒子の腹腔内注射のそれぞれ７日および１４日後の、ネズミの抗体価を示す。
【図１１Ａ】
図１１Ａは、ミクロ微粒子処方のウィルス、ウィルス性対照に対するＴ細胞応答、および処方および未処方ウィルス両方の感染タイターをそれぞれ説明する。
【図１１Ｂ】
図１１Ｂは、ミクロ微粒子処方のウィルス、ウィルス性対照に対するＴ細胞応答、および処方および未処方ウィルス両方の感染タイターをそれぞれ説明する。
【図１１Ｃ】
図１１Ｃは、ミクロ微粒子処方のウィルス、ウィルス性対照に対するＴ細胞応答、および処方および未処方ウィルス両方の感染タイターをそれぞれ説明する。
【図１２】
図１２は、鼻腔内投与後の７日および１４日後で、ミクロ微粒子処方された生存および死滅したインフルエンザウィルスに対するネズミの抗体応答を示す。
【図１３Ａ】
図１３Ａは、対照抗原とともに、ウィルス性ミクロ微粒子または生存ウィルスまたは死滅したウィルス鼻腔内接種後のＴ細胞応答を示す因子のネズミのレベルを示す。
【図１３Ｂ】
図１３Ｂは、対照抗原とともに、ウィルス性ミクロ微粒子または生存ウィルスまたは死滅したウィルス鼻腔内接種後のＴ細胞応答を示す因子のネズミのレベルを示す。
【図１３Ｃ】
図１３Ｃは、対照抗原とともに、ウィルス性ミクロ微粒子または生存ウィルスまたは死滅したウィルス鼻腔内接種後のＴ細胞応答を示す因子のネズミのレベルを示す。
【図１４Ａ】
図１４Ａは、生存および死滅したウィルスの両方を含むミクロ微粒子を、鼻腔内に接種されたマウスにおける、ウィルス性放散（ｓｈｅｄｄｉｎｇ）および体重変化をそれぞれ説明する。
【図１４Ｂ】
図１４Ｂは、生存および死滅したウィルスの両方を含むミクロ微粒子を、鼻腔内に接種されたマウスにおける、ウィルス性放散（ｓｈｅｄｄｉｎｇ）および体重変化をそれぞれ説明する。
【図１５】
図１５は、用量計量型吸入器からウシのガンマグロブリンを含む処方されたミクロスフェアの効率的なデリバリーを示すインビトロのアンデルセンカスケードインパクター研究の結果を示す。[Document name] statement
Patent application title: Particulate delivery system and method of use
[Claim of claim]
1. Use of an immunoactive agent in the manufacture of a medicament for modulation of the immune system of a subject, wherein the medicament comprises a plurality of microparticles associated with one or more immunoactive agents. .
2. The use according to claim 1, wherein the microparticles comprise perforated microstructures.
3. The use according to claim 1 or 2 wherein said microparticles comprise a surfactant.
4. The surfactant according to claim 1, which is selected from the group consisting of phospholipids, nonionic detergents, nonionic block copolymers, ionic surfactants, biocompatible fluorinated surfactants, and combinations thereof. Use of claim 3 to be selected.
5. The use according to claims 3 or 4 wherein said surfactant is a phospholipid.
6. The phospholipids selected from the group consisting of dilauroylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine, dibehenoylphosphatidylcholine, diarachidoylphosphatidylcholine, and combinations thereof. Use of.
7. The use according to any of the preceding claims, wherein the microparticles are dispersed in a non-aqueous suspension medium.
8. The non-aqueous suspension medium comprises a compound selected from the group consisting of hydrofluoroalkanes, fluorocarbons, perfluorocarbons, fluorocarbons / hydrocarbon diblocks, hydrocarbons, alcohols, ethers or combinations thereof. 7 use.
9. The use according to claims 7 or 8, wherein said non-aqueous suspension medium comprises a compound selected from the group consisting of liquid fluorochemicals and hydrofluoroalkane propellants.
10. The use according to any one of the preceding claims, wherein the mean aerodynamic diameter of the microparticles is between 0.5 and 5 μm.
11. The use according to any of the preceding claims, wherein the mean geometric diameter of the microparticles is less than about 5 μm.
12. The immunoactive agent is selected from the group consisting of peptides, polypeptides, proteins, carbohydrates, genetic material including DNA, RNA and antisense constructs, and microorganisms including viruses, phages and bacteria. Use of any of items 1-11.
13. The use according to any of claims 1 to 12, wherein said immunoactive agent comprises a vaccine.
14. The subject's immune system modulation is to induce an immune response to a foreign antigen or pathogenic particle, to induce localized or systemic passive immunity, to stimulate an immune response, or to downregulate an immune response. 14. The use of any of claims 1 to 13 comprising any of the following.
15. The drug is delivered using topical, intramuscular, intradermal, transdermal, intraperitoneal, nasal, pulmonary, vaginal, rectal, aural, oral, or ocular administration. Use of either.
16. Use of an immunoactive agent in the manufacture of a vaccine to be inhaled to induce an immune response in a subject, whereby the vaccine comprises a plurality of microparticles with one or more immunoactive agents. And that the vaccine is to be administered to the subject's respiratory tract.
17. The use according to claim 16, wherein the microparticles comprise perforated microstructures.
18. The use according to claim 16 or 17 wherein said microparticles comprise a surfactant.
19. The immunologically active agent is selected from the group consisting of peptides, polypeptides, proteins, carbohydrates, DNA, RNA, and genetic materials including structures of antisense, viruses, phages and microorganisms including bacteria. The use of any of paragraphs 16-18.
20. The use according to any of claims 16 to 19 wherein the vaccine is administered using a dry powder inhaler.
21. The use according to any of claims 16 to 19 wherein the microparticles are dispersed in a non-aqueous suspension medium.
22. The use according to claim 21, wherein said non-aqueous suspension medium comprises a compound selected from the group consisting of liquid fluorochemicals and hydrofluoroalkane propellants.
23. The use according to claim 21 or 22 wherein the vaccine is administered using a metered dose inhaler, a nebulizer, an atomizer, a nasal pump, a spray bottle, or direct instillation of droplets.
24. The use according to any of claims 16-23, wherein the mean aerodynamic diameter of the microparticles is between 0.5 and 5 μm.
25. The use according to any of claims 16 to 24, wherein the mean geometric diameter of the microparticles is less than about 5 μm.
26. The use according to any of claims 16 to 25 wherein said induced immune response comprises mucosal immunity.
27. A system for administering a bioactive agent to a subject;
A dosing device comprising a reservoir;
A system comprising: powder in the reservoir; the powder comprising a plurality of microparticles with one or more bioactive agents, whereby the powder provides modulation of a subject's immune system upon administration.
28. The system of claim 27, wherein the dosing device comprises a dry powder inhaler or a powder injector.
29. The system of claim 27, wherein the microparticles are dispersed in a non-aqueous suspending medium.
30. The system of claim 29, wherein said non-aqueous suspension medium comprises a compound selected from the group consisting of liquid fluorochemicals and hydrofluoroalkane propellants.
31. The system of claim 29, wherein the dosing device comprises a dose metered inhaler, an atomizer, a spray bottle, an atomizer, a nasal pump, a dropper, or a needleless injector.
32. The system of any of claims 27-31, wherein the particulates comprise perforated microstructures.
33. The system of any of claims 27-32, wherein the microparticles comprise a surfactant.
34. The surfactant according to claim 1, which is selected from the group consisting of phospholipids, non-ionic detergents, non-ionic block copolymers, ionic surfactants, biocompatible fluorinated surfactants, and combinations thereof. 34. The system of claim 33 which is selected.
35. The system of claims 33 or 34, wherein said surfactant is a phospholipid.
36. The system of any of claims 27-35, wherein the microparticles have an average geometric diameter of less than about 5 μm.
37. The system of any of claims 27-36, wherein the bioactive agent comprises an immunostimulatory agent.
38. The immunologically active agent is selected from the group consisting of peptides, polypeptides, proteins, carbohydrates, DNA, RNA, and genetic materials including structures of antisense, viruses, phages and microorganisms including bacteria. Item 37 system.
39. The system of claims 37 or 38, wherein said immunoactive agent comprises a vaccine.
40. A composition for modulating the immune response of a subject in need thereof, the composition comprising a plurality of perforated microstructures with one or more immunoactive agents; said Hole microstructure about 0.5 g / cm³ A composition having a bulk density of less than.
41. The composition of claim 40, wherein said perforated microstructure comprises a surfactant.
42. The surfactant according to any one of the group consisting of phospholipids, non-ionic detergents, non-ionic block copolymers, ionic surfactants, biocompatible fluorinated surfactants, and combinations thereof. 42. The composition of claim 41 which is selected.
43. The composition of claims 41 or 42, wherein the surfactant is a phospholipid.
44. The composition of any of claims 40-43, wherein the microparticles have an average geometric diameter of less than about 5 μm.
45. The composition according to any of claims 40 to 44, wherein the mean aerodynamic diameter of the perforated microstructure is between 0.5 and 5 μm.
46. The composition of any of claims 40-45, wherein the perforated microstructure comprises hollow porous microspheres.
47. The immunologically active agent is selected from the group consisting of peptides, polypeptides, proteins, carbohydrates, DNA, RNA, and genetic materials including structures of antisense, viruses, phages and microorganisms including bacteria. The composition of any one of Items 40 to 46.
48. The composition of any of claims 40-47, wherein the perforated microstructure is dispersed in a non-aqueous suspension medium.
49. The use of a bioactive agent in the manufacture of a medicament for providing an enhanced immune response in a subject, whereby the medicament comprises a plurality of microparticles with one or more immunostimulatory agents. Use wherein the drug elicits an enhanced immune response to a comparable immunoactive agent in an aqueous carrier.
50. The bioactive agent is selected from the group consisting of peptides, polypeptides, proteins, carbohydrates, genetic material including DNA, RNA and antisense constructs, and microorganisms including viruses, phages and bacteria. Use of paragraph 49.
Detailed Description of the Invention
[0001]
Field of the Invention:
[0002]
Kind Code: A1 Abstract: The present invention relates generally to compositions and methods for the administration of microparticles comprising at least one bioactive agent (which in its selected aspect may comprise an immunoactive agent). About. In this regard, the present invention provides for local and systemic delivery of bioactive agents using, for example, airway, gastrointestinal or urogenital routes. In a particularly preferred embodiment, the disclosed composition is used for targeted delivery to the surface of a mucosal membrane, with an inhalation device such as a metered dose inhaler, a dry powder inhaler, an atomizer or a nebulizer. I will.
Background of the invention
[0003]
Vertebrates have the ability to mount an immune response as a defense against environmental pathogens as well as against aberrant cells such as internally expressed tumor cells. This is a form of innate or passive immunity mediated by cells of the neutrophil and monocyte / macrophage lineage, or lymphocyte mediated acquisition or active to specific antigenic sequences. It can take the form of immunity. Active immune responses require the production of specific antibodies that serve themselves to neutralize two arms, antigens exposed to the systemic circulation, and to facilitate uptake by specialized phagocytes. Can be further subdivided into the humoral responses and cellular branches necessary for infected or abnormal cell recognition in the body.
[0004]
In both cases, specific responses are induced by intracellular processes of the antigen. When the antigen is processed through the cellular pathway, the resulting peptide is bound to the initial MHC class I molecule, which facilitates proper presentation to effector T cells. MHC class I development is advantageous for the recognition of cytotoxic lymphocytes. In contrast, intracellular processes via the intracellular uptake pathway result in the emergence of MHC class II molecules that favor T helper responses involved in humoral branch stimulation. The goal of vaccination is to activate both responses and generate memory T cells so that the immune system is primed to respond to pathogenic infections. Such responses are promoted by co-administration of signals that promote costimulatory molecule expression, so called adjuvants. Engagement of both humoral and cellular immune responses results in broad-based immunity and is a preferred goal for intracellular pathogens. Lack of proper costimulatory molecule expression can lead to T cell unresponsiveness.
[0005]
In this regard, modulation of the immune response induces an immune response directed against the two directions, the virulence factor or its antigen, or an inappropriate response that is mounted against the autoepitope leading to chronic inflammation You can take any one direction, to suppress. Chronic responses to such self-epitopes lead to various autoimmune diseases such as diabetes, typically type I, multiple sclerosis, rheumatoid arthritis or lupus erythrematosis. In any case, the active agent often takes the form of other macromolecular structures rather than relatively complex peptides, proteins, RNA, or DNA based factors, or small chemical factors that represent classical drugs. . When administered orally, these complex bioactive agents generally exhibit poor bioavailability and are, therefore, traditionally administered by invasive parenteral injection. Recently, however, it has been suggested that relatively large biomolecules can be delivered by mucosal routes such as, for example, inhalation. Delivery of these agents to the systemic circulation via inhalation is particularly attractive, as administration via the respiratory mucosa bypasses digestive tract enzymes in the GI tract. Furthermore, the large surface area available for exchange with the systemic circulation gives it an increased potential for peptides and proteins for bioavailability. Molecular weight cutoffs for oral bioavailability are generally in the range of 500 daltons, but higher molecular weight peptide hormones or analogs (eg 1.8 kD desmopressin, 5.8 kD insulin, 9.5 kD Parathyroid hormone has been shown to be absorbed across the intact mucous membranes of the nose or lung into the systemic circulation.
[0006]
Besides taking into account the effective delivery of proteins, peptides, viral and DNA formulations without degradation, the targeted delivery to the surface of the mucosa is that which is within the scope of the MALT (mucosal cooperative lymphoid tissue) lymphatic system. If it induces a local immune response, it can provide benefits on its own. Mucosal vaccination is particularly important for vaccines designed against pathogens whose port of entry is typically on one of the mucosal surfaces connecting the body and the external environment. The MALT lymphatic system is within the lamina propria of the mucosa. When external antigens are revealed to local dendritic cells, there is local amplification and maturation of B cell precursors, which are typically lgG antibodies induced by systemic delivery of the antigen. In addition to producing IgA and IgM antibodies. The former is secreted through differentiated transport receptors by a process known as transcytosis into the lumen across the mucosal surface. So they provide the first line of defense against pathogens that invade the surface of the mucous membrane. Recent evidence indicates that, in addition to binding pathogenic antigens, the resulting formation of immune complexes suppresses the viral transmission that is occurring through the transcytotic pathway in and of itself. Show. By providing this first line of immune response against antigens derived from pathogens, mucosal immunization should thereby greatly enhance the efficiency with which organisms first block pathogens that they invade.
[0007]
Several previous attempts have been made to provide effective delivery of peptides or proteins utilizing this uptake mechanism. For example, US Pat. No. 5,756,104 describes the use of liposome formulations for intranasal vaccine formulation. These formulations appear to contain an aqueous carrier with liposomes and free antigenic material. Although their formulations have been found to induce an immune response, they appear to be extremely unstable and susceptible to degradation in time. In a practical sense this is a substantial disadvantage.
[0008]
Attempts to overcome such limitations and further increase delivery efficiency have developed dry powders for the administration of relatively large biomolecules. Unfortunately, conventional powder formulations (ie micronized) often do not provide accurate, reproducible dosing over long periods of time. To some extent this is because powders tend to agglomerate due to hydrophobic or electrostatic interactions between the fines. Such aggregation can be partially overcome by the use of larger carrier particles (ie, lactose) to inhibit aggregation. However, these larger particles and bound drugs often can not reach the target cells, resulting in an uneven delivery profile. In addition, crude mixtures containing carrier molecules provide little, if any, protection for incorporated biomolecules. Thus, like the aqueous compositions described above, such formulations are susceptible to degradation and loss of activity over time.
[0009]
More recently, improved formulation methods have begun to overcome the limitations associated with conventional prior art powders and aqueous formulations. In this regard, U.S. Patent Application Serial Nos. 09 / 218,209 and 09 / 219,736, which are incorporated herein by reference, are methods for producing a formulation comprising a bioactive agent in microparticulate form and Describes the process. The resulting powder, which desirably exhibits a hollow, porous form, is suitable for inhalation devices such as dry powder inhalers (DPIs) or non-aqueous liquids (ie hydrofluoroalkanes) Or suspended in a fluorocarbon), suitable for metered dose inhalers (MDIs) and nebulizers, and the mild conditions used during the formulation process support retention of biological activity Thus, they make them particularly compatible for use with proteins and peptides, as well as more complex macromolecular structures such as viruses. In addition, the resulting powder has a very low residual water content (in short chain fluorocarbons or fluorochemicals such as propellants or in longer chain fluorochemicals such as perfluorooctyl bromide (PFOB) These formulations provide a stable means for the storage of unstable bioactive agents, since they can be further maintained by
[0010]
Besides the enhanced stability, the preferred hollow, porous form of the microparticulates provides aerodynamic properties that are particularly compatible with inhalation therapy. Furthermore, the particulate nature gives the formation of exceptionally stable dispersions and is particularly compatible with hydrofluoroalkane propellants such as HFA-134a as well as others such as PFOB as well as fluorocarbon liquid vehicles To have Thus, whether used in dry form or in non-aqueous dispersions, the microparticulates have good dose repeatability, and excellent plume properties (one measure of propellant or dry powder spray), and respiration The fraction delivered is the dose delivered (a high percentage uniformity criterion for deposition on the device or throat). These properties suggest that the disclosed microparticles provide substantial theoretical advantages deep into the lungs. Such deep deposition is preferred as delivery of large macromolecules such as proteins and peptides is optimal at the alveoli level when delivery to the systemic circulation is desired.
[0011]
Although the use of such microparticulate formulations is a substantial improvement over conventional prior art delivery methods, there is a need to provide targeted delivery of bioactive agents, immunomodulators, or immunoactive agents that produce an enhanced physiological response. Still remains.
[0012]
Accordingly, it is an object of the present invention to provide compositions, systems and methods that provide for the generation of an enhanced immune response.
[0013]
Another object is to provide an effective delivery of immunostimulatory agents, including vaccines and immunomodulators, to the mucosal surface of a patient in need thereof.
[0014]
Yet another object of the present invention is to provide vaccines and other bioactive formulations that do not require refrigeration or freezing to maintain activity.
[0015]
Yet another object of the present invention is to provide establishment of passive and active immunity via inhalation therapy.
[0016]
Yet another object of the present invention is to provide a stable preparation of an immunoactive agent that can be used to down modulate the patient's immune system when conferring or requiring immunity.
Summary of the invention
[0017]
Formulations These and other objects are provided by the claimed invention disclosed herein. To that end, the methods and related compositions of the present invention provide, in a broad aspect, improved delivery of the active agent to selected target sites in powder or particulate form. More particularly, it is surprising that the disclosed methods and compositions can be used to enhance or enhance the activity of incorporated bioactive agents, preferably including immunoactive agents, following administration. It was found especially. In this regard, the vaccine of the present invention exhibits an "adjuvant effect" which can induce an enhanced immune response of order or greater than that induced by the corresponding prior art vaccine formulations. looks like. In addition to the unexpected improvement in this effectiveness, relatively mild formulation techniques can be combined with compositions and particulate forms that protect and enhance the activity of any incorporated agent. This protects the activity of but enhances the composition, giving the formation of a relatively effective formulation that retains their biological activity without the need for refrigeration or freezing. Unlike prior art powders or dispersions for drug delivery, the present invention produces improved flow and dispersibility, preferably using new techniques to reduce attraction between particles. When these powders are incorporated into non-aqueous system suspension media (e.g., liquid fluorochemicals), these same properties may further reduce the rate of agent degradation and reduce flocculation ( Give flocculation), settling or creaming. Finally, administration of the disclosed microparticles or dispersion to selected target sites such as mucosal surfaces can serve to further optimize or enhance biological activity. Thus, the dispersions or powders according to the invention can be used together with dose metered inhalers, dry powder inhalers, atomizers, aerosolizers, nasal pumps, spray bottles, nebulizers or liquid dose instillation (LDI) techniques for liquids. Active agents can be used to deliver effectively.
[0018]
A feature of the disclosed particulate formulation technology that is particularly useful is that a wide range of bioactive structures can be incorporated into the stabilized dispersion or powder regardless of their hydrophobicity or hydrophilicity. is there. In a preferred embodiment, the bioactive powder is produced using a relatively mild spray drying process. Because of such compatible particulate formulation techniques, larger, more unstable biomolecules, such as peptides, proteins or genetic materials, have disclosed compositions without adverse effects or excessive loss of activity. It can be easily incorporated into things. These same formulation techniques and resulting microparticles further provide for relatively high dose (about 10 mg) of bioactive agent uptake and delivery using conventional dosing techniques and systems. Thus, whether administered as a dry powder or a stabilized dispersion, the new particle fabrication technology and enhanced response provided by the disclosed formulation results in the effective delivery of the bioactive agent to the target site, such as a mucosa. Bring
[0019]
In the context of the present invention, the term "bioactive agent" refers to any active peptide and protein such as hormones, cytokines or chemokines, or immunoactive agents. That is, while the disclosed compositions and methods are compatible with almost any bioactive agent, such as immunizing, eg, inducing an immune response to a foreign antigen or pathogen or down-regulating an active immune response. They have been found to be surprisingly effective for the delivery or administration of immunostimulatory agents designed to modulate the response. Thus, as used herein, the terms "immunostimulatory agent" or "immunologically active agent" can be used to induce a physiological or immunological response, or modulate an existing response in a subject Including any molecule that can. Such immunologically active agents or biological preparations include peptides, polypeptides, proteins, carbohydrates, genetic material including DNA, RNA and antisense constructs, and microorganisms including viruses, phages and bacteria. Can.
[0020]
In addition, molecules that can function as cofactors, enhancers or permeation enhancers can be readily coformulated with the microparticles described herein. One skilled in the art will appreciate that any compound that acts to improve uptake, development or bioavailability may also function as an enhancer or permeation enhancer according to the teachings herein. For example, compounds capable of altering or increasing the membrane permeability of cells can function as enhancers or permeation enhancers. Exemplary enhancers or permeation enhancers include chelating agents (eg, EDTA, citric acid), detergents or surfactants (eg, 9-lauryl ether), fatty acids (eg, oleic acid), and bile salts (eg, sodium) (Glycocholate). Particularly preferred permeation enhancers include relatively short chain phospholipids having a chain length of less than about 10 carbons. With bioactive agents, and as discussed in more detail below, selected enhancers or permeation enhancers can be incorporated in or associated with the microparticles at various concentrations.
[0021]
With regard to the microparticles, microparticulates, or perforated microstructures of the present invention, one of ordinary skill in the art would be able to form them with any biocompatible material that they are giving the desired physical properties or morphology. I will admit that I can. In this regard, the perforated microstructures preferably include pores, voids or other gaps that act to reduce attraction by minimizing surface interactions and reducing shear. This form acts to reduce aggregation and improve dispersibility. Furthermore, given these limitations, it is recognized that any medical material or configuration can be used to form the microstructured matrix. For selected materials, it is preferred that the microstructure, in a preferred embodiment, incorporate at least one surfactant which acts as a permeation enhancer. Preferably, the surfactant comprises a phospholipid or other surfactant or amphiphile approved for pharmaceutical use. Likewise, it is preferred that the microstructures incorporate at least one bioactive agent or biological agent. In terms of configuration, selected aspects of the invention are spray-dried hollow microspheres with relatively thin porous walls that define large internal voids, although other void-containing or perforated structures are also contemplated. Including.
[0022]
It is anticipated that the use of hollow and / or porous perforated microstructures can substantially reduce attractive intermolecular forces, such as van der Waals forces governing prior art powder formulations and dispersions. It was found without. In this regard, the powdery composition typically has a relatively low bulk density, which contributes to the flowability of the formulation while providing the desired properties for inhalation therapy. More specifically, the use of relatively low density perforated (or porous) microstructures or microparticulates significantly reduces the interparticle attraction and thereby achieves the resulting powder flowability Reduce the shear force required for The relatively low density of the perforated microstructure provides excellent aerodynamic performance when used in inhalation therapy. In dispersion, the physical properties of these powders give the formation of a stable formulation. Furthermore, by choosing the dispersion components in accordance with the teachings herein, interparticle attraction can be further reduced to provide a formulation or formulation with enhanced stability.
[0023]
Although the preferred embodiment of the present invention comprises perforated microstructures or porous microparticles, the use of relatively non-porous or solid microparticles also to produce powders or dispersions compatible with the teachings herein. Can. That is, powders containing relatively non-porous or solid particulate dispersions are also considered to be within the scope of the present invention. In this regard, such relatively non-porous microparticles can include micronized particles, milled particles or nanocrystals. Thus, as used herein, the term "particulate" is broadly interpreted and maintained to include particles of any porosity and density, including both perforated microstructures and relatively non-porous particles.
[0024]
As mentioned previously, the disclosed powder can be dispersed in a suitable non-aqueous suspension medium to provide a stabilized dispersion comprising a selected bioactive agent. Such dispersions are particularly useful in metered dose inhalers, atomizers, nasal pumps, spray bottles, and nebulizers. Other aspects of the invention include stabilized dispersions that can be administered directly to the lungs or nasal cavity using direct instillation techniques. In any case, particularly preferred suspending media include fluorochemicals (ie, perfluorocarbons or fluorocarbons) that are liquid at room temperature, or fluorinated propellants (ie, hydrofluoroalkanes or chlorofluorocarbons). Because of their beneficial wetting properties, some fluorochemicals can impart more particle dispersion to the surface of the lung or other mucous membranes, which can improve osmotic transfer delivery. Furthermore, such suspension media tend to be anhydrous, thereby delaying the hydrolytic degradation of the incorporated bioactive agent. Finally. Fluorochemicals are generally bacteriostatic and thus reduce the potential for proteolytic degradation associated with microbial growth in compatible formulations.
[0025]
With respect to the delivery of the disclosed powder or stabilized dispersion, another aspect of the present invention is directed to an inhalation system for the administration of one or more bioactive agents or biologicals to a patient. As mentioned above, exemplary inhalation devices compatible with the present invention include atomizers, nasal pumps, nebulizers, spray bottles, dry powder inhalers, metered dose inhalers or nebulizers. In preferred embodiments, these inhalation systems deliver the bioactive agent as an aerosol to the desired physiological site (eg, the surface of the mucous membrane). For the purposes of the present application, the term "aerosolized" is maintained to mean gaseous dispersion of fine solid or liquid particles, unless otherwise defined by context constraints. That is. For example, the aerosol or aerosolized drug can be generated, for example, by a dry powder inhaler, a metered dose inhaler, an atomizer, a spray bottle or a nebulizer. Of course, as described in more detail below, the compositions of the present invention can be delivered directly (eg, by conventional injection or needleless injection) or using techniques such as liquid dose instillation it can. In a particularly preferred embodiment, the composition of the invention is contacted at the surface of the mucosa (e.g. through inhalation) to induce mucosal and systemic immunity.
[0026]
The powders or stabilized dispersions of the invention are particularly suitable for administration of bioactive agents to the surface of mucous membranes, but can be used for local or systemic administration of compounds to any location of the body Is recognized. Thus, in a preferred embodiment, (but not limited to) the gastrointestinal tract, airways, topically, intramuscularly, parenterally, intradermally, transdermally, intraperitoneally, intranasally It should be emphasized that it can be administered using a number of different routes, including vaginally, rectally, in the ear, buccally, orally, or intraocularly. In this regard, one of ordinary skill in the art will recognize that the chosen route of administration is primarily determined by the choice of bioactive agent and the desired subject response.
[0027]
Other objects, features and advantages of the present invention will be apparent to those skilled in the art from consideration of the following detailed description and exemplary preferred embodiments thereof.
Detailed Description of the Preferred Embodiments
[0028]
A. Introduction
[0029]
Although the present invention can be embodied in many different forms, the specific embodiments disclosed and illustrated therein exemplify the principles of the present invention. It should be emphasized that the invention is not limited to the particular embodiments described.
[0030]
As noted above, the present invention provides compositions, methods, and systems comprising powders or microparticulates that can be advantageously used for delivery of bioactive agents. Preferably, the bioactive agent will comprise an active peptide or protein or an immunoactive agent. In the context of the present invention, the immunoactive agent can also comprise any molecule that can be used to induce an immune response or to modulate an existing response, such as a vaccine, an immunoglobulin or a self antigen. . Those skilled in the art will appreciate that the disclosed powder is in a dry state (eg, with a DPI or gas driven powder injector) or in the form of a stabilized dispersion (eg, an atomizer, spray bottle, MDI, LDI, needleless injector, It will be appreciated that it can be advantageously used to deliver the bioactive agent in a syringe, nasal pump or nebulizer). In a particularly preferred embodiment, the powder or microparticulates exhibit a structure that exhibits, defines or contains voids, pores, defects, hollows, spaces, interstitial spaces, apertures, perforations or holes as disclosed herein. It will include perforated microstructures that include a matrix. These perforated microstructured powders have aerodynamic properties that make them particularly useful for inhalation therapy, and a form that gives a form of dispersion stabilized with a propellant or non-aqueous delivery vehicle. Show. More generally, the relatively mild conditions used during formation of the disclosed bioactive powder, and the advantages associated with compatible delivery methods, are the efficiencies of relatively fragile biological preparations Administration of
[0031]
While not wishing to be bound by any particular theory, the relatively mild methods used to form, store, and administer the disclosed compositions are generally biologically active in unstable agents. It is believed to provide effective retention of biological activity. In this regard, preferred formulations do not require refrigeration to maintain their activity. In addition, the selection of appropriate compounds for use in the disclosed powders, and delivery to selected physiological sites (e.g., the surface of the mucous membrane), facilitates the uptake of the incorporated agent or agents, and Its activity can be strengthened. In addition, the compositions and / or delivery techniques of the present invention appear to produce an unexpected "adjuvant effect" that can confer an enhanced immune response or biological activity following administration of the selected agent. . More specifically, as discussed in the examples, the present invention is comparable to that achieved by the administration of the antigen in complete Freund's adjuvant (ie with an order of magnitude or more of conventional drug formulation) It can be used to induce an immune response. Thus, the present invention relates to active peptides, proteins, genetic material or virulence to induce active local or systemic immunization or to achieve passive immunization, immunomodulation, hormonal regulation or gene therapy. Provides effective delivery of particles (either viable or inactivated).
B. Bioactive agent
[0032]
In a broad aspect, the powder or microparticulate compositions of the present invention, including dispersions incorporating such powders, preferably contain at least one bioactive agent. As used herein, the term "bioactive agent" is maintained to include any active peptide or protein or any immunostimulatory agent. Regarding the latter, a particularly preferred embodiment of the present invention will comprise an immunoactive agent designed to be able to modulate the immune response. In accordance with the teachings herein, modulation of the subject's immune response can be by inducing a response to a potential pathogenic infection or foreign antigen or stimulating an existing immune response, either locally or systemically passively. Including inducing or suppressing an autoimmune response or an allergic response. For the purposes of the present application, the term "bioactive agent" or "immunoactive agent" refers to any molecule or organism, or analogues thereof, which give a desired physiological or immunological response in a subject. It is broadly interpreted to include bodies or derivatives. The term "bioactive agent" is maintained to also include "immunoactive agents" and the like unless the context dictates otherwise. Exemplary bioactive agents that can be used with the present invention include peptides, polypeptides, proteins, fusion or chimeric proteins, immunoglobulins, DNA, genetic material including recombinant and antisense constructs in DNA, RNA, viruses Microorganisms, including phage, bacterial carbohydrates and bacteria, as well as potentiators, cofactors or smaller molecules that can function as penetration enhancers. The biologically active compositions according to the invention find use as vaccines, immunomodulators, replicons or effectors for gene therapy applications.
[0033]
It is recognized that the powder or microparticle compositions of the present invention may include only one or more bioactive agents (or agents) (ie, up to 100% weight / weight (weight / weight)) I will. However, in selected embodiments, the perforated microstructure can include much less bioactive agent, depending on its activity. Thus, for highly active materials, the microparticulate or perforated microstructure should be 0.001 wt% (although a concentration above about 0.1 wt% (wt%) (w / w) is preferred) As much as% by weight can be incorporated. Other embodiments of the present invention are: about 5 wt% (wt%), 10 wt% (wt%), 15 wt% (wt%), 20 wt% (wt%), 25 wt% (wt%), 30 Any bioactive agent or biological agent can be included that is greater than wt% (wt%) or even 40 wt% (wt%) (w / w). Even more preferably, the disclosed powder is about 50 wt% (wt%), 60 wt% (wt%), 70 wt% (wt%), 75 wt% (wt%), 80 wt% (wt) % Or even more than 90% by weight (wt%) can be included. The exact amount of bioactive agent incorporated into the powder or perforated microstructure of the present invention depends on the choice of agent, the required dose, the method of administration and the form of the agent actually used. One skilled in the art will recognize that such a determination can be made by using well known pharmacological techniques in combination with the teachings of the present invention.
[0034]
With respect to pharmaceutical formulations, any bioactive agent that can be formulated with the disclosed powder or porous microstructure for the purpose of inducing a physiological response, including an immune response, is also expressly considered within the scope of the present invention Be In accordance with the teachings herein, the selected one or more bioactive agents can be combined with any form of powder or perforated microstructure that provides the desired efficacy and is compatible with the selected manufacturing technology ( or can be incorporated into them. As used herein, the terms "bound" or "bound" refer to particles, microparticulates, structural matrices, or perforated microstructures that contain, absorb, absorb and absorb bioactive agents. Means coated with active agent or formed by bioactive agent. If appropriate, the agents can be used as salts (for example alkali metals or amine salts or acid addition salts) or esters or as solvates (hydrates). In this regard, the form of the bioactive agent can be chosen to optimize the activity and / or stability of the compound, minimize the solubility of the agent in the suspension medium, and / or minimize aggregation. .
[0035]
At least to some extent, the advantages provided by the present invention reside in the unique formulation, storage and delivery provided by the disclosed powders. In this regard, and as discussed in more detail below, the conditions under which the disclosed powder or perforated microstructure can be formed are relatively relaxed. That is, microparticles comprising a bioactive agent can be formed in accordance with the present invention such that the active compound or agent is not exposed to extreme physical or chemical conditions. This is of great importance for relatively large macromolecules or agents such as proteins, genetic material or attenuated viruses which can be easily degraded or inactivated. Furthermore, selected aspects of the invention further serve to maintain the biological activity of the incorporated agent by forming a relatively stable dispersion comprising a non-aqueous suspension medium. The dispersion of the active powder in a suspending medium, preferably a liquid fluorochemical or fluorochemical propellant, tends to be both bacteriostatic and anhydrous, whereby hydrolysis of the incorporated agent or protein Inhibit degradative collapse. It is recognized that such compositions have been found to maintain relatively high levels of activity over long storage periods. Finally, it has surprisingly been found that the disclosed powder composition and its delivery technology can enhance or enhance the activity of the bound bioactive agent. Taken together, these advantages of the present invention provide enhanced delivery and efficacy of the active agent to selected physiological sites.
[0036]
As indicated above, the compositions, methods and systems of the invention are useful for the delivery of bioactive agents such as peptides, polypeptides, bacterial carbohydrates, viruses and genetic material. In this respect, the invention disclosed is a vaccine for active immunization (e.g. mucosal and systemic vaccination), an immunoglobulin for passive immunization, an immunomodulator for the treatment of autoimmune diseases, an activity Useful for administration of peptides or proteins and effectors and expression vectors for gene therapy or vaccination. As described in more detail below, the powder containing the selected agent can be formed by a variety of different means. Preferably, the powder or microparticulates will be in the form of perforated microstructures and will include additional components to enhance the stability and / or efficacy of the incorporated bioactive agent. If desired, the powder can be formulated in a suspending medium to give a stabilized dispersion.
[0037]
Particularly preferred classes of bioactive agents are discussed in more detail immediately below.
B (i). Antigens and vaccines (for active immunization)
[0038]
Particularly preferred bioactive agents can include a vaccine, in accordance with the teachings herein. As discussed throughout the specification and the appended examples, compatible vaccines may be inactivated or killed microorganisms (eg, viruses), attenuated viable microorganisms, phages, proteins, peptides or carbohydrates (eg, bacterial) Subunit vaccines such as carbohydrates, replicons such as fusion proteins or chimeric antibodies, viral vectors, and genetic material including plasmids and recombinant molecules can be included. Regardless of what type of agent or biologic is chosen, the resulting powdered composition can be used to immunize a subject against one or more target antigens. Furthermore, the adjuvant effect or enhanced immunity associated with the disclosed invention provides a particularly effective immunization.
[0039]
As defined herein, "target antigen" refers to an antigen, typically a portion of a protein or peptide against which it is preferred to induce an immune response. Such antigens may be contained in pathogens such as viruses, bacteria, protozoa, fungi, yeast or parasitic antigens, or may be contained in cells (eg cancer cells). Tumor antigens and viral antigens are particularly preferred target antigens. In the case of a genetic vaccine, one or more target antigens will be expressed by the host following transfer or transformation of autologous cells with the genetic material administered. Conversely, in protein or peptide based vaccines, including chimeric or melt proteins or those containing killed or attenuated microorganisms, the target antigen or multiple antigens are given directly to the immune system. In any case, the appearance of target antigens using the powders or dispersions of the invention will induce the desired immune response. Interestingly, it has been found that a particularly vigorous immune response is generated by the subject when a live virus, or a combination of live and dead virus, is used as a vaccine according to the teachings herein.
[0040]
Those skilled in the art will generally recognize that an effective antiviral immune response typically includes both cell mediated responses, including Th1 / CTL cell responses and B cell mediated humoral immunity. Will admit. Generally purified proteins or killed microorganisms induce B, Th2 without CIL response, and certain formulations including subunit or killed vaccine, as well as live vaccines induce B, Th1 with CTL response it can. Preferably, the vaccine compositions of the invention will, upon administration, induce a wide range of immune responses, including B, Th1 and CTL responses. However, surviving viral infections during vaccination can lead to unacceptable side effects. Thus, the goal in a successful vaccination strategy is to guarantee both cellular and humoral branches of immunity without incurring undue side effects. As described below, the compositions of the invention can be used to induce both types of response upon administration.
[0041]
In this regard, a compatible vaccine can include any molecule, organism or compound that produces a B cell response, a T cell response, or a combination thereof against a target antigen. As such, agents that are actually revealed to the host immune system (whether directly or after transformation of the host cell) are analogs, homologs or derivatives, or targets of the naturally occurring target antigen. It can be a molecule that contains an antigen. Furthermore, the immunization can be local or systemic, essentially according to the type of target antigen developed and the form of delivery. For example, in a particularly preferred embodiment, the response of the immunogen will be primarily predominantly mucosal (eg, in the mucosal associated lymphoid tissue [MALT] lymph system). As discussed previously, when foreign antigens are revealed by local dendritic cells, there is local amplification and maturation of T cells and B cells, which are typically induced by systemic delivery of the antigen. In addition to IgG antibodies, they produce IgA and IgM antibodies. Such topical immunization has been found to be particularly effective at preventing infection by airborne pathogens such as influenza virus and respiratory syncytial virus, especially in the nasal passages and sinuses.
[0042]
More generally, the vaccine compositions of the invention can include one or more target antigens from many pathogens. For example (but not limited to) target antigens include influenza virus, t cytomegalovirus, herpes virus (including HSV-l and HSV-II), herpes zoster virus, hepatitis virus (but not limited to A) , Including B, C, or D hepatitis, varicella virus, rotavirus, papilloma virus genus, measles virus, Epstein-Barr virus, coxsackie virus, polio virus, enterovirus, adenovirus, retrovirus, but is not limited thereto (Including HIV-1 or H1V2), RS virus, rubella virus, Streptococcus (Streptococcus pneumoniae etc.), Staphylococcus (Staphylococcus aureus etc.), Hemophilus (eg Hemophilus unfluenzae, etc.) Listeria monocytogenes (for example, Listeria monocytogenes), Klepsula, gram-negative bacillus, Escherichia coli (such as E. coli), Salmonella (such as Salmonella typhimurium), Vibrio (such as cholera), Yersinia (such as Yersinia pestis or Yersinia enterocoliticus) Enterococcus, Neisseria, Neisseria meningitidis, etc., Cornebacterium (Cornebacterium diptheriae, etc.), Clostridium (Clostridial tetani, etc.), Mycoplasma (Mycoplasma tuberculosis, etc.), Candida yeast, Aspergillus, Mucor fungi, Toxoplasma, amoeba, malarial parasite, trypanosome parasite, leishmania parasite, helminth Other possible non-limiting examples of such target antigens include hemagglutinin, nucleoprotein, M protein, F protein, HBS protein, HIV gp120, HIV nef protein And listeriolysin.
[0043]
Regardless of which type of antigen is chosen as the target antigen, it contains at least one relevant epitope. As used herein, the term "related epitope" refers to an epitope contained in a target antigen accessible to the immune system. For example, relevant epitopes can be treated after permeation of the microorganism into cells, or can be recognized by antibodies at the surface of the microorganism or microbial protein. Preferably, the immune response directed to the epitope confers a beneficial effect; eg, when the target antigen is a viral protein, the immune response directed to the relevant epitope of the target antigen is at least partially viral infectivity or pathogenic. Neutralize the sex. One skilled in the art will recognize that the relevant epitope may be a B cell or T cell epitope.
[0044]
As used herein, the term "B cell epitope" refers to a peptide comprising a peptide sequence contained within a larger protein capable of directing antibody production by B cells.
[0045]
For example (but not limited to), it is known that the hypervariable region 3 loop ("V3 loop") of human immunoimmunodeficiency virus ("HIV") type 1 envelope protein is a B cell epitope ing. Other examples of B cell epitopes that can be used according to the present invention have been shown to be (but not limited to) immunodominant B cell epitopes (Li et al. (1992) J. Viol., 66: 399-404. ) An epitope related to influenza virus strains, such as site B of hemagglutinin of influenza HA 1; an epitope of the measles virus F protein (residues 404 to 414, Parlidos et al., 1992, Eur. J. Immunol. 22: 2675) -2680); an epitope of the hepatitis virus pre-S1 region from residues 132-145 (Lclerc, 1991, J. Immunol. 147: 3545-3552); and an epitope of the foot and mouth disease VP1 protein (Residue 1 41-180, Clark et al., 1987, Nature, 330381-3841) and the like. Additional B cell epitopes which can be used are known or can be identified by methods known in the art as described in Caton et al., 1982, Cell, 31: 417-427.
[0046]
In an additional aspect of the invention, the peptide can comprise a T cell epitope. As used herein, the term "T cell epitope" refers to a peptide comprising a peptide sequence within a larger protein that functionally activates T cells when recognized by T cells in association with MHC autoantigens. Say. In this regard, the present invention provides a Th epitope that can be recognized as helper T cells in the context of MHC class II autoantigens, thereby facilitating B cell antibody production through Th cells.
[0047]
For example, without limitation, PR8 strain influenza A hemagglutinin (HA) protein has a Th epitope at amino acid residues 110-120. Examples of other known T cell epitopes include (but are not limited to) two promiscuous epitopes of tetanus toxoid (Ho et al., 1990, Eur. J. Immunol. 20: 477-483); an epitope of cytochrome c (Residues 88-103); epitope of Mycobacterial heat shock protein (residues 350-369, Vordermir et al. Eur. J. Immunol. 24: 2061-2067); epitope of hen egg white lysozyme, (residue 48 61 61, Neilsonet et al., 1992, Proc. Natl. Acad. Sci. U. S. A., 89: 7380-7383); Streptococcus AM protein epitope (residues 308-319, Rossiter et al., 1994, Eur. J. . Immunol. 24: 1 44-1247); and staphylococcal nuclease protein epitope (residues 81~100, de Magistris, 1992, Dell, 68: including 1-20). Additional Th epitopes that can be used with the present invention are known or can be readily identified by methods known in the art.
[0048]
As a further example, the relevant epitope can be a CTL epitope, which can be recognized by cytotoxic T cells in the context of MHC class I autoantigens, thereby being mediated by CTL of cells containing the target antigen Dissolution can be promoted. Non-limiting examples of such epitopes include influenza virus nucleoprotein corresponding to amino acid residues 147-161 and 365-379, respectively (Taylor et al., 1989, Immunogenetics, 26: 267); Townsend et al., 1983. , Nature 348: 674); LSMV peptide (amino acid residues 33 to 41; Zinkernagel et al., 1974, Nature 248: 701 to 702); and egg albumin peptide corresponding to amino acid residues 257 to 264 (Cerbone et al., 1983). J. Exp. Med., 163: 603-612).
[0049]
For genetic vaccines, one or more target antigens are expressed by the host after transfer or transformation in autologous cells with the administered genetic material. The one or more expressed antigens then elicit a desired immune response in the subject. One skilled in the art will recognize that the genetic material can bind to the powder in the form of naked molecules (e.g. DNA or RNA) or in the form of a virus. In any case, a nucleic acid compatible with the present invention preferably encodes one or more relevant epitopes, optionally an element that modulates the expression and / or stability and / or immunogenicity of the relevant epitopes. Can further be included.
[0050]
For example, elements that regulate expression of the encoded epitope within the genetic construct include (but are not limited to) promoter / enhancer elements, transcription initiation site, polyadenylation site, transcription termination site, ribosome binding site, Includes translation start codon, translation stop codon, signal peptide, etc. Specific examples include (but are not limited to) the promoter and intron A sequence of the early early gene of cytomegalovirus (CMV or SV40 virus ("SV40"); Montgomery et al., 1993, DNA and Cell Biology, 12: 777-783). Alternatively, more than one epitope can be expressed in the same open reading frame. Examples of genetic vaccines which can be used according to the invention, and methods for their preparation, are incorporated herein by reference with their internal use, published by Merck & Co. and Vical in an international application WO 94/21797, Mt. No. WO 97/21687, U.S. Pat. Nos. 5,589,466 and 5,580,859 by Sinai and in WO 90/11092 by Vical.
[0051]
It may be preferable to provide the epitopes in the context of larger peptides or proteins in order to confer enhanced stability and / or immunogenicity of the relevant epitopes. For example, related epitopes can be expressed in the variable regions of chimeric antibodies or as part of fusion proteins. In other preferred embodiments, it may be advantageous to administer a full-length protein (eg, a viral coat protein) comprising one or more relevant epitopes. Alternatively, it may be preferable to administer a powder or porous microstructure comprising a combination or cocktail of immunogenic peptides or proteins. In this regard, it is recognized that the relevant epitopes can be from the same or different pathogens. With regard to the latter, opportunistic pathogens can be targeted along with primary disease causing agents. In addition to the broad target range, the disclosed compositions can include various epitope combinations. For example, a composition of the invention can comprise a nucleic acid or peptide or protein comprising a mixture of B cell epitopes, a mixture of T cell epitopes, or a combination of B and T cells.
[0052]
More specifically, administration of compositions containing or expressing more than one relevant epitope can exhibit unexpected synergistic effects. Such combination vaccines are more likely to confer the desired immunity on one or more selected pathogens as compared to a composition comprising a single nucleic acid species encoding a single related epitope. It is recognized that efficiency can be proven. Those skilled in the art will appreciate that such synergy provides an effective immunopreventive or immunotherapeutic response to be generated at lower doses and less frequent administration as compared to single epitope vaccines. You will further recognize that you have done. Furthermore, the use of such multi-epitope vaccine compositions is more resistant to induced multi-site immunity to natural phenotypic changes within the species or rapid mutation of the target antigen by the selected pathogen. Because it tends to, you can give more comprehensive protection. Of course, effective immunity can also be conferred by a vaccine encoding a single B or T cell, and such compositions are clearly considered to be within the scope of the present invention.
[0053]
In addition to the antigen itself, the present invention allows for manipulation of the excipient component of the shell itself of the particle to enhance or modify the immunogenicity of the formulated antigen. For example, it has been shown that efficient antigen capture by dendritic cells is promoted when antigen uptake is promoted by the mannose receptor, thus improving targeting to lysosomal compartments (Salusto et al., J. . Expt. Med. 182: 389-400, 1995). Thus, a low percentage uptake of mannan or other polysaccharides that bind to receptors on cells to the microparticle is expected to enhance the immunogenicity of the microparticle. Furthermore, as discussed in more detail below, the use of cofactors or cytokines to enhance the APC response, as necessary, serves to enhance or suppress the immune response. The present invention is a cofactor capable of enhancing a stimulated local immune response (e.g., transdermally) within the target site of mucosal or other delivery towards localized dendritic cells or other APOs. , Allow for co-formulation of the antigen. By facilitating APC activation within the local environment, and enhancing antigen uptake and development, such combination regimens provided herein provide enhanced efficiency of the resulting immune response. I was able to.
[0054]
More generally, when the subject is immunized or vaccinated, the methods and compositions of the present invention provide an enhanced immune response. This "adjuvant effect" provided by the disclosed microparticles can be used to elicit a comparable immune response induced by an antigen administered with an adjuvant (ie alum or complete Freund's adjuvant). Unlike the present invention, one skilled in the art will recognize that such conventional adjuvants are often unavailable for humans, often in conjunction with undesirable side effects. Conversely, the present invention is generated by comparable antigens developed using technology recognition techniques such as enhanced immune response (ie, CTL levels for antibody titers) without the administration of potentially toxic adjuvants. Can give a larger immune response). While not wishing to be bound by any particular theory, the observed enhancement is, at least in part, the configuration or morphology of the microparticles, the antigen release profile, and the result of possible antigen aggregation within the microparticles. Seems to be. In any case, the effect is to generate a clinically useful immune response with lower levels of antigen and / or fewer inoculations.
[0055]
This adjuvant effect enhances the immune response provided by the compositions and methods of the invention as compared to prior art inoculation techniques. In particular, the immune response induced by the compositions of the invention is generally greater than the immune response generated by intravenous or intraperitoneal administration of the same antigen, solubilized or suspended in an aqueous carrier. Of course, the magnitude of the immune response induced can be measured using any one of a variety of techniques known in the art, including methods of compatibility described in the examples below. Using such comparisons, the formulations of the invention preferably induce an immune response that is 25%, 50%, 75%, or 100% greater than that produced by administration of the same antigen using the prior art methods described above. Do. More preferably, the invention will induce a response that is two, three, four or five times greater than the baseline response obtained with the antigen in aqueous carrier. In more preferred embodiments, the disclosed formulations and methods will induce 10 immune responses that are 6-fold, 7-fold, 8-fold, 9-fold or 10-fold greater than the baseline response. Furthermore, other embodiments can produce responses that are 20, 30, 40, 50, or even 2 orders of magnitude greater than baseline. One skilled in the art will recognize that these new and thus far unexpected properties of the disclosed microparticles make them extremely effective at generating the desired immune response in a subject.
[0056]
In addition to the adjuvant effects described above, other mechanisms may also contribute to the enhanced immune response according to the teachings herein. For example, it has surprisingly been found that the combination of live and dead virus induces a much stronger response than that given by dead virus alone. More specifically, in a preferred embodiment, the powder can be made using viable attenuated virus that has been killed or inactivated to some extent during particle production. As shown in connection with the examples below, this mixture of surviving and dead virus appears to induce a surprisingly strong or enhanced immune response. Furthermore, in keeping with the teachings herein, the selected virus or mixture of viruses can include naturally occurring inactivated or attenuated, or be designed to express one or more foreign antigens. Can. An alternative method of formulating viable virus provided by the present invention is to formulate a viral receptor in the particle matrix and subsequently bind the selected virus to the particles after fabrication (ie after spray drying) Including. A wide variety of cellular viral receptors that are now well defined, eg, retroviral receptors, CCR5, cellular receptors for HIV, prolactin receptors capable of functioning as polio virus receptors, IgG Fc regions that bind to HSV1, And receptors that bind influenza virus.
[0057]
Regardless of the antigen or antigen form (virus, peptide, genetic material, etc.) chosen, one skilled in the art further recognizes that effective immunization of a subject can involve more than one inoculation. I will. As used herein, the terms "immunize" or "immunization" or related terms are used herein to refer to a substantially immune response (immunity of a cell such as an antibody or effector CTL) to a target antigen or epitope. To give the ability to start). These terms do not require that complete protective immunity be created, but rather refer to that a protective immune response is generated that is substantially greater than baseline. For example, a mammal can be considered to be immunized against a target antigen if cellular and / or humoral immune responses to the target antigen are enhanced following application of the methods of the present invention. Assays showing enhancement of both B cell or T cell responses are well known and could easily be performed by one skilled in the art. Preferably, the immunization results in significant resistance to the disease caused or triggered by the pathogen expressing the target antigen.
[0058]
As used herein, the term "inoculation" administers or introduces a composition comprising at least one vaccine capable of producing or expressing the relevant epitope, or which is capable of producing the relevant epitope according to the present disclosure Say that. Although an effective immune response can be elicited by a single inoculation, effective immunization of a subject can include multiple inoculations or subsequent boosters or boosters. Thus, in order to achieve the desired immunopreventive efficacy, the method of the invention may comprise one, two, three, four or even five inoculations. Furthermore, as mentioned above, it is preferred that the vaccine administered is in surface contact with the mucosa and / or absorbed thereby. In a particularly preferred embodiment, the mucosal surface is associated with the oral or nasal path or cavity, the airways of the lungs. Those skilled in the art will appreciate that the vaccine composition (i.e., powder or dispersion) of the present invention may be newborn (0 to 6 months), infant (6 months to 2 years), child (2 to 13 years) or adult (13 years). It will be further recognized that it can be used to vaccinate over a year).
[0059]
B (ii). Immunoglobulin (passive immunotherapy)
[0060]
Although the methods and compositions of the invention provide an effective means for inducing localized and systemic active immunity, they may also be used for induction of localized or systemic passive immunity. It is. In particular, the disclosed powders and microparticles can also be used to administer immunoglobulins or fragments or portions thereof to provide rapid prophylaxis or therapy for infection or disease. The administered immunoglobulin, which may be monoclonal or polyclonal, recognizes at least one antigen on the target pathogen. Preferably, the recognized antigen or antigens comprise one or more relatively conserved epitopes. For the purposes of the present invention, the composition administered can comprise neutralizing, therapeutic or prophylactic antibodies or combinations thereof. In a particularly preferred embodiment, the administered composition will comprise one or more species of monoclonal antibodies or immunologically active fragments.
[0061]
Following administration, the active immunoglobulin or immunoglobulins function at the site of delivery or are taken into systemic circulation. The antibody held at the site of administration can rapidly bind to any target infectious agent (eg airborne virus) that contacts the treated site (ie mucosal surface) and prevents subsequent infection Or remove the microbes. Alternatively, the relatively high levels of circulating antibody conferred by the preferred embodiments of the invention allow for rapid realism of target pathogens from the bloodstream, thereby preventing or at least ameliorating the symptoms associated with infection. Of course, unlike active immunity, which can last for the life of the subject, passive immunization is relatively transient and is recognized as continuing as long as the delivered immunoglobulin dose remains in circulation.
[0062]
As mentioned above, any immunoglobulin or immunologically active fragment thereof that recognizes the antigen or antigens on the target pathogen can be used to confer the desired immunity on the subject. The ability to confer monoclonal and polyclonal antibodies to particular pathogens and / or antigens and / or epitopes is well known in the art. With regard to the form of the antibody actually administered, both natural and engineered antibodies are compatible with the teachings herein, which are different classes of antibodies including IgA, IgG, IgE, IgG and IgM. Be recognized. Similarly, F (ab ')₂ , Any immunologically active fragment, including Fab or Fv, or immunoglobulin domains may be used to provide the desired protection. For the antibodies designed, humanized constructs (ie, chimeric antibodies) are particularly preferred. Such immunoglobulins typically comprise the antigen binding complementarity determining regions (CDRs) of the murine antibody, but the remainder of the molecule comprises human antibody sequences which are not recognized as foreign. See, for example, Jones et al., Nature 321: 522-525, 1986, which is incorporated herein by reference. Because human polyclonal IgGs are typically not recognized as foreign by the subject, they are rarely administered and do not have the tendency to produce undesirable side effects unless they are rapidly cleared by the body.
[0063]
Passive immunity is effective in preventing or reducing the chance of infection by easily transmitted pathogens, particularly those that are air or water borne. Thus, the powders and dispersions of the present invention containing suitable immunoglobulins are particularly effective against respiratory viruses and pathogens such as influenza or RS virus. For example, stabilized dispersions comprising laden perforated microstructures in a liquid fluorocarbon medium could be provided to the nasal passage via an atomizer or spray bottle. The composition, which can be easily administered as needed, provides local and systemic passive immunity with respect to target pathogens such as cold virus (Orthomyxovirus, Paramyxo virus, Rhino virus). Similarly, easily administered compositions can be given according to the present invention to provide protection against waterborne agents such as Vibrio cholerae. The passive immunity disclosed herein is used to provide at least some protection for various organisms including, but not limited to, rabies virus, hepatitis (A, B, C) viruses, HIV and tetanus I was able to. Other infectious agents to which passive immunity can be conferred by the disclosed compositions can be readily ascertained by one of ordinary skill in the art.
[0064]
B (iii). Tumor antigen
[0065]
In other embodiments, the target antigen can be a tumor antigen. One skilled in the art will appreciate that tumor antigens are often peptide fragments derived from cellular proteins either restricted to the type of tissue from which the tumor is induced or mutated in the course of malignant transformation. Will recognize. Other tumor antigens are often "neoplastic" antigens that are aberrantly expressed by tumor cells and / or resulting from errors in transcription, RNA processing for mutations characteristic of tumor cells. Instead, changes in post-translational modification (eg glycosylation) of normal proteins reveal previously hidden (negative) epitopes that are not normally recognized by the immune system (eg MUC1 in the case of mucins) Can help. B cell epitopes associated with tumor antigens are expressed on the surface of tumor cells and recognized by specific antibodies. In contrast, T cell epitopes are of two types: CTL epitopes which are MHC class I-restricted peptides derived from tumor associated antigens, and MHC class II-restricted peptides derived from tumor antigens It is a Th epitope. While the Th epitope is revealed by an antigen that gives mostly cells (APCs) to CD4 + T cells, CT epitopes are revealed by APC as well as tumor cells and are recognized by tumor-specific CD8 + T cells. Exemplary tumor antigens include (but are not limited to) carcinoembryonic antigen ("CEA") antigen, melanoma associated antigen, alphafetoprotein, papilloma virus genus antigen, Epstein-Barr antigen, MUC1, p53, etc. Including. Several other tumor antigens, as reported, are incorporated herein by reference in their entirety, see Boon et al. Exp. Med. 183: 725-729, 1996; Disis. M. L. Et al, Curr. Opin. Immunol. 8: 637-642 (1996): Robins, P .; F. Et al. Curr. Opin. Immunol. 8: 628-636, 1996; Salgaller et al. Surg. Oncol. , 68122-136 (1998), it is recognized by autologous cytotoxic T lymphocytes.
[0066]
B (iv) Immunomodulation
[0067]
Autoimmune diseases are mediated by autoreactive T and B cells, as well as other immune cell subtypes that can exert regulatory or effector roles. T cells that recognize organ-specific self epitopes appear to be important elements in the pathogenesis of autoimmune diseases such as diabetes type I, multiple sclerosis (MS) or rheumatoid arthritis (RA). CD4 +, which recognize antigens developed at certain locations in the body, and in certain cases, CD8 + T cells, can infiltrate tissues and cause destruction of various cell types and induction of persistent inflammation. CD4 + Th1 cells producing IL-2, IFN, TNF- and LT- are considered pathogenic but CD4 + Th2 cells producing IL-4, IL-10, IL-5, IL-13 and IL-9 It is considered nonpathogenic compared to autoimmunity and can suppress disease in certain situations. Furthermore, Th3 cells induced by mucosal exposure to antigen to secrete TGF- and IL-10 appear to be important mediators of mucosal induction resistance.
[0068]
As a strategy to prevent or suppress autoimmune disease, autoreactive T cells provide good therapeutic targets. Some means of inactivation (generally not necessarily limited to "elimination", indication of "resistance") of pathogenic autoreactive T cells responsible for autoimmune disease are: (1) high levels of Directly arrest or anogenize pathogenic cells by providing years of exposure to the antigen; (2) exposing them to the antigen in the context of non-professional APCs or certain regulatory factors Slows or switches the function of pathogenic T cells by: and (3) transitioning to the site of the disease to suppress the function of pathogenic T cells. Antigen-specific Th suppression of Ph2 / Th3 phenotype May induce cells.
[0069]
It has been surprisingly found that, in accordance with the present invention, tolerance can be induced through the use of inhalation therapy. The advantage of the airway as a target site for induction of immune tolerance is doubled: first, it is a non-invasive route to deliver complex antigens locally and systemically: and secondly and mucosally Immunity may include Th2 / Th3 suppressors to the administered antigen. Such antigens are peptides limited to whole autoantigens (recombinant or purified), antigenic fragments (obtained by molecular biology or biochemical techniques well known in the art) or epitopes. be able to. In other embodiments, they are incorporated as viral components, phage, chimeric antibodies, fusion proteins, replicons, bacteria, or delivered on a nucleic acid basis or via viral vectors. They can be incorporated with self molecules, such as immunoglobulins for receptors on somatic cells or any natural or synthetic ligand. They can be administered as isolated individual components or in mixtures. Examples for diabetes type I (not limited to these peptides and antigens): GAD 65 (glutamate decarboxylase 65, Baekkeskov et al., Nature, 1990, 347: 151), insulin (Parmer et al., Science, 1983, 222) : 1337), ICA5I₂/ IA-2 (islet cell antigen 512; Rabin et al., J. Immunol., 1994, 152: 3183). In the case of MS. Such proteins and peptides are described in MBP (Myelin Basic Protein, Steinman et al., 1995, Mol. Med. Today., 1: 79; Warren et al. (1995) Proc. Natl. Acad. Sci. USA, 92: 11061; PL_P . Transaldolase. 2 ', 3 cyclic nucleotides 3' phosphodiesterase (CNP), MOG and MAG (Steinman, L., 1995, Nature (375: 739). Besides autoimmune diseases, the compositions and methods of the invention It is recognized that it can also be used to downregulate the immune response generated by the antigen.
[0070]
B (v). Active peptides and proteins
Certain peptides and proteins are known to have the ability to modulate, upregulate, or downregulate the immune response to foreign or autoantigens. Such peptides or proteins can act by engaging the endogenous receptor leading to activation or inhibition of certain processes, or by interfering with ligand-receptor binding of endogenous elements. An example of such a protein or peptide is a cytokine that exerts an immunoregulatory function leading to the suppression of autoimmunity: an interferon, a natural, or bound to, taken up by, or other molecule, or a complex fragment of IL- 4, IL-10, IL-13, and IL-9. Other cytokines can act as immunostimulatory agents that lead to increased immunity to microorganisms or tumor cells: IL-12, IL-2, interferons, interferons, TNF-, TNF, lymphotoxin and GM. -MSF. For example, co-administration of GC-MSF, IFN-a, IL-2, IL-12 or TNF-α has been shown to enhance immune response and antigen expression. However, systemic delivery of such agents in many cases resulted in unacceptable side effects and resulted in a concerted effort directed towards targeted delivery of these pluripotent agents. The present invention advantageously enhances local immune response stimulation within the target site of mucosal or other delivery (e.g., transdermal or intradermal) directed at local dendritic cells or other APC emergence. Allow co-formulation of the selected antigen or antigens with cofactors that can be By facilitating APC activation in the local environment and enhancing antigen uptake and development, such combination formulations of the present invention were able to provide increased efficiency of the resulting immune response.
[0071]
Other active proteins or peptides which can be used according to the invention are fragments in their natural form or with other molecules capable of increasing, modulating or suppressing lymphocyte recruitment. Or contains the chemokine as a complex. For example, eotaxin-1, eotaxin-2, TARC, MIP-3b, SLC are thought to mediate the recruitment of Th2 cells, while MIG, IP-10, MIP-1, IP-1 and RANTES are It is thought to mediate the recruitment of Th1 cells (Sallusto et al. (1998) J. Exp. Med. (187: 875); Ward et al. (1998) Immunity, 9: 1). Similarly, a cytokine or chemokine receptor, in its native form or as a fragment, recombinant construct or complex with other molecules, can inhibit recruitment or activation of certain lymphocytes. it can. Examples of cytokines and chemokine receptors that may suppress ongoing Thl responses include IL-12 receptor, IFN-receptor, IL-2 receptor, TNF-receptor, CXCR3 or CCR5. Examples of cytokines and chemokine receptors that may suppress ongoing Th2 responses include IL-4 receptor, IL-13 receptor, IL-9 receptor, IL-10 receptor, CCR3, CCR⁴ Or CCR7. Of course, highly compatible compounds are not limited to cytokines, chemokines or their receptors, but also other ligands or receptors such as integrins and homing receptors (natural, fragments with other molecules, structures or complexes It is recognized that can be included in the form of the body). In preferred embodiments, categories of these compounds can be formulated and administered locally or systemically via the airway to enhance, suppress or modulate the immune response.
[0072]
It is further recognized that the perforated microstructure in accordance with the present invention can optionally include a combination of two or more active ingredients. The agents can be provided as a single species of foraminous microstructure or separately in combination of discrete species of foraminous microstructure. For example, two or more active or bioactive agents can be incorporated into a single feedstock formulation and spray dried to provide a single microstructured species comprising multiple bioactive agents. Conversely, individual agents could be added to individual stocks and spray dried separately to give multiple microstructured species with different compositions. These individual species could be added to the suspending medium or the dry powder dispensing compartment in any desired ratio and placed in the aerosol delivery system as described below.
[0073]
Based on the foregoing, it will be appreciated by those skilled in the art that a wide variety of bioactive agents can be incorporated into the disclosed powders. Thus, the above list of preferred bioactive agents is exemplary only and not intended to be limiting. It will be appreciated by one skilled in the art that the appropriate amount of bioactive agent and timing of administration can be determined for the formulation according to information already existing and without undue experimentation.
C. Powder composition
[0074]
As can be appreciated from the above discussion, the present invention can be used effectively to deliver a wide variety of bioactive agents. Although the microparticles can be formed solely by the bioactive agent, they are preferably, in selected embodiments, one or more additional materials that can include absorption enhancers, enhancers, excipients or structural components. including. More generally, the microparticles (i.e. structural matrix) can be formed from or include any material having physical and chemical properties compatible with any incorporated active agent. A wide variety of materials can be used to form the powder, but in a particularly preferred pharmaceutical embodiment, the microparticles are combined with or comprise a surfactant such as a phospholipid or fluorinated surfactant. Although not required, incorporation of compatible surfactants can improve powder flow, increase aerosol efficiency, improve dispersion stability, and facilitate formulation of dispersions. . Furthermore, selected surfactants can also function as absorption enhancers, which can increase uptake and improve the biological activity of the selected agent. Of course, it is recognized that the powders of the present invention can also be formed using nothing but traditional non-surfactant excipients, and one or more bioactive agents incorporated. Will be done.
[0075]
As indicated, the disclosed powder can optionally be associated with or contain one or more surfactants. In accordance with the teachings herein, these compounds stabilize any incorporated biological activity, facilitate stabilization of microparticles suspended in non-aqueous media, or enhance agent uptake at the target site Can be helpful. Aside from the disclosed microparticles and their associated surfactants, compatible surfactants can be optionally combined if the microparticles are formulated in the dispersion medium liquid phase. Although not essential to the practice of the present invention, it will be appreciated by those skilled in the art that the use of surfactants can further increase dispersion stability, powder flowability, simplify the procedure or increase the efficiency of delivery. It will be recognized. Of course, combinations of surfactants, including one or more in the liquid phase, and one or more uses associated with perforated microstructures, are considered to be within the scope of the present invention. By "associated with or comprising" is meant that the particulate or porous microstructure can take up, adsorb, absorb, be coated with, or be formed by the surfactant.
[0076]
Broadly, surfactants suitable for use in the present invention promote the formation of perforated microparticles, any of which provide enhanced suspension stability, improved powder dispersibility, or reduced particle aggregation. Or a compound or composition of The surfactant can also comprise a single compound or any combination of compounds (eg, in the case of co-activators). Particularly preferred surfactants are selected from the group consisting of non-fluorinated, saturated and unsaturated lipids, non-ionic detergents, non-ionic block copolymers, ionic surfactants, and combinations thereof. In those embodiments that include a stabilized dispersion, such non-fluorinated surfactants are preferably relatively insoluble in the suspending medium. It should be emphasized that, in addition to the surfactants described above, suitable fluorinated surfactants are compatible with the teachings herein and can be used to provide the desired formulation.
[0077]
Lipids from natural and synthetic sources, including phospholipids, are particularly compatible with the present invention and can be used at various concentrations to form microparticles or structural matrices. Commonly compatible lipids include those having a gel-liquid crystal phase transition of greater than about 40 <0> C. Preferably, the incorporated lipid has a relatively long chain (ie, C₁₆~ C₂₂) Is a saturated lipid, and more preferably contains a phospholipid. Exemplary phospholipids useful in the disclosed stabilized formulations are dipalmitoyl phosphatidyl choline, disteroyl phosphatidyl choline, diarachidoyl phosphatidyl choline, dibehenoyl phosphatidyl choline, short chain phosphatidyl choline, long chain saturated phosphatidyl ethanolamine, long chain saturated Phosphatidyl serine, long chain saturated phosphatidyl glycerol, long chain saturated phosphatidyl inositol, glycolipid, ganglioside GMl, sphingomyelin, phosphatidic acid, cardiolipin; lipid having a polymer chain such as polyethylene glycol, chitin, hyaluronic acid or polyvinyl pyrrolidone; sulfonated mono , Di- and polysaccharide-bearing lipids; fatty acids such as palmitic acid, stearic acid and oleic acid; cholesterol, cholesterol Esters and cholesterol, including hemisuccinate.
[0078]
Compatible non-ionic detergents: Sorbitan trioleate (Span® 85), sorbitan sesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, and Sorbitan ester comprising polyoxyethylene (20) sorbitan monooleate; including oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, glycerol ester and sucrose ester . Other suitable non-ionic detergents are easily identified using McCutcheon's emulsifying agent and detergent (Mc Publishing, Glen Rock, NJ), which is incorporated herein by reference in its entirety. can do. Preferred block copolymers include polyoxyethylene and polyoxypropylene diols, including poloxamer 188 (Pluronic® F-68), poloxamer 407 (Pluronic® F-127) and poloxamer 338. Includes block and triblock copolymers. Ionizable surfactants such as sodium sulfosuccinates and fatty acid soaps can also be utilized. In a preferred embodiment, the microstructure can comprise oleic acid or an alkali salt thereof. Because of their excellent biocompatibility properties, phospholipids, and combinations of phospholipids and poloxamer are particularly suitable for the pharmaceutical embodiments disclosed herein.
[0079]
In addition to the above mentioned surfactants, cationic surfactants or lipids are particularly preferred for the delivery of RNA or DNA. Examples of suitable cationic lipids are: DOTMA, N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride; DOTAP, 1,2-dioleyloxy-3- (trimethylammonio And DOTB, 1,2-Dioleyl-3- (4'-trimethylammonio) butanoyl-sn-glycerol. Polycationic amino acids such as polylysine and polyarginine are also contemplated.
[0080]
It is further recognized that a wide range of surfactants can optionally be used with the present invention, in addition to the surfactants listed above. Furthermore, for a given application, the optimum surfactant or combination thereof can be readily determined by empirical studies that do not require undue experimentation. As discussed in more detail below, surfactants comprising particulates or structural matrices are the oil-in-water (oil-in-) type of precursors used during processing to form a perforated microstructure. Water) may also be useful in the formation of emulsions (ie, spray dried feedstocks).
[0081]
Unlike prior art formulations, relatively high levels of incorporation of surfactants or biocompatible wall-forming materials (eg, phospholipids) improve powder dispersion and increase suspension stability of the disclosed application. It has surprisingly been found that it can be used to reduce powder agglomeration. That is, on a mass to mass (weight to weight) basis, the microporous microstructured particulate or structural matrix can comprise relatively high levels of surfactant. In this regard, the microparticles are preferably about 1 wt.% (Wt.%), 5 wt.% (Wt.%), 10 wt.% (Wt.%), 15 wt.% (Wt.%), 18 wt.% (Wt.%) Or even 20 wt% (wt%) w / w will contain surfactant. More preferably, the microparticulates or microstructures are about 25 wt% (wt%), 30 wt% (wt%), 35 wt% (wt%), 40 wt% (wt%), 45 wt% (wt%) Or 50 wt% (wt%) w / w surfactant will be included. In yet another exemplary embodiment, the surfactant or surfactants are contained in an amount of about 55% by weight (% by weight), 60% by weight (% by weight), 65% by weight (% by weight), 70% by weight (by weight) %), 76% by weight (% by weight), 80% by weight (% by weight). It will include microparticles present in excess of 85 wt% (wt%), 90 wt% (wt%) or even 95 wt% (wt%) w / w. In selected embodiments, the powder will comprise essentially 100% w / w of surfactant (such as phospholipid) w / w. Those skilled in the art will recognize that in such cases, the remainder of the microparticles or structural matrix may include (if applicable) bioactive agents, excipients or other additives. I will.
[0082]
As discussed below, surfactants can be incorporated into any type of microparticles. That is, while the surfactant levels described above are preferably used in perforated microstructures, they are relatively non-porous, or to provide a powder or stabilized dispersion comprising substantially solid particulates. It is usable. Although selected embodiments of the invention will include perforated microstructures with high levels of surfactant, compatible powders use relatively low porosity particulates of equivalent surfactant concentration. Can be formed. Preferably, such microparticles will comprise relatively high levels of surfactant, on the order of greater than about 5% w / w. In this regard, such embodiments are considered to be particularly within the scope of the present invention.
[0083]
In another preferred embodiment of the invention, the microparticles optionally comprise synthetic or natural polymers or combinations thereof. In this respect, useful polymers are polylactic acid, polylactic acid-co-glycolide, cyclodextrin, polyacrylate, methylcellulose, carboxymethylcellulose, polyvinyl alcohol, polyanhydride, polylactam, polyvinyl pyrrolidone, monosaccharide, disaccharide or polysaccharide (dextran , Starch, chitin, chitosan etc.), hyaluronic acid, proteins (albumin, collagen, gelatin etc.). Examples of polymeric resins which can prove useful for the production of microparticles are: styrene-butadiene, styrene-isoprene, styrene-acrylonitrile, ethylene-vinyl acetate, ethylene-acrylate, ethylene-acrylic acid, ethylene-methyl acrylate Ethylene-ethyl acrylate, vinyl-methyl methacrylate, acrylic acid-methyl methacrylate and vinyl chloride-vinyl acetate. One skilled in the art can tailor the delivery efficiency and / or dispersion stability of the microparticles to optimize the activity or effectiveness of the bioactive agent by choosing the appropriate polymer. You will recognize that you can.
[0084]
In addition to the polymeric materials and surfactants described above, various types of particulates may be added in or in the microparticles to provide structure and to form perforated microstructures (i.e. microspheres such as latex particles) in preferred embodiments. Excipients can be incorporated. In this regard, it will be appreciated that post-production techniques, such as selective solvent extraction, can be used to remove rigidified components. Compatible excipients can include carbohydrates including (but not limited to) monosaccharides, disaccharides and polysaccharides. For example, monosaccharides such as dextrose and the like (anhydrides and monohydrates), galactose, mannitol, D-mannose, sorbitol, sorbose and the like; disaccharides such as lactose, maltose, sucrose, trehalose and the like; raffinose and the like Trisaccharides; and carbohydrates such as starch (hydroxyethyl starch), cyclodextrins, and maltodextrins and the like. Amino acids are also suitable excipients, with glycine being preferred. Mixtures of carbohydrates and amino acids are further maintained as within the present invention. The inclusion of both inorganic salts (e.g. sodium chloride, calcium chloride, etc.), organic salts (e.g. sodium citrate, sodium ascorbate, magnesium gluconate, sodium gluconate, tromethamine hydrochloride, etc.) and buffering agents are also conceivable. The inclusion of organic solids and salts such as ammonium carbonate, ammonium acetate, ammonium chloride or camphor is also conceivable.
[0085]
Along with the compounds described above, it is preferred to add other excipients to the microspheres in order to improve particle stiffness, manufacturing yield, delivery efficiency, and deposition, shelf life and patient acceptability. Such optional excipients include (but are not limited to): colorants, taste masking agents, buffers, hygroscopic agents, antioxidants, and chemical stabilizers. Furthermore, as mentioned above, the microparticles can include compounds that can enhance, induce or modulate the uptake of the relevant bioactive agent. In addition, the microparticles of the invention may include antibodies, cofactors, receptors, ligands, and targeting molecules such as substrates that preferentially allow binding to molecules associated with cells towards the microparticles or at the target site. it can. For example, microparticles could be formed to contain an immunoactive compound and an antibody that targets mucosal cell receptors. Such targeting molecules will probably increase the concentration of bioactive particles at the target mucosal site and further enhance any local immune response. It will be appreciated that ligands directed to preferentially expressed receptors on the surface of mucosal or other target cells could also be used to increase microparticle binding at the desired site.
[0086]
Also, preferred embodiments include perforated microstructures that can include or be coated with charged species that extend the residence time at the point of contact or enhance permeation through the mucosa. For example, although a microparticle having a cationic charge can be used to bind a negatively charged bioactive agent such as genetic material, it is known that an anionic charge is advantageous for mucoadhesion. The charge can be imparted through the binding or uptake of polyanionic or polycationic materials such as polyacrylic acid, polylysine, polylactic acid and chitosan.
[0087]
D. Powder form
[0088]
Those skilled in the art will recognize that powders or microparticles of various compositions, configurations and forms can be used in accordance with the present invention, as long as they provide the desired stability and delivery properties. In this regard, it is advantageous to use relatively dense solid particles or powders for some applications (eg for intradermal administration of the stabilized dispersion via an air gun or needleless injector) In other embodiments (e.g., DPI administration), a relatively porous, aerodynamically light perforated microstructure may be preferred. Thus, although the invention will be described in the following with reference to preferred embodiments, it should be emphasized that it is not limited to any particular particle composition, configuration or form. Rather, the choice of microparticle properties (charge, density, composition, etc.) is mainly based on the form of administration, the targeted delivery site, and the choice of bioactive agent.
[0089]
Although various particulate configurations (including micronized and milled particulates) can be used in accordance with the teachings herein, the present invention provides for reducing cohesion between dry particles. Unique methods and compositions can be provided to minimize particulate aggregation which can result in improved delivery efficiency. Thus, the selected disclosed formulations are efficiently aerosolized and uniformly delivered within the pulmonary or nasal path to provide a highly fluid, dry powder that can penetrate deeply. Furthermore, it has been found that the selected powder configuration and form give a relatively stable dispersion when combined with the non-aqueous suspension medium. In any event, the disclosed microparticles can be made to produce surprisingly low throat deposition upon administration.
[0090]
As previously discussed, a particularly preferred embodiment of the present invention incorporates powders or microparticles in the form of porous or perforated microstructures comprising a structural matrix. As used herein, the terms "structural matrix" or "microstructural matrix" are equivalent and are perforated microstructures that define multiple voids, apertures, hollows, defects, pores, holes, crevices, etc that provide the desired properties. Is maintained to mean any solid component that forms In selected embodiments, the perforated microstructure defined by the structural matrix comprises spray dried hollow porous microspheres incorporating at least one surfactant. It is further recognized that the density of the structural matrix can be adjusted to increase dispersion stability or delivery efficiency by altering the matrix components.
[0091]
The absolute form (relative to the morphology) of the particulate or perforated microstructure is generally not critical, and any overall configuration that provides the desired properties is considered within the scope of the present invention. Thus, preferred embodiments can include substantially microsphere-like forms. However, crushed or deformed or broken microparticles are also compatible. With this warning, it will be further appreciated that a particularly preferred embodiment of the present invention comprises spray dried hollow, porous microspheres. In any case, the disclosed powders of the perforated microstructure have increased (but not limited to) suspension stability, improved dispersibility, excellent sampling properties, detachment of carrier particles, and reinforcement. It offers several advantages, including aerodynamics.
[0092]
The mean geometric particle size of the particulate or perforated microstructure is preferably about 0.5 to 50 μm, more preferably 1 to maximize dispersibility, dispersion stability, and to optimize the distribution on administration. -30 μm. In applications where valves or small orifices are used, large particles are likely to be clumped or separated from the dispersion, potentially causing the device to clog. It will be recognized that there is a possibility that it is not preferable. In a particularly preferred embodiment, the average geometric particle size (or diameter) of the perforated microstructure is less than 20 μm or 10 μm. More preferably, the average geometric diameter is less than about 7 μm or 5 μm, more preferably less than about 4 μm or 2.5 μm. Another preferred embodiment comprises a formulation wherein the mean geometric diameter of the perforated microstructure is between about 1 μm and 5 μm. In a particularly preferred embodiment, the perforated microstructure is a dry, hollow, porous powder of about 1 to 10 μm, or 1 to 5 μm in diameter, having a shell thickness of about 0.1 to about 0.5 μm. Will have a microsphere-like shell. It is a particular advantage of the present invention that the particle concentration of the dispersion and structural matrix components can be adjusted to optimize the delivery characteristics of the selected particle size.
[0093]
As mentioned throughout the specification, microstructure porosity plays a significant role in establishing dispersibility (e.g. at DPI) or dispersion stability (e.g. for MDis, jet guns or nebulizers) be able to. At this point the average porosity of the perforated microstructure can be determined via electron microscopy combined with modern imaging techniques. More specifically, to determine the porosity of the formulation, electron micrographs of representative samples of the perforated microstructure can be obtained and analyzed digitally. Such a methodology is well known and can be accomplished without undue experimentation.
[0094]
For purposes of the present invention, the average porosity of the particulate or perforated microstructure (ie, the percentage of particle surface area open to internal and / or central voids) is in the range of about 0.5% to about 80%. Can be. In more preferred embodiments, the average porosity is in the range of about 2% to about 40%. Based on the chosen manufacturing parameters, the average porosity may be greater than about 2%, 5%, 10%, 15%, 20%, 25% or 30% of the surface area of the microstructure. In other embodiments, the average porosity of the microstructure can be greater than about 40%, 50%, 60%, 70% or even 60%. For the pores themselves, they are typically in the range of about 5 nm to about 400 nm, preferably in the range of about 20 nm to about 200 nm, in average pore size. In a particularly preferred embodiment, the average pore size is in the range of about 50 nm to about 100 nm. As discussed in more detail below, it is an important advantage of the present invention that pore size and porosity can be closely controlled by careful selection of incorporated components and production parameters.
[0095]
In this regard, the particulate form and / or hollow design of the particulate or perforated microstructure plays an important role on the dispersibility or cohesion of the dry powder formulation disclosed herein. That is, it is surprising that the inherent binding properties of fine powders can be overcome by reducing the van der Waals, electrostatic attraction and liquid bridging forces typically present between dry particles. It was discovered. More specifically, in accordance with the teachings herein, improved powder dispersibility can be provided by designing particle morphology and density, as well as humidity and charge controls. To that end, preferred embodiments of the present invention include perforated microstructures with pores, voids, hollows, defects or other interstitial spaces that reduce surface contact area between particles, thereby minimizing interparticle forces. It is like that. In addition, the use of surfactants such as phospholipids and fluorinated blowing agents in accordance with the teachings herein contributes to the improvement of powder flow properties by relieving the strength of the charge and electrostatic forces and the water content. Can.
[0096]
Most flours (e.g. <5 [mu] m) exhibit poor dispersibility which can be problematic when trying to deliver, aerosolize and / or pack powders. The main forces that contrast particle interactions can typically be divided into long and short range forces. Long distance forces include gravitational attraction and electrostatic forces, while interactions vary as separation distance or particle size squared. Important short-range forces for dry powders include van der Waals interactions, hydrogen bonds and liquid bridges. The latter two short distance forces differ from others in that they occur when there is already contact between the particles. The ability to substantially reduce or reduce these attractive forces through the use of the perforated microstructures described herein is a major advantage of the present invention.
[0097]
One skilled in the art will recognize that van der Waals (VDW) attraction occurs in a short range and depends at least in part on surface contact between interacting particles. As the two particles approach each other, the VDW force increases with the increase in contact area. VDW interaction force magnitude, F, for two dry particles⁰ _VDWCan be calculated using the following formula:
[Equation 1]

Where h is Planck's constant, ω is angular frequency, d 0 is the distance at which adhesion is maximal, and r 1 and r² Is the radius of the two interacting particles). Thus, it will be appreciated that one way to minimize the magnitude and strength of the VDW force for the dry powder is to reduce the interparticle area of contact. It is important that the size of d0 is a reflection of this area of contact. The smallest area of contact between two opposing objects will occur if the particle is a perfect sphere. In addition, if the particles are very porous, the area of contact will be further minimized. Thus, the perforated microstructure of the present invention acts to reduce interparticle contact and the corresponding VDW force. It is important to note that this reduction in VDW force is primarily the result of the unique particle morphology of the powder of the invention rather than the increase in geometric particle size. In this regard, it is recognized that a particularly preferred embodiment of the present invention provides a powder having an average or small particle size (eg, an average geometric diameter <10 μm) exhibiting relatively low VDW attraction.
[0098]
Furthermore, as indicated above, electrostatic forces that affect the powder occur when either or both of the particles are electrically charged. This phenomenon will occur due to attraction or repulsion between particles, depending on charge similarity or dissimilarity. In the simplest case, the charge can be described using Coulomb's law. One way to adjust or reduce the electrostatic forces between particles is whether one or both of the particles have a non-conductive surface. Thus, if the porous microstructured powder contains relatively nonconductive excipients, surfactants or activators, any charge generated on the particles will be randomly distributed on the surface. As a result, the charge half-life of the powder containing the non-conductive component is relatively short, since high charge retention is noted by the resistivity of the material. The resistive or nonconductive component is a material that does not function as either an efficient electron donor or acceptor.
[0099]
Derjaguin et al. (Muller, V. M., Yushchenko, VS., and Derjaguin, B. V., J. Colloid Interface Sci., 1980, 77, 115-119), which is incorporated herein by reference, accepts electrons. Provides a ranked list of molecule groups for those functions that do or donate. In this regard, an exemplary group can be ranked as follows:
[0100]
Donor: -NH₂> -OH> -OR> COOR> -CH₃> -C₆H₅>-Halogen>-COOH>-CO>-CN acceptor:
[0101]
The present invention provides reduced electrostatic effects in the disclosed powders through the use of relatively non-conductive materials. Using the above rankings, preferred non-conductive materials comprise halogenated and / or hydrogenated components. Materials such as phospholipids and fluorinated blowing agents, which can be held to some extent with spray dried powders, are preferred as they are resistant to particle charge. Residual effervescent agents (eg fluorochemicals) in the particles are typically applied during spray drying and cyclone separation even at relatively low levels, thus minimizing particulate or porous microstructure charge It will be recognized as promoting Based on the general electrostatic principle and the teachings herein, one of ordinary skill in the art can identify additional materials that help to reduce the electrostatics of the disclosed powder without undue experimentation. I will. In this regard, highly charged agents can be electrostatically modified by simple pH adjustment or chelation with oppositely charged compounds (eg, nucleic acids bound to cationic lipids). Furthermore, if necessary, the electrostatic force can also be manipulated and minimized using energization and charging techniques.
[0102]
In addition to the surprising advantages described above, the present invention further provides for the attenuation or reduction of hydrogen and liquid bonding. As known to those skilled in the art, both hydrogen bonding and liquid bridging result from the moisture absorbed by the powder. In general, higher humidity results in higher interparticle forces on hydrophilic surfaces. This is an essential problem in prior art formulations for inhalation therapy which tends to use relatively hydrophilic compounds such as lactose. However, in accordance with the teachings herein, adhesion with adsorbed water can be adjusted or reduced by increasing the hydrophobicity of the contact surface. One skilled in the art will recognize that an increase in particle hydrophobicity can be achieved by the choice of excipients and / or the use of post-production spray dry coating techniques, such as those used with fluid beds. Thus, preferred excipients include hydrophobic surfactants such as phospholipids, fatty acid soaps and cholesterol. In light of the teachings herein, it will be appreciated that one of ordinary skill in the art will be able to identify materials exhibiting similar desirable properties without undue experimentation.
[0103]
Microparticles or porous microstructures will preferably be provided "dry" whether they are used as a dry powder or in combination with a non-aqueous suspension medium. That is, the microparticles will have a moisture content that allows the powder to remain chemically and physically stable during storage at ambient temperature and to be easily dispersible. Thus, the water content of the microparticles is typically less than 6% by weight (% by weight), preferably less than 3% by weight (% by weight). In some instances, the water content is as low as 1% by weight (wt%). Of course, the water content is at least partially defined by the formulation and recognized as being controlled by the process conditions used, such as inlet temperature, feed concentration, pump speed, blowing agent type, concentration and post drying Will.
[0104]
As known by those skilled in the art, methods such as angle of repose or shear can be used to evaluate the flow properties of the dry powder. The angle of repose is defined as the angle formed when pouring a powder cone onto a flat surface. Powders having a repose angle in the range of 45 ° to 20 ° are preferred and exhibit a suitable powder flow. More specifically, powders having a repose angle of between 33 ° and 20 ° flow with relatively low shear and are particularly useful in pharmaceutical formulations used for inhalation therapy (eg, DPIs). The shear index takes longer than the angle of repose to measure, but is considered more reliable and easier to determine. Those skilled in the art will appreciate that the experimental procedures outlined by Amidon and Houghton (G.E. Amidon and M.E. Houghton, Pharm. Manuf., 2, 20, 1985), which are incorporated herein by reference, refer to the present invention. It will be recognized that it can be used to evaluate the shear index for the purpose of Here, too, the S. Kocova and N.A. Pipel, J.M. Pharm. Pharmacol. The shear index is evaluated from powder parameters such as yield stress, effective angle of internal friction, tensile strength, and specific cohesion, as described in U.S. 8, 33-35, 1973. In the present invention, powders having a shear index of less than about 0.98 are preferred. More preferably, the powders used in the disclosed compositions, methods and systems will have a shear index of less than about 1.1. In particularly preferred embodiments, the shear index will be less than about 1.3, or even less than about 1.5. Of course, powders with different shear index can be used as a result of effective deposition of active or bioactive agent at critical sites.
[0105]
It will also be appreciated that powder flow properties have been shown to correlate well with bulk density measurements. In this regard, conventional prior art considerations (CF Harwood, J. Pharm. Sci., 60, 161-163, 1971)IncreaseWas considered to correlate with improved flow properties, as predicted by the shear index of the material. Conversely, it has surprisingly been found that, due to the perforated microstructure of the present invention, powders with relatively low bulk density exhibited superior flow properties. That is, the hollow porous powder of the present invention exhibited excellent flow properties as compared to a powder having substantially no pores. For this reason, 0.5 g / cm showing particularly good flow properties³ It has been found that it is possible to provide powders having a bulk density of less than. More surprisingly, 0.3 g / cm showing excellent flow properties³ Less than about 0.1 g / cm³Less than 0.05 g / cm³It has been found that it is possible to provide a perforated microstructured powder having a bulk density of less than an order of magnitude. The ability to produce low bulk density powders with excellent flowability further exaggerates the novel and unexpected properties of the present invention.
[0106]
These low bulk densities are particularly advantageous when using the powders disclosed with DPIs. Specifically, by providing a powder having an unusually low bulk density, the present invention provides the lowest commercially viable reduction in fill weight for dry powder inhalers. That is, the most designed unit dose containers for DPIs are filled using fixed volume or gravity techniques. Contrary to many prior art, the present invention provides a powder in which the bioactive agent and the incipient or bulking agent make up the whole inhaled particle. By providing particles with very low bulk density, the minimum powder mass that can be filled into a unit dose container is reduced, which eliminates the need for carrier particles. That is, the relatively low density of the particles of the present invention provides for reproducible administration of relatively low dose drug compounds without the use of carrier particles. In addition, the removal of carrier particles acts to minimize throat deposition and any "gag" effects as the large lactose particles of the prior art tend to impact the throat and upper airways due to their size. Do.
[0107]
The reduced attraction (eg, van der Waals, electrostatic, hydrogen and liquid bridging, etc.) and excellent flow provided by perforated microstructured powders can be used for inhalation therapy (eg, DPIs, MDIs, nebulizers etc.) It will be appreciated that they are particularly useful in formulations for (in an inhalation device). Along with the excellent flowability, the microstructured, porous and / or hollow design also plays an important role in the resulting aerosol properties of the powder when released. This phenomenon is also effective for the delivery of perforated microstructures in dry form as in the case of MDI or nebuliser, or in the case of DPI, for particulates or perforated microstructures that are aerosolized as dispersions. In this regard, the porous structure and relatively high surface area of the dispersed microparticles are more easily compared to non-porous particles of comparable size, along the gas flow during inhalation at longer distances. Enable to be carried.
[0108]
More specifically, due to their high porosity, the density of particles is substantially 1.0 g / cm.³Less than, typically 0.5g / cm³Less than 0.1 g / cm more often³Girder, and 0.01 g / cm³Low enough. Unlike geometrical grain size, aerodynamic grain size of perforated microstructure, d_aer Is substantially the particle density, ρ: d_aer= D_geoρ (where d is_geoIs the geometric diameter). 0.1 g / cm³For particle density of d_aerIs about 3 times d_geoIt becomes smaller, resulting in increased particle deposition in the peripheral area of the lung and correspondingly less deposition in the throat. In this regard, the average aerodynamic diameter of the perforated microstructure is preferably less than about 5 μm, more preferably less than about 3 μm, and in a particularly preferred embodiment less than about 2 μm. Such particle distribution acts to increase the deep lung deposition of the bioactive agent, regardless of whether it is administered using DPI, MO or a nebulizer.
[0109]
As shown in the following examples, the particle size distribution of the aerosol formulation of the present invention can be measured by conventional techniques such as cascade impaction or by time of flight analysis. In addition, the dose released from the inhalation device was determined according to the proposed United States Pharmacopoeia method (Pharmacopeial Previews, 22 (1996) 3065), which is incorporated herein by reference. These and related techniques allow the "particulate fraction" of the aerosol to be calculated that corresponds to those particulates that can be deposited efficiently in the lungs. As used herein, the term "particulate fraction" refers to the total amount of active drug delivered per actuation from the DPI, MDI or nebulizer mouthpiece onto the plate 2-7 of the 8-stage Andersen cascade impactor. Say a percentage. Based on such measurements, the formulations of the present invention will preferably have a particulate fraction of about 20% by weight (wt%) (w / w) or more based on the weight of the perforated microstructure, more preferably , They exhibit a fine particle fraction of about 25 wt% (wt%) to 90 wt% (wt%) (w / w), more preferably about 30-80 wt% (wt%) (w / w) I will. In selected embodiments of the present invention, about 30 wt% (wt%), 40 wt% (wt%), 50 wt% (wt%), 60 wt% (wt%), 70 wt% (wt%), It preferably comprises particulate fractions in excess of 80% by weight (% by weight) or even 90% by weight (% by weight).
[0110]
Furthermore, it has also been found that the formulation of the invention exhibits a relatively low deposition rate on the induction port and on the impactor plates 0 and 1 when compared to prior art formulations. Deposition on these components is linked to deposition in the throat in humans. More specifically, while the most commercially available MDIs and DPIs simulated throat deposition of about 40-70% by weight (w / w) of the total dose, while the formulations of the present invention Typically, less than about 20% by weight (wt%) (w / w) is deposited. Therefore, preferred embodiments of the present invention are: about 40% by weight (% by weight), 35% by weight (% by weight), 30% by weight (% by weight), 25% by weight (% by weight), 20% by weight (% by weight), Even throat deposition less than 15 wt% (wt%) or 10 wt% (wt%) (w / w) was simulated. One skilled in the art will recognize that the significant reduction in throat deposition provided by the present invention results in a corresponding reduction in associated local side effects such as throat irritation.
[0111]
With regard to the advantageous deposition profiles provided by the present invention, it is well known that MDI propellants push the suspended particles out of the device at high speed, typically in the posterior direction of the throat. As the prior art formulations typically contain a significant percentage of large particles and / or aggregates, more than as much as 2/3 of the dose released can impact the throat. Furthermore, the unfavorable delivery profile of conventional powder formulations is also shown under low particle velocity conditions, as occurs with DPI devices. In general, this problem is inherent when aerosolizing solid, dense particles that are prone to aggregation. Nevertheless, as mentioned above, the novel and unexpected properties of the stabilized dispersions of the present invention result in surprisingly low throat deposits upon administration from inhalation devices such as DPI, MDI atomizers or nebulizers.
[0112]
While not wishing to be bound by any particular theory, the reduced throat deposition provided by the present invention is due to the reduction in particle aggregation and from the incorporated microstructured hollow and / or porous form It seems to occur. That is, the hollow and porous nature of the dispersed microstructure results in particles in the propellant stream (or gas stream in the case of DPIs) such that the hollow / porous whiffle ball decelerates more quickly than the base ball. Slow down the speed of Thus, rather than impacting and sticking to the back of the throat, relatively slowly moving particles are more likely to be inhaled by the patient. In addition, the highly porous nature of the particles allows the propellant in the perforated microstructure to be removed quickly, reducing the particle density before impacting the throat. Thus, a substantially higher percentage of the bioactive agent administered is deposited in the airways of the lungs that it can absorb efficiently.
[0113]
E. Powder formation
[0114]
As understood from the above section, various components can be bound or incorporated into the microparticle of the present invention. Similarly, several techniques can be used to provide microparticles having the desired morphology (eg, a perforated or hollow / porous configuration), dispersibility and density. Among other methods, microparticles compatible with the present invention can be formed by techniques including spray drying, vacuum drying, solvent extraction, emulsification, lyophilization, and combinations thereof. Many of the basic concepts of these techniques are well known in the art and, in view of the teachings herein, excessive experimentation has been done to adapt them to give the desired particle configuration and / or density. It will be further appreciated that it is not necessary.
[0115]
Although some procedures are generally compatible with the present invention, particularly preferred embodiments typically include particulate or perforated microstructures formed by spray drying. As is well known, spray drying is a one-step process of converting a liquid feed into the form of dried particulates. It will be appreciated that spray drying has been used to provide powder materials for various administration routes, including inhalation, for pharmaceutical applications. For example, inhaled aerosols, which are incorporated herein by reference: "Physical and biological basis of treatment", J. Hicley, ed., Mercer Decker, Inc., New York, M., in 1996. M. Sachetti and M. M. See Van Oort.
[0116]
In general, spray drying consists of combining the highly dispersed liquid with a sufficient volume of hot air to provide evaporation and drying of the droplets. The formulation to be spray dried or feed (or feedstock) can be any aqueous solution, passage dispersion, slurry, colloid dispersion or paste that can be atomized using a selected spray dryer. In a preferred embodiment, the feedstock comprises a colloidal system, such as an emulsion, inverse emulsion, microemulsion, multiple emulsion, particulate dispersion or slurry. Typically, the feed is sprayed onto a stream of warm filtered air which evaporates the solvent and carries the dried product to a collector. The spent air is then evacuated with the solvent. One skilled in the art will recognize that several different types of devices can be used to provide the desired product. For example, a commercial spray dryer manufactured by Buchi or Niro will effectively produce particles of the desired size, shape and density.
[0117]
It is further recognized that these spray drying devices, and in particular their atomizers, can be adapted or customized for specialized applications, ie simultaneous spraying of two solutions with overlapping nozzle technology) Will. More specifically, a W / O type (water-in-oil) emulsion can be atomized from one nozzle, and a solution containing an antiadherent such as mannitol can be co-atomized from a second nozzle Can. In other cases, it may be preferable to push the feed solution through a custom designed nozzle using a high pressure liquid chromatography (HPLC) pump. The choice of device is not important if the microstructure comprising the desired form and / or composition is manufactured, and will be readily apparent to one of ordinary skill in the art in view of the teachings herein.
[0118]
The resulting spray-dried powder is typically approximately spherical in shape, nearly uniform in size, and often hollow, but depending on the drug incorporated and the spray-drying conditions, some degree in shape Irregularities in In many instances, the dispersion stability, and the dispersibility of the particulate or perforated microstructure, appear to be improved if they are used in their manufacture of expanding agents (or blowing agents). An especially preferred embodiment can comprise an emulsion having the expanding agent as the dispersed or continuous phase. The expansion agent is preferably dispersed with the surfactant using, for example, a commercially available microfluidizer at a pressure of about 5,000 to 15,000 psi. This process forms an emulsion, preferably one that is stabilized by the incorporated surfactant and typically comprises submicron droplets of water immiscible swelling agent dispersed in the aqueous continuous phase . The formation of such emulsions using this and other techniques is common in the art and well known to those skilled in the art. The blowing agent is preferably evaporated in a spray-drying process, and in selected embodiments, a fluorine compound (eg, perfluorohexane, etc.) leaving behind relatively hollow, porous aerodynamically light microspheres. Perfluorooctyl bromide, perfluorodecalin, perfluorobutyl ethane). As discussed in further detail below, other suitable liquid blowing agents include non-fluorinated oils, chloroform, freons, ethyl acetate, alcohols and hydrocarbons. Nitrogen and carbon dioxide are also considered as suitable blowing agents.
[0119]
In addition to the compounds mentioned above, inorganic and organic substances which can be removed under reduced pressure by sublimation in post-production steps are also compatible with the present invention. These sublimable compounds are soluble or dispersible as micronized crystals in the spray dry feed solution and can include ammonium carbonate and camphor. Other compounds compatible with the present invention include rigidizable solid structures that can be dispersed in the feed solution or can be manufactured in-situ. These structures are then extracted after initial particle formation using post-production solvent extraction steps. For example, latex particles can be dispersed and then dried with other wall forming compounds and then extracted with a suitable solvent.
[0120]
The microparticles are preferably manufactured using the blowing agents described above, but in some instances additional blowing agents are not required, and an aqueous dispersion of the drug and / or excipient and one or more surfactants. It will be appreciated that the liquid is spray dried directly. In such cases, the formulation can follow process conditions (eg, high temperature) that can result in the formation of hollow, relatively porous microparticles. Furthermore, the drug can have special physicochemical properties (eg high crystallinity, high melting temperature, surfactant, etc.) which make it particularly suitable for use in such techniques.
[0121]
When a blowing agent is used, the degree of porosity and dispersibility of the resulting microparticles depend, at least in part, on the nature of the blowing agent, its concentration in the feed (eg, as an emulsion), and spray drying conditions It seems to be. It is surprising that, with regard to controlling porosity and dispersion in dispersion, the use of compounds which were previously regarded as undesirable as blowing agents can give porous microstructures with particularly favorable properties. It was found. More specifically, in this novel and unexpected aspect of the invention, the use of a fluorine compound having a relatively high boiling point (ie above about 40 ° C.) is particularly for producing microparticles that are porous. It was found to be usable. Such perforated microstructures are particularly suitable for inhalation therapy. In this regard, fluorinated or partially fluorinated blowing agents having boiling points above about 40 ° C., 50 ° C., 60 ° C., 70 ° C., 80 ° C., 90 ° C. or even 95 ° C. can be used. Particularly preferred blowing agents have the boiling point of water, ie a boiling point above 100 ° C. (eg perflubron, perfluorodecalin). In addition, they have relatively low water solubility (<10, as they facilitate the production of stable emulsion dispersions with mean-weighted particle sizes of less than 0.3 μm.^-6Preference is given to blowing agents having M).
[0122]
As previously described, these blowing agents are preferably incorporated into the emulsified feed prior to spray drying. For the purpose of the present invention, this feedstock will also preferably contain one or more bioactive agents, one or more surfactants or one or more excipients. Of course, combinations of the components described above are also within the scope of the present invention. Although high boiling (> 100 ° C.) fluorinated blowing agents comprise one preferred aspect of the present invention, non-fluorinated blowing agents with similar boiling points (> 100 ° C.) are also used to provide compatible microparticles. It will be recognized as possible. Exemplary non-fluorinated blowing agents suitable for use in the present invention have the formula:
[0123]
R¹-X-R²Or R¹-X
(In the formula, R¹Or R²Is hydrogen, alkyl, alkenyl, alkynyl, aromatic, cyclic or a combination thereof, and X is any group containing carbon, sulfur, nitrogen, halogen, phosphorus, oxygen, and a combination thereof.
including.
[0124]
Without limiting the invention in any way, it is hypothesized that it will leave a thin envelope on the particle surface as the aqueous feed component evaporates during spray drying. The resulting particle wall or envelope formed at the beginning of the spray appears to trap any high boiling blowing agent as hundreds of emulsion droplets (about 200-300 nm). As the drying process continues, the pressure inside the particulate buildup increases, thereby evaporating at least a portion of the incorporated blowing agent and extruding it through the relatively thin skin. Venting or degassing of this leads to the formation of pores or other defects in the apparently microstructure. At the same time, the remaining particulate components (possibly with some blowing agent) migrate from the inside to the surface as the particles solidify. This transition is apparently delayed during the drying process as a result of the increased resistance to mass transfer due to the increased internal viscosity. Once migration occurs, the particles solidify leaving voids, holes, defects, hollows, spaces, apertures, perforations, or holes. The number of pores or defects, their size, and the resulting wall thickness depend on the chosen foaming agent formulation and / or properties (e.g. boiling point), its concentration in the emulsion, total solids concentration, and spray drying conditions. Rely mainly on. As mentioned throughout the specification, this preferred particle morphology appears to contribute, at least in part, to improved powder dispersibility, suspension stability and aerodynamics.
[0125]
It has surprisingly been found that substantial amounts of these relatively high boiling blowing agents can be retained in the resulting spray-dried product. That is, the spray-dried particulates described herein represent 1% by weight (% by weight), 3% by weight (% by weight), 5% by weight (% by weight), 10% by weight (% by weight), 20% by weight of the residual blowing agent. % (Wt%), 30 wt% (wt%) or 40 wt% (wt%) (w / w) can be included as high as possible. In such cases, the increased particle density produced by the retained blowing agent resulted in a higher production yield. It will be recognized by those skilled in the art that the retained fluorinated blowing agent can alter the surface properties of the microparticles, thereby minimizing particle aggregation during processing and further increasing dispersion stability. Residual fluorinated blowing agents in the powder can also reduce interparticle cohesion by providing a barrier or by reducing attraction (eg, electrostatic) during manufacture. When using the disclosed microstructure with a dry powder inhaler, this reduction in cohesion can be particularly advantageous when using the disclosed microstructure in combination with a dry particle inhaler.
[0126]
Furthermore, the amount of residual blowing agent can be controlled by the process conditions (eg outlet temperature), blowing agent concentration or boiling point. If the outlet temperature is boiling point or higher, the blowing agent escapes the particles and the production yield decreases. Preferred outlet temperatures will generally be operated 20, 30, 40, 50, 60, 10, 80, 90 or 100 ° C. below the blowing agent boiling point. More preferably, the temperature difference between the outlet temperature and the boiling point is in the range of 50-150 ° C. Particle porosity, production yield, electrostatics and dispersibility are firstly optimized by identifying the range of process conditions (eg outlet temperature) that are suitable for the chosen active agent and / or excipient Those skilled in the art will recognize that it is possible. Preferred blowing agents can be selected with the highest outlet temperature, such that the temperature difference is at least 20 ° C. up to 150 ° C. In some cases, for example, under supercritical conditions or when producing microparticles using lyophilization techniques, the temperature difference may be outside of this range. One skilled in the art will further recognize that the preferred concentration of blowing agent can be determined without undue experimentation using techniques similar to those described in the Examples herein.
[0127]
Although residual blowing agents may be advantageous in selected embodiments, it may be preferable to substantially remove any blowing agent from the spray-dried product. In this regard, residual blowing agent can be easily removed in a post-production evaporation step in a vacuum oven. Furthermore, such post-manufacturing techniques can also be used to provide porosity in particulates. For example, the pores can be formed by spray drying a bioactive agent and an excipient that is removable from particulates formed under reduced pressure.
[0128]
In any case, an exemplary concentration of blowing agent in the feedstock is between 2% and 50% (v / v), more preferably between about 10% and 45% (v / v). In other embodiments, it may be preferable for the blowing agent concentration to preferably exceed about 5%, 10%, 15%, 20%, 25% or even 30% (v / v). Nevertheless, other feedstock emulsions can contain 35%, 40%, 45% or even 50% (v / v) of selected compounds.
[0129]
In a preferred embodiment, another way of ascertaining the concentration of the blowing agent used in the feed is as a ratio of the concentration of the blowing agent in the precursor or feed emulsion to that of the stabilizing surfactant (eg phosphatidyl choline or PC) , Is to give it. This ratio was called the PFC / PC ratio because of the fluorocarbon blowing agent (eg perfluorooctyl bromide) and for the purpose of illustration. It will be appreciated that, more generally, compatible blowing agents and / or surfactants can be substituted for the exemplary compounds without being outside the scope of the present invention. In any event, typical PFC / PC ratios are in the range of about 1 to about 60, more preferably about 10 to about 50. For preferred embodiments, the ratio will generally be greater than about 5, 10, 20, 25, 30, 40 or even 50. It should be appreciated that the use of a higher PFC / PC ratio generally gives a more hollow and porous nature of the structure. More specifically, methods using a PFC / PC ratio greater than about 4.8 tended to provide a structure that is compatible with the dry particle formulations and dispersions disclosed herein.
[0130]
While relatively high boiling blowing agents comprise one preferred aspect of the present invention, it will be appreciated that other blowing or expanding agents can also be used to provide compatible microstructures. Thus, the blowing agent can also include any volatile material that can be incorporated into the feed solution to produce the desired microstructure. The blowing agent can be removed during the initial drying process or in post-production steps such as vacuum drying or solvent extraction. Suitable agents are:
[0131]
1. Dissolved low-boiling (less than 100 ° C.) agents that can be mixed with aqueous solutions such as methylene chloride, acetone, ethyl acetate and alcohols used to saturate the solution;
[0132]
2. CO used at high pressure₂Or N₂Etc., or liquids such as freons, CFCs, HFAs, PFCs, HFCs, HFBs, fluoroalkanes, and hydrocarbons;
[0133]
3. Emulsions of immiscible low boiling (less than 100 ° C.) liquids suitable for use in the present invention generally have the formula:
[0134]
R¹-X-R²Or R¹-X
(Wherein, Rl or R²Is hydrogen, alkyl, alkenyl, alkynyl, aromatic, cyclic or a combination thereof, and X is any group containing carbon, sulfur, nitrogen, halogen, phosphorus, oxygen and combinations thereof)
It is.
[0135]
4. Dissolved or dispersed salts or organics which can be removed under reduced pressure by sublimation in post-production steps such as ammonium salts, camphor, etc.
[0136]
5. Dispersed solids that can be extracted after initial particle formation using post-production solvent extraction
including.
[0137]
For lower boiling expanders, they are typically added to the feed in an amount of about 1% to 40% (v / v) of the surfactant solution. About 15% (v / v) of expansion agent has been found to produce a spray-dried powder that can be used in the method of the present invention.
[0138]
It has been found that compatible fine particles can be produced using commercially available equipment such as a Büchi mini spray dryer (type B-191, Switzerland), whatever blowing agent is finally chosen. As recognized by those skilled in the art, the inlet and outlet temperatures of the spray drying apparatus can be adjusted to provide the desired particle size and maintain the activity of the incorporated bioactive agent. At this point, the inlet and outlet temperatures are adjusted according to the melting characteristics of the formulation and feed composition. The inlet temperature may thus be between 60 ° C. and 170 ° C. and the outlet temperature may be between about 40 ° C. and 120 ° C., depending on the composition of the feed and the desired particulate properties. Preferably, these temperatures are 90 ° C. to 120 ° C. for the inlet and 60 ° C. to 90 ° C. for the outlet. The flow rate used in the spray drying facility will generally be about 3 ml per minute to about 15 ml per minute. The atomizer air flow rate varies between 25 liters per minute to about 60 liters per minute. Commercially available spray-drying devices are well known to the person skilled in the art and the appropriate settings for any particular dispersion are easy via experimental tests, with corresponding reference to the examples which follow. It can be decided.
[0139]
Microparticulates are preferably formed using a fluorinated blowing agent in the form of an emulsion, but non-fluorinated oils can be used to increase the loading capacity of the bioactive agent without reducing the microstructure. It will be recognized. In this case, the choice of non-fluorinated oil is based on the solubility of the active or bioactive agent, water solubility, boiling point and flash point. The bioactive agent will be dissolved in the oil and then emulsified in the feed solution. Preferably, the oil has substantial solubilization capacity, lower water solubility (<10 for selected agents)^-3M), having a boiling point higher than water, and a flash point higher than the drying outlet temperature). Also within the scope of the present invention is the addition of co-solvents to non-fluorinated oils and of surfactants to increase the solubilization capacity.
[0140]
In a particularly preferred embodiment, non-fluorinated oils can be used to solubilize bioactive agents having limited solubility in aqueous compositions. The use of non-fluorinated oils is a particular application for increasing the loading capacity of hydrophobic peptides and proteins. Preferably, the oils or oil mixtures for solubilizing them have a refractive index between 1.36 and 1.41 (e.g. ethyl butyrate, butyl carbonate, dibutyl ether). In addition, process conditions such as temperature and pressure can be adjusted to boost the solubility of the selected agent. Selection of appropriate oils or oil mixtures and processing conditions to maximize agent loading volume is within the purview of one of ordinary skill in the art, given the teachings herein, and recognized as achievable without undue experimentation. Will be done.
[0141]
Particularly preferred embodiments of the invention comprise surfactants such as phospholipids and at least one bioactive agent. Other embodiments can further include an excipient that can further include a hydrophilic moiety such as, for example, a carbohydrate (ie, glucose, lactose or starch) in addition to any chosen surfactant. Contains spray-dried formulations. In this regard, various starches and derivatized starches are suitable for use in the present invention. Other optional ingredients include buffers such as viscosity modifiers, phosphate buffers or other conventional biocompatible buffers or pH adjusters such as acids or bases, and osmotic agents (isotonic, To provide a high molarity or low molarity). Examples of suitable salts include sodium phosphate (monobasic and dibasic), sodium chloride, calcium phosphate, calcium chloride and other physiologically acceptable salts.
[0142]
Whatever component is chosen, the first step in particulate production typically involves feedstock production. Preferably, the selected drug is dissolved in water to produce a concentrated solution. The drug can also be dispersed directly in the emulsion, especially in the case of water insoluble agents. Alternatively, the drug can be incorporated in the form of a solid particle dispersion. The concentration of active or bioactive agent used depends on the amount of agent needed in the final powder, and the performance of the delivery device used (e.g. fine dose for MDI or DPI). Optionally, cosurfactants such as poloxamer 188 or span 80 may be dispersed into this additional solution. In addition, excipients such as sugar and starch can also be added.
[0143]
In selected embodiments, an oil-in-water emulsion is then formed in a separate container. The oil used is preferably a fluorocarbon (eg perfluorooctyl bromide, perfluorodecalin) which is emulsified with a surfactant such as a long chain saturated phospholipid. For example, 1 gram of phospholipid is 8000 rpm of an appropriate high shear mechanical mixer (eg, Ultra-Turrax T-25 type mixer) in 150 g of hot distilled water (eg, 60 ° C.) It can be used for 5 minutes and homogenized. Typically, 5 to 25 grams of fluorocarbon is added dropwise to the dispersed surfactant solution while mixing. The resulting perfluorocarbon in the water emulsion is then treated with a high pressure homogenizer to reduce particle size. For example, the emulsion can be processed in two separate passes at 82,737 to 103,421 kPa (12,000 to 18,000 psi) and can be maintained at 50-80 ° C.
[0144]
The bioactive agent solution and the perfluorocarbon emulsion can then be combined and fed to a spray dryer. The two formulations will typically be miscible, as the emulsion preferably comprises an aqueous continuous phase. Although bioactive agents are separately solubilized for the purpose of this discussion, in other embodiments it is recognized that the active or bioactive agent can be directly solubilized (or dispersed) in the emulsion Will. In such cases, the active or bioactive emulsion is simply spray dried without combining the separated drug formulations.
[0145]
In any case, operating conditions such as inlet and outlet temperature, feed rate, atomization pressure, flow rate of dry air and configuration of the nozzle, to give the required particle size of the resulting dry structure, and the production yield , Can be adjusted according to the manufacturer's guidelines. An exemplary setting is as follows: air inlet temperature between 60 ° C. and 170 ° C .; air outlet between 40 ° C. and 120 ° C .; feed rate between about 3 ml and 15 ml per minute; Minute suction air flow rate, and atomization air flow rate between 25-50. Selection of appropriate equipment and processing conditions is within the scope of one of ordinary skill in the art in view of the teachings herein, and can be accomplished without undue experimentation. In any event, the use of these and substantially equivalent methods results in the formation of hollow porous aerodynamically light microspheres having a particle size suitable for aerosol deposition to the lungs. The microstructures, which are hollow and porous, are generally honeycomb or foamy in appearance. In a particularly preferred embodiment, the perforated microstructure comprises hollow, porous, spray-dried microspheres.
[0146]
With spray drying, porous microstructures useful in the present invention can be formed by lyophilization. One skilled in the art will recognize that lyophilization is a freeze-drying process in which water is sublimed from the composition after it has been frozen. A particular advantage associated with the lyophilization process is that biological agents and other agents that are relatively unstable in aqueous solution are dried without elevated temperatures (thereby eliminating deleterious thermal effects). It can then be stored dry, with little stability problems. In the context of the present invention, such techniques do not compromise the physiological activity, without particulates or porous microstructures, with peptides, proteins, genetic material and other natural and synthetic macromolecules. It is particularly compatible with uptake. Methods for providing lyophilized microparticles are known to those skilled in the art, and providing compatible microstructures in accordance with the teachings herein obviously does not require undue experimentation. Lyophilized cakes containing fine cellular structures can be micronized using techniques known to those skilled in the art to provide particles having an average diameter of less than 5 μm or 10 μm. Thus, to the extent that a lyophilization process can be used to provide a microstructure having the desired properties, they are expressly considered to be within the scope of the present invention.
[0147]
In addition to the above techniques, the particulate and perforated microstructures of the present invention also provide that the feed solution (emulsion or aqueous) comprising the wall-forming agent can be rapidly heated under reduced pressure heating oil (eg Perflubron or other high boiling point FCs) Can be formed using the method added to the reservoir of. The water and volatile solvents of the feed solution are rapidly boiled and evaporated. This process, as well as puffed rice or popcorn, can be used to provide a foraminous structure from a wall former. Preferably, the wall former is insoluble in the heating oil. The resulting particles can then be separated from the heated oil using filtering techniques and then dried under reduced pressure.
[0148]
In addition, the particles or perforated microstructures of the present invention can also be formed using a double emulsion method. In the double emulsion method, the drug is first dispersed in the polymer dissolved in an organic solvent (eg methylene chloride) by sonication or homogenization. This first emulsion is then stabilized by forming a multiple emulsion in a continuous aqueous phase comprising an emulsifying agent such as polyvinyl alcohol. The organic solvent is then removed by evaporation or extraction using conventional techniques and equipment. The resulting microparticles are then washed, filtered and dried prior to combining or using them with a suitable suspension medium according to the invention.
[0149]
F. Administration
[0150]
Whatever method is ultimately chosen for the production of the microparticulates, the resulting powders are to be effectively used in powder form or as dispersions containing non-aqueous suspending media. Have a number of advantageous properties. In a particularly preferred embodiment, the bioactive composition will be administered to the surface of the mucous membranes of the respiratory tract (i.e. lung and / or nasal passages), either in dry powder or dispersion form, via inhalation therapy. . Such administration can be performed using MDIs, DPIs, nebulizers, nasal pumps, atomizers, spray bottles, or by direct instillation in the form of drops. However, while inhalation therapy is highly compatible with the present invention, other forms and / or routes of administration will be recognized as useful.
[0151]
In this regard, the powders and stabilized dispersions of the present invention may also be used for local or systemic administration of the compound to any location of the body. Thus, in a preferred embodiment, many including but not limited to topical, intramuscular, transdermal, intradermal, intraperitoneal, nasal, pulmonary, buccal, vaginal, rectal, aural, or to the eye (including, but not limited to) It should be emphasized that the formulation can be administered using different routes of Preferred target sites can be found, for example, in the gastrointestinal tract, urogenital tract or respiratory tract. More generally, the stabilized dispersions of the invention can be used to deliver agents locally, or by administration to any body cavity. In a preferred embodiment, the body cavity is selected from the group consisting of peritoneal cavity, sinus cavity, rectum, urethra, stomach, nasal cavity, vagina, ear canal, oral cavity, buccal capsule and capsule. One skilled in the art will recognize that the chosen route of administration is primarily determined by the choice of bioactive agent and the desired response of the subject.
[0152]
With respect to the delivery of the disclosed powder or stabilized dispersion, another aspect of the invention is directed to a system for administration of one or more bioactive agents, or biologicals, to a patient. As noted above, exemplary inhalation devices compatible with the present invention can include an atomizer, a nasal pump, a nebulizer or spray bottle, a dry powder inhaler, a metered dose inhaler or a nebulizer. In preferred embodiments, these inhalation systems will deliver the bioactive agent as an aerosol to the desired physiological site (eg, the surface of the mucous membrane). Unless otherwise defined by context constraints, for the purposes of the present application, the term "aerosolized" is maintained to mean a gaseous dispersion of fine solid or liquid particles. That is, the aerosol or aerosolized drug can be generated, for example, by a dry powder inhaler, a metered dose inhaler, an atomizer, a spray bottle or a nebulizer. Of course, as described in more detail below, the compositions of the present invention are delivered directly (eg, by conventional injection or needleless injection) or using techniques such as liquid dose instillation be able to. Thus, a further aspect of the present invention is directed to a needleless injector (eg, a pressurized gas gun) comprising the disclosed powder or dispersion.
[0153]
F (i). Dry powder inhaler
[0154]
With regard to inhalation therapy, those skilled in the art will recognize that the powders of the present invention, particularly those comprising perforated microstructures, are particularly useful in DPIs. A conventional DPIs, or dry powder inhaler, delivers a predetermined dose of drug as a fine mist or aerosol of dry powder for inhalation, in powder form, alone or as a blend of lactose carrier particles And the apparatus to be Useful DPI drugs are typically formulated such that they are easily dispersed in individual particles having a size of between 0.5 and 20 μm. The powder is actuated by inhalation or by some external delivery force such as pressurized air. DPI formulations use reservoir systems that can measure multiple doses, typically packaged in a single dose unit or by manual transfer of doses to a device.
[0155]
DPIs are generally classified based on the dose delivery system used. In this regard, the two major types of DPIs include unit dose delivery devices and bulk reservoir delivery systems. As used herein, the term "reservoir" is used in a general sense and is maintained to encompass both configurations unless the context dictates otherwise. In any case, a unit dose delivery system requires a dose of powder formulation to be provided to the device as a single unit. With this system, the formulation is foil-packaged or pre-filled into dosing wells that can be provided with blister strips to prevent water ingress. Other unit dose packages include hard gelatine capsules. Most unit dose containers designed for DPIs are filled using fixed volume technology. As a result, there is a physical limit (here density) to the minimum dose that can be weighed into a unit package defined by powder flow and bulk density.
[0156]
As mentioned above, the powders of the present invention eliminate many of the difficulties associated with prior art carrier formulations. That is, improvements in DPI performance can be obtained by controlling particle size, aerodynamics, morphology and density, humidity, and charge, as disclosed herein. In this regard, the present invention provides a formulation in which the drug, excipient or bulking agent is preferably combined with the perforated microstructure or contains the perforated microstructure. As mentioned above, preferred compositions according to the invention typically have a content of 0.1 g / cm.³ Less than, often less than 0.05 g / cm³ Produces a powder with a bulk density of By providing a powder having a bulk density of the order of magnitude lower than conventional DPI, a much lower dose of selected bioactive agent can be loaded into a unit dose container, or via a reservoir based DPIs It becomes possible to measure. The ability to meter small amounts effectively is important for relatively efficacious bioactive agents such as hormones. In addition, the ability to effectively deliver microparticles without associated carrier particles simplifies product formulation and loading and reduces undesirable side effects.
[0157]
It will be appreciated that the powders of the present invention are particularly effective in delivering relatively high doses of bioactive agent in a single operation. Unlike the prior art, the powdered formulation does not require the use of a bulking agent for effective loading and delivery, and thus can include higher levels of bioactive agent on a weight-to-weight basis. Importantly, the disclosed compositions can be used to deliver as much as about 10 mg of bioactive agent in a single operation. Such advantages would probably be particularly important, for example, in delivering immunomodulators or antibodies for passive immunization that may not be as potent as other compatible agents. Of course, although this discussion is specifically directed to the use of DPIs, this same advantage is equally applicable to dispersions and other forms of administration such as MDIs, nasal pumps and needleless injectors.
[0158]
In addition to the aforementioned advantages, preferred embodiments of the present invention exhibit good aerodynamic properties that make them effective, especially for DPIs. More specifically, the perforated structure of the microparticles and the relatively high surface allow the gas to be more easily and longer distance during inhalation as compared to relatively non-porous particles of comparable size. Allow them to be carried in a stream of Because of their high porosity and low specific gravity, administration of perforated microstructures by DPI is associated with increased particulate deposition at target sites such as mucosal surfaces around the nasal passages and lungs, and correspondingly Give less deposition at. Such particle distribution can be used to increase the deep lung deposition of the administered agent, which is preferred for systemic administration. Furthermore, in a substantial improvement over prior art DPI formulations, the low density, highly porous powders of the present invention preferably eliminate the need for carrier particles.
[0159]
F (ii). Stabilized dispersion
[0160]
It will be appreciated that the powders of the present invention, along with their use in dry powder configurations, can be incorporated in suspension media to provide a stabilized dispersion. Preferably, the stabilization dispersion comprises a non-aqueous dispersion. Among other uses, stabilized dispersions can be delivered to the airways of the patient's lungs using MDIs, atomizers or spray bottles, nasal pumps, needleless injectors, nebulizers or liquid dose instillation (LDI technology). Provides effective delivery of the bioactive agent.
[0161]
Those skilled in the art will appreciate that the disclosed dispersion or enhanced stability of the dispersion is primarily due to the reduction of van der Waals attraction between suspended particles, and between the suspending medium and the particles in density. It will be appreciated that this is achieved by reducing the differences. In accordance with the teachings herein, increased suspension stability can be imparted by designing a porous microstructure that is then dispersed in a compatible suspension medium. As mentioned above, perforated microstructures include pores, voids, hollows, defects, or other interstitial spaces that allow fluid suspension media to freely penetrate or perfuse particulate boundaries. A particularly preferred embodiment comprises a substantially honeycomb or foam-like porous microstructure in appearance that is both hollow and porous. In a particularly preferred embodiment, the perforated microstructure comprises hollow, porous, spray-dried microspheres. Of course, in other embodiments involving relatively non-porous, solid particulates, enhanced stability can be imparted through the selection of particulate components (eg, surfactants).
[0162]
The porous medium can be infiltrated into the particles, where the porous microstructure is disposed in the suspension medium (ie, hydrofluoroalkane propellant or liquid fluorocarbon), so that the continuous and dispersed phases can be discerned. Not make homodispersity. Particle aggregation (flocculation) as drawn or “virtual” particles (ie containing a volume circumscribed by a microparticulate matrix) are made almost entirely in the medium in which they are suspended The power to drive) is minimized. In addition, the difference in density between fractionated particles and the continuous phase is minimized by packing the microstructure with media, thereby effectively slowing particle creaming or centrifugation. Thus, the microparticles and stabilized dispersions of the present invention are particularly compatible with many aerosolization techniques (eg, MDI, atomization via spray bottle, nasal pump, nebulization, etc.) Furthermore, stabilized disper. John is compatible with other routes of administration, including (but not limited to) liquid dose instillation, needleless injection, conventional infusion and topical application.
[0163]
Unlike the prior art compositions, the preferred dispersions of the present invention are designed to not increase the repulsion between the particles, but rather to reduce the attraction between the particles. In this regard, it should be recognized that the primary advantage of driving flocculation in non-aqueous media is van der Waals attraction. As mentioned above, the VDW force is a quantum mechanical origin and can be visualized as attraction between fluctuating dipoles (i.e. induced dipole-induced dipole interactions). Dispersion forces are very short distances and scale as the sixth power of the distance between atoms. When two macroscopic objects approach one another, the dispersion attraction between atoms is summed up. The resulting force is dependent on the geometry of the interacting objects at a much greater distance.
[0164]
More specifically, for two spherical particles, the magnitude of the VDW potential (VA) is:
[0165]
[Equation 2]

(In the formula, A_effIs an effective Hamaker constant that describes the nature of the particles and media, H₀ Is the distance between particles, R¹And R²Is the radius of the spherical particles 1 and 2)
It can be approximated by The effective Hamaker constant is proportional to the difference in polarizability of the dispersed particles and the suspending medium:
[Equation 3]

(In the formula, A_SMAnd A_PARTAre the Hamaker constants for the suspending medium and the particles, respectively). When the suspended particles and the dispersion medium are essentially similar,_SMAnd A_PARTBecome closer in size, A_effAnd V_A Is smaller. That is, by reducing the difference between the Hammer constant associated with the suspending medium and the Hamman constant associated with the dispersed particles, the effective Hamser constant (and corresponding van der Waals attraction) can be reduced.
[0166]
One way to minimize the differences in the Hammer constant is to create a "homodispersion" in which both the continuous and the dispersed phase are essentially indistinguishable, as described above. Besides utilizing the morphology of the particles to reduce the effective Hamerzer constant, the components of the structural matrix (which define the perforated microstructure) are preferably such that they exhibit Hamerzer constants relatively close to that of the suspending medium. Will be chosen. In this regard, it is possible to use the actual values of the Hamer's constant of the suspending medium and of the particulate component to determine the compatibility of the dispersion component and to give a good indication as to the stability of the formulation. Instead, a relatively compatible particulate or perforated microstructure component, and a suspending medium, with characteristic physical values that are more indicative of the measurable Hamerman's constant but are more easily identifiable. And you will be able to choose.
[0167]
In this regard, it has been found that the refractive index values of many compounds tend to scale with the corresponding Hammer constant. Thus, the refractive index value of the substantial, easily, is that the combination of the suspending medium and the particle excipient will give a dispersion having a relatively low effective Hamermant's constant, and the stability associated therewith. It can be used to give a fairly good indication of wax. It will be appreciated that the use of such values allows for the formation of a stabilized dispersion according to the invention without undue experimentation, as the refractive index of the compound is widely available or easily derivable. I will. For purposes of illustration only, the indices of refraction of some compounds compatible with the disclosed dispersion are given in Table I immediately below:
[0168]
[Table 1]

[0169]
Consistent with the compatible dispersion components described above, one of ordinary skill in the art will recognize that the formation of dispersions in which the components have refractive index differences less than about 0.5 is preferred. That is. The refractive index of the suspending medium is preferably in the range of about 0.5 of the refractive index associated with the particle or perforated microstructure. It will further be appreciated that the refractive index of the suspending medium and the particles can be measured directly or can be approximated using the refractive index of the main component in the respective respective phase. For particulate or perforated microstructures, the major components can be determined on a weight percent basis. For the suspending medium, the main components are typically derived on a volume percent basis. In selected embodiments of the invention, the value of the refractive index difference will preferably be less than about 0.45, about 0.4, about 0.35 or even less than about 0.3. A particularly preferred embodiment is less than about 0.28, about 0.25, about 0.2, about 0.15 or less than about 0.28, provided that low index differences imply higher dispersion stability. Includes exponential difference. One skilled in the art can, given the present disclosure, be able to determine which excipients are particularly compatible without undue experimentation. The final choice of preferred excipients will also be influenced by other factors including biocompatibility, regulatory context, ease of manufacture, cost.
[0170]
As mentioned above, the minimization of the density difference between the particles and the continuous phase can be achieved by using perforated and / or hollow microstructures such that the suspending medium constitutes the majority of the particle volume it can. As used herein, the term "particle volume" refers to the volume of suspension medium that will be replaced by the incorporated hollow / porous particles if they are solid (ie, the volume defined by the particle boundaries). Corresponds to For purposes of illustration and as noted above, the volume of these fluid-filled particulates can be referred to as "virtual particulates." Preferably, the average volume of the bioactive agent / excipient shell or matrix (ie, the volume of medium actually displaced by the perforated microstructure) is less than 80% of the average particle volume (or less than 80% of the virtual microparticles) )including. More preferably, the volume of the microparticulate matrix comprises less than about 50%, 40%, 30% or even 20% of the average particle volume. More preferably, the average volume of the shell / matrix comprises less than about 10%, 5%, 3% or 1% of the average particle volume. One skilled in the art will recognize that such matrix or shell volumes typically contribute little to the virtual particle density predominantly defined by the suspending medium found therein.
[0171]
The use of such microstructures will be further recognized as the apparent density of virtual particles approximates that of a suspending medium that is substantially eliminating attractive van der Waals forces. As further mentioned above, it is preferred to choose the components of the microparticulate matrix to approximate as much as possible other given considerations in order to approximate the density of the suspending medium. Thus, in a preferred embodiment of the invention, the virtual particles and the suspending medium are about 0.6 g / cm³ It will have a density difference of less than. That is, the average density of the virtual particles (as defined by the matrix boundaries) is about 0.6 g / cm of the suspending medium³ In the range of More preferably, the average density of virtual particles is 0.5, 0.4.0.3 or 0.2 g / cm of the chosen suspension medium³ Would be within the scope of In more preferred embodiments, the density difference is less than about 0.1, 0.05. 0.01, or 0.005 g / cm.³ It will be in the range below.
[0172]
In addition to the above advantages, the use of the disclosed microparticles results in the formation of a dispersion comprising a much higher volume particle fraction in suspension. It should be appreciated that formulation of prior art dispersions at volume fractions that approach close packing generally results in a dramatic increase in dispersion visco-elastic behavior. This type of rheological behavior is not suitable for MDI or nebulizer applications. One skilled in the art will recognize that the volume fraction of particles can be defined as the ratio of the apparent volume of the particles (ie, the particle volume) to the total volume of the system. Each system has a maximum volume fraction or fill fraction. For example, particles in a single cubic configuration reach a maximum fill fraction of 0.52, while those in a face-centered cubic / hexagonal close-packed configuration reach a maximum fill fraction of about 0.74 Do. For non-spherical particles or polydispersed systems, the derived values are different. Therefore, the maximum filling fraction is often considered to be an empirical parameter for a given system.
[0173]
It has now surprisingly been found that the preferred microparticles of the invention do not exhibit undesirable visco-elastic behavior even at high volume fractions approaching close packing. Conversely, they remain free flowing, low viscosity suspensions with little or no yield stress as compared to similar suspensions containing solid particles. The low viscosity of the disclosed suspensions appears to be at least in large part due to the relatively low van der Waals attraction between the fluid-filled hollow, porous particles. Thus, in selected embodiments, the volume fraction of the disclosed dispersion is greater than about 0.3. Other embodiments may have packing values on the order of 0.3 to 0.8, or on the order of 0.5 to about 0.8, with higher values approaching close packing conditions. Furthermore, the formation of a relatively concentrated, dense dispersion can further increase the stability of the formulation, as particle sedimentation tends to decrease as the volume fraction approaches close packing.
[0174]
Although the methods and compositions of the present invention can be used to form relatively concentrated suspensions, the stabilizing agent works equally well at much lower packing volumes and such dispersions Is considered to be within the scope of the present disclosure. In this regard, it will be appreciated that dispersions containing low volume fractions are extremely difficult to stabilize using prior art techniques. Conversely, dispersions incorporating microparticles containing bioactive agents, as described herein, are particularly stable even at low volume fractions. Thus, the invention forms a stabilized dispersion, in particular a volume fraction of less than 0.3, which gives the dispersion for the breath to be used. In some preferred embodiments, the volume fraction is about 0.0001 to 0.3, more preferably about 0.001 to 0.01. Still other preferred embodiments include stabilized dispersions having a volume fraction of from about 0.01 to about 0.1.
[0175]
The perforated microstructures of the present invention can also be used to stabilize dilute dispersions of micronized bioactive agents. In such embodiments, porous microstructures can be added to increase the volume fraction of particles in the suspension, thereby increasing the suspension stability to creaming or sedimentation. Furthermore, in these embodiments, the incorporated microstructures can also act to prevent the close approach (aggregation) of the micronized drug particles. It should be appreciated that the perforated microstructure incorporated in such an embodiment may not necessarily include the bioactive agent. Rather, they can be formed solely with various excipients, including surfactants.
[0176]
One skilled in the art will further appreciate that the stabilized suspension or dispersion of the present invention can be prepared by dispersion of the microstructure in a chosen suspension medium which can then be placed in a container or reservoir. In this regard, the stabilized formulations of the present invention can be made by simply mixing the ingredients in sufficient amounts to produce the final desired dispersion concentration. The microstructures are easily dispersed without mechanical energy, but the application of mechanical energy to facilitate dispersion (eg, using sonication) is also contemplated. Alternatively, the components can be mixed by simple shaking or other types of agitation. The process is preferably carried out under anhydrous conditions to eliminate the adverse effects of water on suspension stability. Once formed, the dispersion has a reduced sensitivity to flocculation or sedimentation.
[0177]
As indicated throughout the specification, the dispersions of the invention are preferably stabilized. Broadly, the term "stabilized dispersion" means any dispersion that is resistant to aggregation, flocculation or creaming to the extent required to provide effective delivery of the bioactive agent. To be maintained. Furthermore, it is an important advantage of the present invention that the disclosed dispersions and powders are stable at room temperature and do not require refrigeration or freezing to effectively maintain their activity. Besides extending shelf life, notable temperature stability significantly simplifies shipping and dosing.
[0178]
One skilled in the art will recognize that there are several methods that can be used to assess the stability of a given dispersion, but the preferred method for the purposes of the present invention is dynamic light precipitation. Including determination of creaming or settling time using A preferred method involves subjecting the suspended particles to centrifugal force and measuring the absorbance of the suspension as a function of time. The rapid decrease in absorbance confirms the suspension with poor stability. It can be said that the person skilled in the art can adapt the procedure to a particular dispersion without undue experimentation.
[0179]
For the purposes of the present invention, creaming time is defined as the time for the suspended drug microparticles to be creamed to half the volume of the suspension medium. Similarly, settling time can be defined as the time required for the microparticles to settle to half the volume of the liquid medium. In addition to the light precipitation techniques described above, a relatively simple method of determining the creaming time of a formulation is to provide a particulate dispersion in a sealed glass vial. The vials are agitated or shaken to give a relatively uniform dispersion, which is then placed aside and observed using a suitable instrument or by visual inspection. The suspended particles either cream to half of the volume of the suspension medium (ie rise to the upper half of the suspension medium) or settle to half of the volume (ie half of the medium) The time required to settle to the bottom is then recorded. Dispersion formulations with creaming times longer than 1 minute are preferred and exhibit adequate stability. More preferably, the stabilized dispersion comprises a creaming time longer than 1, 2, 5, 10, 15, 20 or 30 minutes. In a particularly preferred embodiment, the stabilized dispersion exhibits a creaming time of greater than about 1, 1.5, 2, 2.5 or 3 hours. Substantially equal time periods for the settling time indicate compatible dispersions.
[0180]
It will be understood that other ingredients can be included in the stabilized dispersion of the present invention. For example, osmotic agents, stabilizers, chelating agents, buffers, viscosity modifiers, salts and sugars can be added to the stabilized dispersion to fine-tune maximum lifetime and ease of administration. Such components can be added directly to the suspension medium or can be combined with or incorporated into the perforated microstructure. Considerations such as sterility, isotonicity and biocompatibility may govern the use of conventional additives in the disclosed compositions. The use of such agents will be understood to those skilled in the art, and specific amounts, ratios and types of agents can be determined empirically without undue experimentation.
[0181]
F (iii). Metered dose inhaler
[0182]
As mentioned above, the stabilized dispersion may be administered to the airways of the patient's nose or lung, preferably via aerosol application, such as a dose metered inhaler. The use of such a stabilizing formulation provides excellent dose reproducibility, and improved deposition at the target site as described above. MDIs are well known in the art and could easily be used for the administration of the claimed dispersion without undue experimentation. Respiratory activated MDIs, as well as those containing other types of improvements that may already be developed or developed in the future, are also compatible with the stabilized dispersion and the present invention and are considered within their scope.
[0183]
MDI canisters are generally of a plastic or plastic-coated glass bottle, or preferably of a propellant such as an optionally anodised, lacquer-coated and / or plastic-coated metal (eg aluminum) A container or reservoir capable of withstanding vapor pressure, the container being closed with a metering valve. The metering valve is designed to deliver a metered amount of prescription per actuation. The valve incorporates a gasket to prevent propellant leak through the valve. The gasket can include any suitable elastomeric material such as, for example, low density polyethylene, chlorobutyl, white and black butadiene-acrylonitrile rubbers, butyl rubber, and neoprene. Suitable valves include, for example, Valois of France (e.g. DFIO, DF30, DF31 / 50ACT, DF60), Bespak plc (LTK) (e.g. BK300, BK356), and 3M-neotechnic LTK (e.g. Are commercially available from manufacturers well known in the aerosol industry, such as, for example, Spraymiser.
[0184]
Individually filled canisters are conveniently adapted channeling devices or actuators prior to use to form a dose-metering inhaler for administration of the drug to the patient's lungs or cavity in the mouth or nose. It is put in. Suitable channeling devices can thereby deliver the drug, for example, from the filled canister to the patient's nose or mouth (eg, mouthpiece actuator) via a metering valve, such as valve actuators and cylinders or Includes conical shaped paths. Metered dose inhalers are designed to deliver a fixed unit dose per actuation, such as a range of 10 to 5000 micrograms per actuation, such as a bioactive agent. Typically, single-packed canisters give dozens or hundreds of shots or doses.
[0185]
It is an advantage of the present invention that, for MDIs, any biocompatible suspending medium having a suitable vapor pressure to act as a propellant can be used. Particularly preferred suspending media are compatible for use with dose-metering inhalers. That is, they will be able to form an aerosol upon actuation of the metering valve and release of the associated pressure. In general, the chosen suspending medium should be biocompatible (i.e. relatively non-toxic) and non-reactive with respect to the suspended porous microstructure containing the bioactive agent. Preferably, the suspending medium does not act as a substantial solvent for any component incorporated into the porous microspheres. Selected embodiments of the present invention include suspending media selected from the group consisting of fluorocarbons (including those substituted with other halogens), hydrofluoroalkanes, perfluorocarbons, hydrocarbons, alcohols, ethers or combinations thereof. Including. It will be appreciated that the suspending medium can comprise a mixture of various compounds selected to be given specific properties.
[0186]
Particularly suitable propellants for use in the MDI suspension media of the present invention are liquefiable under pressure at room temperature, safe for inhalation or topical use, toxicologically harmless, and have no side effects. Agent gas. In this regard, compatible propellants can be any hydrocarbon, fluorocarbon, hydrogen-containing fluorocarbon or fluorocarbon having a vapor pressure sufficient to efficiently form an aerosol upon operation of the dose metering inhaler. A mixture of them can be included. Their propellants, typically called hydrofluoroalkanes or HFAs, are particularly compatible. For example, suitable propellants are short chain hydrocarbons, CH.₂ClF, CCl₂F₂CHClF, CF₃CHClF, CHF₂CClF₂, CHClFCHF₂, CF₃CH₂Cl and CClF₂CH₃Etc C₁~ C₄Hydrogen-containing chlorofluorocarbons; CHF₂CHF₂, CF₃CH₂F, CHF₂CH₃ And CF₃CHFCF₃Etc C₁~ C₄Hydrogen-containing fluorocarbons (eg HFAs); and CF₃CF₃And CF₃CF₂CF₃ And other perfluorocarbons. Preferably, a single perfluorocarbon or hydrogen-containing fluorocarbon is used as the propellant. Particularly preferred as a propellant is 1,1,1,2-tetrafluoroethane (CH 2₃CH₂F) (HFA-134a), and 1,1,1,2,3,3,3-heptafluoro-n-propane (CF₂CHFCF₃(HFA-227), perfluoroethane, monochlorodifluoromethane, 1,1-difluoroethane, and combinations thereof. Preferably, the formulation does not contain components that deplete the stratosphere ozone. CCl₃F, CC₁₂F₂And CF₃CCl₃It is particularly preferable to be substantially free of chlorofluorocarbons such as
[0187]
Preferred embodiments of the present invention include ecologically friendly suspension media, but conventional chlorofluorocarbons and substituted fluorine compounds can also be used as suspension media in accordance with the teachings herein. Although there may be environmental concerns in this regard, FC-11 (CCl₃F), FC-11 B1 (CBrCl₂F), FC-1 162 (CBr₂ClF), FC 12 B 2 (CF₂Br₂), FC 21 (CHCl 3)₂F), FC-21 B1 (CHBrClF), FC-21 B2 (CHBr₂F), FC-31B1 (CH₂BrF), FC 113A (CCl)₃CF₃), FC-122 (CClF)₂CHCl₂), FC-123 (CF₃CHCl₂), FC-132 (CHClF CHClF), FC-133 (CHClFCHF)₂), FC-141 (CH₂ClCHClF), FC-141B (CCl)₂FCH₃), FC-142 (CHF₂CH₂Cl), FC-151 (CH₂FCH₂Cl), FC-152 (CH₂FCH₂F), FC-1112 (CClF = CClF), FG-1121 (CHCl = CFCl) and FC1131 (CHCl = CHF) are all compatible with the teachings herein. Thus, each of these compounds, alone or in combination with other compounds (ie less volatile fluorocarbons) can be used to stabilize the respirable dispersion according to the invention.
[0188]
In addition to the above embodiments, the stabilized dispersions of the present invention can also be used with a nebulizer to provide an aerosolized drug that can be administered to the airways of the patient's lungs as needed. Nebulizers are well known in the art and could easily be used for administration of the claimed dispersion without undue experimentation. Respiratory activation nebulizers, as well as those including other types of modifications already developed or will be developed, are compatible with the stabilized dispersion and are considered within the scope of the present invention.
[0189]
The nebulizer is powered by an aerosol that converts bulk liquid into small droplets suspended in a breathable gas. Here, the aerosolized drug to be administered (preferably to the airways of the lung) comprises small droplets of suspending medium combined with a perforated microstructure comprising a bioactive agent. In such embodiments, the stabilized dispersions of the present invention are typically disposed in a fluid reservoir operably coupled to the nebulizer. The particular volume of formulation given, the means of filling the reservoir, and the like depend primarily on the choice of the individual nebulizer and is well within the skill of the art. Of course, the present invention is completely compatible with single dose nebulizers and multiple dose nebulizers.
[0190]
The present invention overcomes these and other difficulties by providing a stabilized dispersion, preferably with a suspending medium comprising a fluorochemical (ie fluorochemical, fluorocarbon or perfluorocarbon). A particularly preferred embodiment of the invention comprises fluorochemicals which are liquids at room temperature. As mentioned above, the use of such compounds, either as a continuous phase or as a suspending medium, offers several advantages over prior art liquid inhalation formulations. In this regard, it is well established that many fluorochemicals have a proven history of safety and biocompatibility in the lung. Furthermore, in contrast to aqueous solutions, fluorochemicals do not negatively impact gas exchange following pulmonary administration. Conversely, their ability to actually improve gas exchange, and their unique wettability properties, allow aerosolized streams of particles to be carried deep into the lungs, thereby providing systemic delivery of the desired drug compounds. Improve. In addition, the relatively non-reactive nature of the fluorochemical acts to retard any degradation (by proteolysis or hydrolysis) of the incorporated bioactive agent.
[0191]
In any event, nebulizer-mediated aerosol application typically provides increased delivery surface area of droplets, and in some cases delivery of micronized or aerosolized drug, , Require energy input. One common method of aerosol application is to push the fluid stream out of the nozzle and release it, thereby forming droplets. For nebulized administration, additional energy is generally applied to the droplets so that they are small enough to be deeply transported into the lungs. Thus, additional energy is required, such as provided by a high velocity gas flow or a piezoelectric crystal. Two popular types of nebulizers, jet nebulizers and ultrasonic nebulizers, rely on the aforementioned method of applying additional energy to the fluid during atomization.
[0192]
Recent work has focused on the use of mobile hand-held ultrasound nebulizers, also referred to as metered solutions, for pulmonary delivery of bioactive agents to the systemic circulation via nebulization. These devices, known as single bolus nebulizers, aerosolize single boluses of a drug in aqueous solution having an efficient particle size for deep pulmonary delivery in one or two breaths. These instruments fall into three broad categories. The first category includes pure piezoelectric single volas nebulizers such as those described by Muetterlein et al. (J. Aerosol Med. 1988; 1: 231). In another category, the desired aerosol cloud can be generated by a microchannel extrusion single bolus nebulizer, such as that described in US Pat. No. 3,812,854. Finally, the third category includes the device exemplified by Robertson et al. (WO 92/11050) which describes a circular pressurized single-bolus nebulizer. Each of the above references is incorporated herein in its entirety. While most instruments are manually actuated, some of the instruments present are breath activated. When the device senses breathing through the patient's circuit, releasing the aerosol moves the respiratory actuated device. A breath actuated nebulizer may be placed inline with the ventilator circuit for releasing the aerosol into the air stream, including the inhaled gas for the patient.
[0193]
It is an advantage of the present invention that regardless of what type of nebulizer is used, or that biocompatible non-aqueous compounds can be used as the suspending medium. Preferably, they are able to form aerosols by the application of energy thereto. In general, the chosen suspension medium should be biocompatible (i.e. relatively non-toxic) and non-reactive to the suspended porous microstructure containing the bioactive agent. Should be. A preferred embodiment is that the suspending medium is from the group consisting of fluorochemicals, fluorocarbons (including those substituted with other halogens), perfluorocarbons, fluorocarbons / hydrocarbon diblocks, hydrocarbons, alcohols, ethers or combinations thereof. Contains the chosen suspending medium. It will be appreciated that the suspending medium can comprise a mixture of various compounds chosen to impart particular properties.
[0194]
In accordance with the teachings herein, the suspending medium can comprise any one of many different compounds including hydrocarbons, fluorocarbons or hydrocarbon / fluorocarbon diblocks. In general, contemplated hydrocarbons or highly fluorinated perfluorinated compounds can be linear or branched or cyclic, saturated or unsaturated compounds. Conventional structural derivatives of these fluorochemicals and hydrocarbons are also considered to be within the scope of the present invention. Selected embodiments involving these fully or partially fluorinated compounds can include one or more heteroatoms and / or atoms of bromine or chlorine. Preferably, these fluorochemicals contain, but are not limited to, 2 to 16 carbon atoms, linear, cyclic or polycyclic perfluoroalkanes, bis (perfluoroalkyl) alkenes, perfluoroethers, Perfluoroamines, perfluoroalkyl bromides and perfluoroalkyl chlorides (eg dichlorooctane). Particularly preferred fluorine compounds used for the suspension medium are perfluorooctyl bromide C₈F₁₇Br (PFOB or Perflubron), dichlorofluorooctane C₈F₁₆Cl₂And hydrofluoroalkanes, perfluorooctylethane C₈F₁₇C₂H₅(PFOE) can be included. For other embodiments, the use of perfluorohexane or perfluoropentane as a suspending medium is particularly preferred.
[0195]
More generally, exemplary fluorochemicals contemplated for use in the present invention are halogenated fluorochemicals (ie, C_nH_{2n + 1}X, XC_nF_2nX, wherein n = 2-10, X = Br, Cl or I), and in particular 1-bromo-F-butane, nC₄F₉Br, 1-bromo-F-hexane (n-C)₆F₁₃Br), 1-bromo-F-heptane (n-C)₇F₁₅Br), 1,4-dibromo-F-butane and 1,6-dibromo-F-hexane. Other useful brominated fluorochemicals are disclosed in US Pat. No. 3,975,512 to Long, which is incorporated herein by reference. Perfluorooctyl chloride (n-C₈F₁₇Cl), 1, 8-dichloro-F-octane (n-ClC)₈H₁₆Cl), 1,6-dichloro-F-hexane (n-ClC)₆F₁₂Cl), and 1,4-dichloro-F-butane (n-ClC)₄F8C₁Particular fluorochemicals having a chloride substituent, such as) are also preferred.
[0196]
Halogenated fluorochemicals, fluorocarbons, fluorocarbon-hydrocarbon compounds are also suitable for use as the suspending medium in the present invention, including esters, thioethers and other linkages such as amines. For example, the general formula C_nF_{2n + 1}OC_mF_{2m + 1}Or C_nF_{2n + 1}CH = CHC_mF_{2m + 1}, Wherein n and m are the same or different, and n and m are integers of about 2 to about 12 (eg, C₄F₉CH = CHC₄F₉(F-44E), i-C₃F₉CH = CHC₆F13 (F-i36E), and C₆F13 CH = CHC₆F13 (as F-66E) is compatible with the teachings herein. Useful fluorochemical hydrocarbon diblock and triblock compounds have the general formula C_nF_{2n + 1}-C_mF_{2m + 1}Or C_nF_{2n + 1}-C_mF_2m-1(Wherein, n = 2 to 12; m = 2 to 16), or C_pF_{2p + 1}-C_nF_2n-C_mF_{2m + 1} (Wherein, p = 2 to 12; m = 1 to 12; n = 2 to 12). Preferred compounds of this type are C₈F₁₇C₂H₅, C₆F₁₃C₁₀H₂₁, C₈F₁₇C₈H₁₇, C₆F₁₃CH = CHC₆H₁₃, And C₈F₁₇CH = CHC₁0H₂Including one. Substituted ether or polyether (ie XC_nF_2nOC_mF_2mX, XCFOC_nF_2nOCF₂X (where n and m = 1 to 4, X = Br, Cl or I), and fluorochemical-hydrocarbon ether diblock or triblock (ie C_nF_{2n + 1}-OC_mF_{2m + 1}H (in the formula, n = 2 to 10; m = 2 to 16), C_pF_{2p + 1}-OC_mF_2m-OC_pF_{2p + 1} (Wherein, p = 2 to 12; m = 1 to 12; n = 2 to 12)_nF_{2n + 1}-OC_nF_2n-OC_mF_{2m + 1} (Wherein n, m and p are 1 to 12) can be used in the same manner. Furthermore, depending on the application, the perfluoroalkylated ether or polyether can be compatible with the claimed dispersion.
[0197]
C₁₀F₁₈And polycyclic fluorocyclic chemicals such as F-decalin or perfluorodecalin, perfluoroperhydrophenanthrene, perfluorotetramethylcyclohexane (AP-144) and perfluoro-butyldecalin are also within the scope of the present invention. is there. Additional useful fluorochemicals include F-tripropylamine ("FTPA") and F-tributylamine ("FTBA"), F-4-methyloctahydroquinolizine ("FMOQ"), FN-methyl Decahydroisoquinoline ("FMIQ"), FN-methyl decahydroquinoline ("FHQ"), FN-cyclohexyl pyrrolidine ("FCHP") and F-2-butyltetrahydrofuran ("FC-75" or "FC And perfluorinated amines such as -77 "). Still other useful fluorine compounds include perfluorophenanthrene, perfluoromethyldecalin, perfluorodimethylethylcyclohexane, perfluorodimethyldecalin, perfluorodiethyldecalin, perfluoromethyladamantane, perfluorodimethyladamantane. Other contemplated fluorochemicals with non-fluorinated substituents, such as perfluorooctyl hydride, and similar compounds with different numbers of carbon atoms are also useful. One of ordinary skill in the art will further appreciate that various other modified fluorochemicals are included within the broad definition of fluorochemicals as used in this application and are suitable for use in the present invention. Thus, each of the foregoing compounds can be used alone or in combination with other compounds to form the stabilized dispersions of the present invention.
[0198]
Additional illustrative fluorocarbon or fluorine compound classes that may be useful as suspending media include (but are not limited to) fluoroheptane, fluorocycloheptane, fluoromethylcycloheptane, fluorohexane, fluorocyclohexane, fluoro Includes pentane, fluorocyclopentane, fluoromethylcyclopentane, fluorodimethylcyclopentane, fluoromethylcyclobutane, fluorodimethylcyclobutane, fluorotrimethylcyclobutane, fluorobutane, fluorocyclobutane, fluoropropane, fluoroether, fluoropolyether, and fluorotriethylamine . Such compounds are generally environmentally sound and biologically non-reactive.
[0199]
Any liquid capable of producing an aerosol upon application of energy may also be used with the nebulizer, the chosen suspending medium being less than about 506,625 Pa (5 atm), more preferably about 202,650 Pa (2 atm) It will preferably have a vapor pressure below. Unless stated otherwise, all vapor pressures described herein are measured at 25 ° C. In other embodiments, preferred suspending medium compounds have vapor pressures on the order of about 5 Torr to about 760 Torr, with more preferred compounds having a vapor pressure of about 1067 Pa to about 79993 Pa (about 8 Torr). Particularly preferred compounds have a vapor pressure on the order of about 10 Torr to about 350 Torr. Such suspension media can be used with a compressed air nebulizer, an ultrasonic nebulizer or a mechanical atomizer to provide effective ventilation therapy. In order to provide a suspending medium with selected specific physical properties that further enhance the stability or bioavailability of the dispersed bioactive agent, the more volatile compounds can be converted to components of lower vapor pressure It can be mixed.
[0200]
Another aspect of the invention directed to a nebulizer comprises a suspension medium boiling at a selected temperature under ambient conditions (i.e. 1 atmosphere). For example, preferred embodiments will include suspension media compounds boiling above 0 ° C., above 5 ° C., above 10 ° C., above 15 ° C., or above 20 ° C. In other embodiments, the suspending medium compound can boil above 25 ° C. or above 30 ° C. Furthermore, in other embodiments, the selected suspension vehicle compound boils above 45 ° C., 55 ° C., 65 ° C., 75 ° C., 85 ° C., or 100 ° C. above human body temperature (ie, 37 ° C.). Can.
[0201]
F (v). Direct administration
[0202]
It will be appreciated that the stabilized dispersions of the present invention, along with MDI and nebulizers, can be used to administer bioactive agents to different target sites using different routes. For example, the disclosed compositions can be delivered directly to the lung with liquid dose instillation (LDI) technology. Instead, the stabilized dispersion could be effectively delivered to the mucosal surface in the nasal passage using a nasal pump, spray bottle or atomizer. In still other embodiments, the disclosed dispersion can be administered (eg, intramuscularly or intradermally) to the target site using conventional injection or by a needleless injector using compressed gas The The latter is particularly preferred in the case of needleless inoculation. Still other embodiments are directed to the topical delivery of dispersions to the target site of the mucosal surface such as the eye or ear, or more preferably those in the urogenital or gastrointestinal tract. Such techniques can further use iontophoresis to enhance permeation of incorporated bioactive agents. In any case, the stabilized dispersion provides excellent dose repeatability while preserving the activity of the incorporated agent.
[0203]
One skilled in the art will recognize that suspension media compatible with the above described delivery techniques are similar to those described above for use with the nebulizer. That is, the stabilized dispersion is selected from the group consisting of fluorochemicals, fluorocarbons (including those substituted with other halogens), perfluorocarbons, fluorocarbons / hydrocarbon diblocks, hydrocarbons, alcohols, ethers or combinations thereof. Preferably, the suspension medium is Particularly preferably, for the purposes of the present application, suspension media compatible with such delivery techniques are equivalent to those listed in the receiver with use in the nebuliser. In a particularly preferred embodiment, the chosen suspending medium comprises the fluorochemical which is liquid under ambient conditions.
[0204]
Furthermore, it should be appreciated that liquid dose instillation involves direct administration of the stabilized dispersion to the lungs. In this regard, direct pulmonary administration of biologically active compounds is particularly effective in the treatment of diseases, particularly where poor vascular circulation of the diseased portion of the lung reduces the effectiveness of intravenous drug delivery. For LDI, the stabilized dispersion is preferably used with partial liquid ventilation or conventional liquid ventilation. In addition, the present invention introduces a therapeutically beneficial amount of a physiologically acceptable gas (eg, nitric oxide or oxygen) into the drug microparticle dispersion prior to, during, or after administration. Can be further included.
[0205]
For LDI, the dispersions of the present invention can be administered to the lungs using a pulmonary delivery conduit. Those skilled in the art, as used herein, will be interpreted broadly to include any device or device or portion thereof that provides for instillation or administration of fluid into the lung, as the term "pulmonary delivery conduit" Will recognize. In this regard, the pulmonary delivery conduit or delivery conduit may be any bore, lumen, catheter, tube, or the like that provides for administration or instillation of the disclosed dispersion to at least part of the lung airways of the patient in need thereof. Maintained to mean a conduit, syringe, actuator, mouthpiece, endotracheal tube or bronchoscope. It will be appreciated that the delivery conduit may or may not be accompanied by a liquid or gas ventilator. In a particularly preferred embodiment, the delivery conduit comprises an endotracheal tube or bronchoscope.
[0206]
While the stabilized dispersions of the present invention can be administered to the functional residual capacity of the patient's lungs, the chosen embodiment has a much smaller volume (eg, on the order of 1 milliliter or less) It will be appreciated to include pulmonary administration. For example, depending on the disease to be treated, the volume administered can be on the order of 1, 3, 5, 10, 20, 50, 100, 200 or 500 milliliters. In preferred embodiments, the volume of liquid is less than 0.25 or 0.5 percent FRC. For particularly preferred embodiments, the volume of liquid is less than or equal to 0.1 percent FRC. It will be appreciated that the wettability and spread properties of the suspending medium (especially the fluorochemicals) facilitate uniform distribution of the active agent in the lung, for administration of relatively small volumes of the stabilized dispersion. However, in other embodiments, it may be preferable to administer a volume of dispersion greater than 0.5, greater than 0.75 or 0.9 percent FRC. Of course, the extraordinary wetting and spreading properties associated with at least some fluorochemicals make them particularly compatible for administration to the surface of other mucous membranes such as the nasal passages.
[0207]
With regard to the powders and the stabilization and dispersion disclosed herein, one skilled in the art can advantageously supply them to a physician or other health care professional in the form of a sterile, prepackaged kit. Will recognize. More specifically, the formulation can be supplied as a stable powder that can be administered immediately to the patient, or as a preformed dispersion. Conversely, they can also be provided as separate, readily mixable components. When provided in a ready-to-use form, the powder or dispersion can be packaged in a single-use container or reservoir, as well as in a multi-use container or reservoir. In any case, the container or reservoir can be combined with and used with the chosen inhalation or administration device as described herein. When provided as individual components (e.g. as powdery microspheres and as a neat suspension medium), it is then always by simply combining the contents in the container as directed prior to use Can form a stabilized formulation. In addition, such a kit can include many pre-packaged dosing units ready for mixing so that the user can then administer them as needed.
[0208]
G. Example
[0209]
The above description will be more fully understood by reference to the following examples. Such examples, however, are merely representative of preferred methods of practicing the present invention and should not be read or interpreted as limiting the scope of the present invention in any manner.
[0210]
I. Production of hollow porous particles of HA peptide by spray drying
[0211]
Suction: 100%, inlet temperature: 85 ° C, outlet temperature: 51 ° C; feed pump: 10%: N₂ Hollow porous HA 110-120 peptide (amino acid residue of hemagglutinin of influenza virus particles by spray drying technology using a B-191 mini spray drier (Buechi, Flavil, Switzerland) under conditions of flow: 800 L / hr Groups 110-120) Particles ("PulmoSheres") were prepared. The feed was made by mixing the two formulations (A and B) just prior to spray drying. A 150 mesh stainless steel screen was placed at the cyclone outlet port to facilitate particle collection.
[0212]
Formulation A: 5 g deionized water to dissolve 18 mg of the HA 110-120 peptide (Chiron, Emeryville, CA) and 1 mg of hydroxyethyl starch (Ajinomoto, Japan) Was used.
[0213]
Formulation B: A phospholipid-stabilized fluorocarbon-in-water emulsion was prepared as follows. The Phospholipid (0.3 g EPC-100-3 (Lipoid KG, Ludwigshafen, Germany), Ultra-Turax Mixer (T-25 type) in 33 g hot deionized water (T = 50-60 ° C.) Was homogenized at 8000 rpm for 2 to 5 minutes (T = 60-70 ° C.) 8 grams of perflubron (perfluorooctyl bromide: Atochem, Paris, France) was added dropwise during mixing. After the fluorocarbon was added, the emulsion was mixed for at least 4 minutes The resulting crude emulsion was then passed through a high pressure homogenizer (Avestin, Ottawa, Canada) in 5 passes at 124,106 kPa (18,000 psi).
[0214]
One eighth of Formulation B was separated and added to Formulation A. The resulting emulsion feed solution of HA peptide / perflubron was fed to the spray dryer under the conditions described above. The powder was collected in a cyclone and the sieving screen was washed into the collection jar using a perflubron. The suspension of HA in perflubron was then frozen at -60 <0> C and lyophilized. A white powder with free flowing was obtained.
[0215]
II. In vitro activity of hollow porous microparticles containing HA peptide
[0216]
The functionality of the HA peptide in Purmo sphere (HA-Pul) to activate antigen-expressing cells was compared to the neat HA peptide. The Purmo sphere of the HA peptide from Example 1 was incubated with sterile PBS at a concentration of 5 mg / ml (formulation weight / volume). Serial dilutions of the resulting HA-Pul-PBS solution were performed on cells (1 × 10 6) expressing M12 antigen in complete RPMI-10% FCS.⁴/ Well) and HA specific TcH (2 x 10)⁴Added to the microwells containing TcH cell lines carry a reporter gene controlled by the IL-2 promoter (IL-2 / -gal).
[0217]
After incubation for 12 hours at 37 ° C., centrifuge the microwell plate, fix the cells with paraformaldehyde-glutaraldehyde for 5 minutes at 4 ° C., wash with PBS and add X-gal substrate overnight The The number of active TcH per 500 cells per well was counted using a light microscope. The total number of active TcH per well was estimated by multiplying the total number of cells by the percentage of blue cells. The results shown in FIG. 1 indicate the presence of active peptide in the formulation. Comparison with a standard activation curve (HA saline) showed that the concentration of active peptide was about 5% mass / mass (weight / weight), which was consistent with reverse phase-HPLC measurements.
[0218]
III . Mechanism of action of HA peptide pulsosphere
[0219]
The need for internalization and processing of Purmo sphere microparticles containing the T cell epitope HA110-120 (HA-Pul) was investigated. Air-dried HA-Pul (500 nM / well HA110-120 peptide) suspended in perflubron and unfixed or paraformaldehyde fixed M12 antigen presenting cell (APC) cells, complete with Incubated in the presence of specific TcH cells in RPMI-10% FCS, and compared to HA-Pul suspended in PBS at similar concentrations, and neat HA peptide. Sucrose-purified A / PR / 8/34 (H1N1) virus (15 g / ml) was used as a positive control since it does not require intracellular processing. Negative controls consisted of the NP147-155 peptide, the non-formulated NP peptide and the unrelated unrelated virus. The cell numbers and growth conditions described in Example II followed. Cells were fixed and exposed to X-gal substrate. The results were expressed as% of active TcHl.
[0220]
FIG. 2 shows that fixed and non-fixed APC were able to reveal neat HA peptide and HAPul. In contrast, only surviving APCs were able to reveal HA peptides from the viral context. Furthermore, formulated and neat NP peptides, as well as B / Lee virus, did not activate specific TcH. The results show that internalization and processing of HA-Pul is not a requirement for TcH activation. Rather, the HA-peptide is readily released from Purmo spheres, and MHC class II molecules (IE^d ), Resulting in TCR engagement and TcH activation. This processing step was observed for HA-Pul delivered in PBS or perflubron as well as the neat HA peptide. Furthermore, these results demonstrate that the peptide of HA 110-120 formulated in Purmo spheres and stabilized in perflubron retains its immunogenicity.
[0221]
IV. Production of fluorescently labeled hollow porous HA peptide particles by spray drying
[0222]
Suction: 100%, inlet temperature: 85 ° C, outlet temperature: 51 ° C; feed pump: 10%: N₂ Flow: Hollow porous HA-fluoroscein 110-120 peptide / Texas Red DHPE by spray drying technology using a B-191 mini spray dryer (Buechi, Flavil, Switzerland) under conditions of 800 L / hr. The particles were produced. The feed was made by mixing the two formulations (A and B) just prior to spray drying. A 150 mesh stainless steel screen was placed at the cyclone outlet port to facilitate particle collection.
[0223]
Formulation A: 5 g of deionized water to dissolve 20 mg of the HA-Fluoroscein 110-120 peptide (Chiron, Emeryville, CA) and 1 mg of hydroxyethyl starch (Ajinomoto, Japan) Water was used.
[0224]
Formulation B: A phospholipid-stabilized fluorocarbon-in-water emulsion was prepared as follows. The phospholipid (0.3 g EPC-100-3 (Lipoid KG, Ludwigshafen, Germany), and 0.3 mg fluorescent dye, Texas Red DHPE (Molecular Probe, Eugene, OR, 3 mg) were first dissolved in chloroform Chloroform was then removed using Buchi RotoVap The El00-3 / Texas Red DHPE film was then dispersed in 33 g of hot deionized water (T = 60-70 ° C.) The surfactant was removed. , And further processed with an Ultra-Tulax Mixer (type T-25) at 10,000 rpm for about 2 minutes (T = 50-60 ° C.) Mix 8 grams of perflubron (Atochem, Paris, France) The addition of the fluorocarbon was followed by the addition of Emissions were mixed for at least 4 minutes. The resulting crude emulsion was then passed high-pressure homogenizer at 5 path 124,106kPa (18,000psi) (Avestin, Ottawa, Canada) to.
[0225]
One eighth of Formulation B was separated and added to Formulation A. The resulting HA peptide / Texas Red DHPE / Perflubron emulsion feed solution was fed to the spray dryer under the conditions described above. The powder was collected in a cyclone and the sieving screen was washed into the collection jar using a perflubron. The suspension of HA in perflubron was then frozen at -60 <0> C and lyophilized. A free-flowing fluorescent fuchsia-colored powder was obtained.
[0226]
V. Bioavailability of Fluorescently Labeled HA Pulmospheres
[0227]
The fluorescein-HA peptide (20% wt / wt (wt / wt%)) prepared as in Example IV was suspended in perflubron. Metofan-anesthetized mice were inoculated intranasally (in) with a 70 l volume of f-HAPul in perflubron, corresponding to a 70 g peptide dose. Blood samples were collected by eye breathing into heparinized tubes, plasma was separated, and the concentration of peptide was determined by fluorescence analysis. As a control, an intravenous (i.v.) inoculation of 70 g of f-HA peptide in 70 1 of sterile saline (n = 4 for all groups) was used.
[0228]
FIG. 3 shows serum concentrations of f-HA peptide versus time. i. n. The absolute bioavailability of the delivered f-HA peptide is about 5%, T_max Occurred in 20 minutes. i. v. Continuous log reduction for dosing i. n. Pharmacokinetic profiles differed between the two routes of administration, with a transient increase upon administration and a subsequent exponential decrease. With a total clearance of up to 6 hours, removal of f-HA occurs via urine (not shown).
[0229]
An example of this is the i.v. of T cell epitopes (molecular weight of approximately 1.4 kDa) formulated in Pul. n. Show that administration is compatible with systemic delivery.
[0230]
VI. Production of human IgG hollow porous microparticles by spray drying
[0231]
Suction: 100%, inlet temperature: 85 ° C, outlet temperature: 61 ° C; feed pump: 10%: N₂ Hollow porous human IgG particles were produced by spray drying technique using a B-191 mini spray dryer (Buechi, Flavil, Switzerland) under flow conditions: 800 L / hr. The feed was made by mixing the two formulations (A and B) just prior to spray drying.
[0232]
Formulation A: 2 mg normal saline (Baxter, Chicago, IL) to dissolve 55 mg of human IgG (Sigma Chemical, St. Louis, MO) and 3.2 mg of hydroxyethyl starch (Ajinomoto, Japan) Was used.
[0233]
Formulation B: A phospholipid-stabilized fluorocarbon-in-water emulsion was prepared as follows. The phospholipid, 0.415 g of EPC-100-3 (Lipoid KG, Ludwigshafen, Germany), in 40.3 g of hot deionized water (T = 50-60 ° C.) Ultra-Turax Mixer (T-25) The mold is homogenized at 8000 rpm for 2 to 5 minutes (T = 60 to 70.degree. C.). 5.2 g of perflubron (Atochem, Paris, France) was added dropwise during mixing. After the fluorocarbon was added, the emulsion was mixed for at least 4 minutes. The resulting crude emulsion was then passed through a high pressure homogenizer (Avestin, Ottawa, Canada) in 5 passes at 124,106 kPa (18,000 psi).
[0234]
One eighth of Formulation B was separated and added to Formulation A. The resulting emulsion feed solution of IgG / perflubron was fed to the spray dryer under the conditions described above. The powder was collected in a cyclone and the sieving screen was washed into the collection jar using a perflubron. The IgG suspension in perflubron was then frozen at -60 <0> C and lyophilized. A free-flowing white powder was obtained. Hollow porous IgG particles have a volume-weighted mean aerodynamic diameter of 2.373 ± 1.88 μm as determined by time-of-flight analysis (Aerosizer, Amherst Process Instruments, Amherst, Mass.) The
[0235]
VII. In vitro activity of polyclonal human IgG Purmo spheres
[0236]
Formulations of polyclonal human IgG Purmo spheres (hIgG-Pul) from Example IV were confirmed for activity using a captured hIgG enzyme-linked immunosorbent assay (ELISA). A 5 mg / mL hIgG-PuL suspension in perflubron was prepared, pipetted into the wells and air dried. PBS was added to the dried hIgG-Pul and allowed to incubate overnight. Dilute the hydrated hIgG-Pul solution and transfer to the enzyme immunosorbent assay plate coated with mouse anti-human k chain monoclonal antibody in coating buffer (dil. 1: 1000, Sigma Immunochemical), then Blocked with PBS containing 15% goat serum for 2 hours at room temperature. The wells were washed and assayed by using a conjugate of goat anti-human IgG alkaline phosphatase (PBS-15% goat serum, 1: 1000 in 0.05% Tween) followed by addition of pNPP substrate. Optical density (OD) was read using an automatic plate reader set at 405 nm. HIgG in saline (standard) and hIgG mixed with blank Purmo spheres were used as controls to rule out lipid effects on the assay. The blank Purmo sphere consisted only of phospholipid and starch.
[0237]
FIG. 4 shows calibration curves for hlgG-Pul, hIgG and hIgIG + blank Purmo spheres. The hIgG-Pul formulation was determined to contain approximately 20% by weight (wt%) hIgG. In addition, hIgG-Pul retained the expression of k light and heavy chain epitopes.
[0238]
VIII. Dissolution Kinetics of HA 110-120 Peptide and Human IgG from Pulmosphere Formulation
[0239]
The kinetics of antigen and hIgG release from Purmo spheres was determined as 0. Measurements were made using a lysis chamber equipped with a lum diameter filter and a fitted 24-well flat bottom cell culture plate. Approximately 3 mg of Purmo sphere powder from Examples 1 and VI were placed in the lower compartment of the lysis chamber and simultaneously exposed to sterile PBS (1.3 ml / well). The plate was placed on a horizontal shaker (30 RPM) at 37 ° C. to simulate the breathing pattern. 25 μl samples were collected from the upper compartment and analyzed by capture ELISA (FIG. 5A) for hIgG or bioassay (FIG. 5B) for fluorescein labeled HA peptide formulation (f-HA). Results for f-HA were independently confirmed by fluorescence analysis (not shown). The dissolution kinetics of hIgG and HA peptide Purmo spheres were compared to their respective aqueous controls.
[0240]
The results shown in FIGS. 5A and 5B were expressed as percent release. A rapid diffusion controlled release was observed for the HA peptide formulation and there was no difference between the aqueous control. Complete dissolution occurred within 2 hours. In contrast, slower erosion control kinetics were observed for hIgG. Complete lysis required more than 6 hours as compared to 1 hour for water IgG control. The results described here demonstrate that the dissolution kinetics from Purmo spheres depend, at least in part, on the molecular weight of the formulated compound (1.4 kDa and 150 kDa, respectively). It will also be appreciated that differences in hydrophilicity and hydrophobicity may have similar effects.
[0241]
IX. Bioavailability of hIoG plume spheres
[0242]
From Example VI via intratracheal route (20 g hIgG in 20 l of perflubron) in mice anesthetized with ketamine / xylazine or by nasal route (70 gh IgG in 70 μl of perflubron) to mice anesthetized with metophane Human IgIG Pulmosphere (hIgG-Pul) was administered. The same volume of hIgG in sterile PBS was administered intravenously in the control group. Mice were bled at various time intervals and serum concentrations of hIgG were assessed by capture enzyme immunosorbent assay in all groups (n = 3). i. v. Absolute bioavailability was determined from the area under the serum concentration-time curve (AUC) compared to control. AUC values were calculated using the trapezoidal rule.
[0243]
Plasma hIgG concentration curves are shown in FIG. 6A (i.t.) and FIG. 6 GB (i.n.). The absolute bioavailability for intratracheal delivery of hIgG-Pul was 27% and 1.5% for intranasal inoculation. In both cases T_maxOccurred in about 2 days. Western blotting showed that the molecular weight for circulating hIgG after delivery through the airway was indistinguishable from the neat material. The hIgG was observed to persist in circulation for more than 14 days.
[0244]
X. Antibody response to hIgG Purmospheres delivered via the tracheal route
[0245]
Intratracheal administration (20 g dose of hIgG) in blood and bronchial-alveolar lavage (BAL) in mice treated with hIgG-Pulmo spheres (hIgG-Pul) from Example VI suspended in perflubron Fluid response. Mice were also treated with the following controls: 20 μg hIgG in saline, i. t. 100 μg of hIgG in saline, i. v. And i. t. 100 μg in Complete Freund's Adjuvant (CFA), subcutaneously, and saline, i. t. . Individual groups were performed in triplicate. Blood and BAL were collected two weeks after immunization.
[0246]
The titer of antigen hIgG-mouse IgG was measured using an enzyme-linked immunosorbent assay plate coated with hIgG or 0.1% BSA. The wells were blocked with PBS-15% goat serum and incubated for 2 hours with various dilutions of serum or BAL. After washing, the assay was performed with a conjugate of anti-mouse IgG alkaline phosphatase, followed by the addition of PNPP substrate. And followed. The optical density (OD) of the plate was analyzed at 550 nm using an automatic plate reader and the results were expressed as end point dilution titers for serum IgG (FIG. 7A) or as mean OD against BAL IgG (FIG. 7B).
[0247]
These results show increased systemic and regional fluid in mice treated with hIgG-Pul via the endotracheal route, as compared to the dose / route matched group receiving hIgG in saline. Show sexual responses. Furthermore, the response was enhanced via the intratracheal or intravenous route as compared to mice receiving higher doses of hIgG in saline. The titer of serum antibodies is as follows: hIgG in CFA. c. It was similar to that measured in immunized mice. Interestingly, the humoral response did not correlate with systemic bioavailability (data not shown), implying the involvement of local immunity.
[0248]
XI. T cell response to hIgG Purmospheres delivered via the tracheal route
[0249]
Level of T cell immunity induced in the spleen of mice immunized by the tracheal route with hIgG-Pulmo sphere (hIgG-Pul) from Example VI suspended in perflubron. The spleen was separated into single cell suspensions and treated with hypotonic buffer to remove red blood cells. Splenocytes were resuspended at 4 × 10 6 cells / ml in complete RPMI-10% FCS and incubated in 24-well flat bottom plates (1 ml / well) in the presence of 6 g / ml hIgG. After 72 hours of incubation, the supernatants were collected and the concentrations of IL-2, IFN- and IL-4 were determined by enzyme-linked immunosorbent assay (Biosource International, Camarillo, CA).
[0250]
The results (FIG. 8) are expressed as the mean of cytokine concentration in individual mice in each group, and all three cytokines enhanced in hIgG-Pul immunized mice compared to hIgG saline controls Showed the produced. Cytokine production by splenic T cells against the hIgG-Pul treated group was determined by the i.g. v. It was comparable to that observed for the group. These results strongly suggest systemic migration of lung-activated memory T cells.
[0251]
XI. Antibody response to hIgG Purmospheres delivered via the nasal route
[0252]
Liquidity of mice receiving hIgG via any intranasal instillation (20 g) formulated as Purmo sphere (hIgG-Pul) from Example VI suspended in perflubron or dissolved in saline Confirmed the response. Sera were obtained at various time intervals after immunization and titers of specific mouse IgG to hIgG were determined using the enzyme-linked immunosorbent assay procedure described in Example X. The results (FIG. 9) are expressed as mean endpoint titers (n = 3), with faster onset kinetics and higher size in mice treated with hIgG-Pul compared to saline It showed lower intersubject reproducibility of responses.
[0253]
X III . Antibody response to hIgG Purmo spheres delivered via the peritoneal route
[0254]
Humoral response of mice treated with hIgG-Pulmo sphere (hIgG-Pul) from Example VI suspended in perflubron via the peritoneal (ip) route (100 g dose of hIgG). The mice were treated with 100 μg of hIgG i. p. Treated: in saline, in multilamellar dipalmitoylphosphatidylcholine (DPPC) liposome saline solution (+ ml lip), in monolayer (unilamellar) DPPC liposome saline solution (+ ul lip), and in blank Purmo sphere saline solution (+ empty Pul) During. An additional control group of blank Purmo sphere solution without any hIgG was also tested. Particle median sizes of ml lip (> 10 μl) and ul lip (90 nm) were determined using laser light scattering techniques. Each group was performed in triplicates. The IgG humoral immune response in serum was measured at 7 and 14 days using the same enzyme-linked immunosorbent assay technology as in Example X.
[0255]
Results are expressed as mean endpoint titers, showing a consistent increase in antibody titers to animals inoculated with hIgG-Pul. In particular, FIGS. 10A and 10B show endpoint titers at 7 days and 14 days, respectively. IgG added to empty Puh induced titers similar to hIgG in saline. In addition, addition of any DPPC liposome formulation to hIgG did not restore the increased immunity observed with hIgG-Pul. Thus, these results are: (1) Enhanced immunity of hIgG-Pul is not a pathway dependent phenomenon (see Examples X and XII); (2) hIgG-Pul formulation was enhanced of hIgG It is a prior requirement for immunogenicity; and (3) other components of DPPC or Pul demonstrate that they do not have an independent adjuvant effect. Furthermore, these results elucidate the importance of delivery pathways responsible for the enhanced immunity induced by hIgG-Pul, as well as other factors.
[0256]
XIV. Production of hollow porous particles of influenza virus A / WSN / 32 (H1N1) by spray drying
[0257]
Suction: 100%, inlet temperature: 85 ° C, outlet temperature: 61 ° C; feed pump: 10%: N₂ Flow: 8 structural protein complexes and 8 negatively charged RNA segments by spray drying using a B-191 mini spray dryer (Buechi, Flavil, Switzerland) under conditions of 800 L / hr. The hollow porous influenza virus A / WSN / 32 (H1N1) containing the relatively complex enveloped virus was well incorporated into the microparticles. The feed was made by mixing the two formulations (A and B) just prior to spray drying. Prior to formulation, the virus was alive and purified by sucrose gradient centrifugation.
[0258]
Formulation A: lmg of hydroxyethyl starch (Ajinomoto, Japan) was weighed and transferred to a tube containing 0.6g of influenza virus in saline.
[0259]
Formulation B: A phospholipid-stabilized fluorocarbon-in-water emulsion was prepared as follows. The Phospholipid 0.111 g EPC-100-3 (Lipoid KG, Ludwigshafen, Germany) in a 20 g hot deionized water (T = 50-60 ° C.) Ultra-Turax Mixer (type T-25) Were homogenized at 8000 rpm for 2 to 5 minutes (T = 60 to 70.degree. C.). 4.4 g of perflubron (Atochem, Paris, France) was added dropwise during mixing. After the fluorocarbon was added, the emulsion was mixed for at least 4 minutes. The resulting crude emulsion was then passed through a high pressure homogenizer (Avestin, Ottawa, Canada) in 5 passes at 124,106 kPa (18,000 psi).
[0260]
One eighth of the volume of Formulation B was separated and added to Formulation A. The resulting influenza virus / perflubron emulsion feed solution was fed to a spray dryer under the conditions described above. The powder was collected in a cyclone and the sieving screen was washed into the collection jar using a perflubron. The suspension of HA in perflubron was then frozen at -60 <0> C and lyophilized. A free-flowing white powder was obtained.
[0261]
XV. In vitro activity of influenza virus A / WSN / 32 (H1N1) Purmo sphere
[0262]
Uptake of viable viral antigen into spray-dried particles was confirmed using the following technique: Influenza virus A / WSN / 32 (H1N1) Purmo sphere (WSN-Pul) from Example XIV at 40 ° C. It was dissolved in sterile PBS at a concentration of 5 mg / ml for 6 hours. The hydrated WSN-Pul was then incubated with unfixed and paraformaldehyde-solidified M12 antigen-expressing cells (APC) at various dilutions at 37 ° C. for 1 hour in a 96-well plate. After antigen pulsing, the APCs were washed and incubated with TcH for 4 hours. Formaldehyde-glutaraldehyde fixed cells were incubated with X-gal substrate and positive cells were counted.
[0263]
The results were expressed as percent activated TcH (FIG. 1 IA). Various concentrations of sucrose-purified viable WSN virus were used as controls (FIG. 1 1 B). The WSN-Pul formulation was determined to contain approximately 5% by weight (wt%) influenza virus. Only non-fixed APC's were able to activate the virus, indicating that the antigen was not degraded. Titration of infectious virus was determined by the MDCK (Medin-Darby kidney cancer cell) assay (FIG. 11C) and showed that about 1% of the total virus was still able to infect and replicate in permissive cells. Together, these results show the successful uptake of relatively large influenza virus antigens into Purmo sphere powder.
[0264]
XVI. Antibody Response to Influenza Virus AIWS NI 32 (H1N1) Purmosphere Delivered Via Nasal Route
[0265]
5g virus and 2 x 10 of live virus³TCID₅₀After intranasal inoculation of BALB / c mice with Influenza virus A / WSN / 32 (H1N1) Purmo sphere (WSN-Pul) formulation containing (1% of the total antigen load corresponding to the amount of viable virus), WSN The induction of virus specific IgG antibody response to the virus was measured. Control mouse, 2 × 10³ TCID₅₀Immunized with surviving virus (corresponding to 0.05 g of total virus) or UV-killed WSN virus (5 g). Sera from mice treated with hIgG was used as a negative control. Antibody responses were measured in serum using the following enzyme-linked immunosorbent assay technology: wells were coated with sucrose purified WSN virus in coating buffer and blocked with non-mammalian protein (SeraBlok) Incubated with serial dilutions of serum samples. The samples were washed and assayed with a biotin-conjugated rat anti-mouse mAb, followed by streptavidin-alkaline phosphatase and pNPP substrate. Results were expressed as the geometric mean of the endpoint titer reciprocal. The number of mice was 3 per inoculated group.
[0266]
The results shown in FIG. 12 show the induction of high titer IG antibodies in mice immunized with WSN-Pul or viable WSN virus (WSN / Io) in saline at 7 and 14 days. In contrast, only small titers of specific IgG were detected in mice immunized with virus killed in saline.
[0267]
XVII. T cell response to influenza virus A / WSN / 32 (H1N1) Purmo spheres delivered via the nasal route
[0268]
T cell responses were defined in terms of virus and epitope-specific cytokine production of lymphocytes from mice (Example XVI) immunized as described above. 5g virus and 2 x 10 of live virus³TCID₅₀After intranasal inoculation of BALB / c mice with Influenza virus A / WSN / 32 (H1N1) Purmo sphere (WSN-Pul) formulation containing (1% of the total antigen load corresponding to the amount of viable virus) T The induction of cellular responses was measured. Control mouse, 2 × 10³TCID₅₀Immunized with surviving virus (corresponding to 0.05 g of total virus) or UV-killed WSN virus (5 g). The antigens tested were sucrose purified WSN virus, HA110-120 peptide, and NP147-155 peptide. An untreated saline group was included as a control.
[0269]
Ten days after immunization, peripheral blood mononuclear cells (PSMCs) were isolated from blood by Ficoll gradient centrifugation. Different numbers of responder cells were added to 3 × 10 6 in complete RPMI-10% FCS.⁵ Cells / well were incubated with nitrocellulose / anti-IFN or anti-IL-4 (PharMingen) ELISPOT plates (Millipore). Stimulating cells (mitomycin treated splenocytes, 5 × 10⁵ / Well), antigen, and human rIL-2 (20 U / ml) were added and the plates were co-incubated for 48 hours. The cells are then washed with PBS-0.05% Tween, the anti-cytokine antibody (PharMingen) is incubated overnight, and the assay is performed using HRP-streptavidin conjugate followed by an insoluble substrate (vector laboratory) The The assay was stopped with water, the wells were air dried, and the spots were counted using a stereomicroscope.
[0270]
IFN- or IL-4 / 10 after subtracting the background signal from the results⁶It was expressed as the frequency of specific cells producing PBMC. The background is reproduced 6/10⁶ It was less than. PBMCs were pooled from each group of mice. Figures 13A, 13B and 13C show that vaccination with WSN-Pul and WSN viruses generally induced HA-, NP- and WSN-specific T cells producing IFN- and IL-4. Show. In contrast, immunization with dead virus mainly induced T cells producing IL-4. Furthermore, immunization with dead virus induced an enhanced subpopulation of IL-4 producing Tc2 cells specific for the peptide of NP147-155. These data show that cell responses elicited by survival controls and formulated virus (ie, including viable and killed viruses) are elicited by killed virus controls corresponding to typical conventional vaccines. It shows that it was more effective.
[0271]
XV III . Nasal route. Against Infectious Attacks of Mice Immunized with Influenza Virus A / WSN / 32 (H1N1) Purmosphere Delivered Via Viruses
[0272]
Mice immunized as described in Example XVII were delivered 1.2 x 10 3 via the nasal route 3 weeks after immunization.⁶ Attacked by the flu virus. Protection in terms of virus spread and weight change was defined 4 days after the attack. The results are shown in FIGS. 14A and 14B.
[0273]
Viral titer measurements in nasal washes were determined by titrating viable virus in the MDCK assay. The results showed that there was no infectious virus in mice previously immunized with influenza virus A / WSN / 32 (H1IN1) Purmo sphere (WSN-Pul) or control surviving WSN virus (FIG. 14A). Mice immunized with UV killed WSN virus, or non-dosed mice showed significant titers of influenza virus in the nasal washes. In addition, mice immunized with WSN-Pul or WSN virus (low dose of survival virus) retained their weight after challenge (FIG. 14B). Non-immunized mice, and mice immunized with UV-killed WSN virus showed a marked reduction in body weight, and subsequent death (7 days, 2/3 in each group). These results demonstrate that WSN-Pul provides effective vaccination efficiency in mucosal delivery.
[0274]
XIX. Production of hollow porous particles of TA7 retrovirus by spray drying
[0275]
Suction: 100%, inlet temperature: 85 ° C, outlet temperature: 61 ° C; feed pump: 10%: N₂ TA7 retrovirus particles were produced by spray drying technology using a B-191 mini spray dryer (Buechi, Flavil, Switzerland) under flow conditions: 800 L / hr. The feed was made by mixing the two formulations (A and B) just prior to spray drying.
[0276]
Formulation A: 2 g of deionized water was used to dissolve 1 mg of TA7 retrovirus.
[0277]
Formulation B: A phospholipid-stabilized fluorocarbon-in-water emulsion was prepared as follows. The Phospholipid (0.3 g EPC-100-3 (Lipoid KG, Ludwigshafen, Germany), Ultra-Turax Mixer (T-25) in 16.5 g hot deionized water (T = 50-60 ° C.) The mold was homogenized at 8000 rpm for 2 to 5 minutes (T = 60-70 ° C.) 8.0 g of perflubron (Atochem, Paris, France) was added dropwise during mixing, the fluorocarbon was added. After that, the emulsion was mixed for at least 4 minutes The resulting crude emulsion was then passed through a high pressure homogenizer (Avestin, Ottawa, Canada) in 5 passes at 124,106 kPa (18,000 psi).
[0278]
One-eighth (by volume) of formulation B was separated and added to formulation A. The resulting TA7 retrovirus / perflubron emulsion feed solution was fed to the spray dryer under the conditions described above. The powder was collected in a cyclone and the sieving screen was washed into the collection jar using a perflubron. The TA7 retrovirus suspension in perflubron was then frozen at -60 ° C and lyophilized. A free-flowing white powder was obtained.
[0279]
Example XIX. In vitro activity of spray dried particles of TA7 retrovirus
After incorporation into the spray-dried particles produced in Example XIX, the activity of the TA7 retrovirus was examined. Spray dried TA7 retroviral particles were dissolved in saline and added to Hela cells for 1 hour. Twenty-four hours after inoculation, the cells were then analyzed for transgenic expression using β-gal. No differences were observed between neat and spray dried TA7 retroviral particles. These results indicate that the relatively large and complex TA7 retrovirus can be effectively incorporated into spray dried particles without loss of apparent activity.
[0280]
XXI. Of hollow porous particles of bovine gamma globulin by spray drying Manufacturing
Suction: 100%, inlet temperature: 85 ° C, outlet temperature: 61 ° C; feed pump: 10%: N₂ TA7 retrovirus particles were produced by spray drying technology using a B-191 mini spray dryer (Buechi, Flavil, Switzerland) under flow conditions: 800 L / hr. The feed was made by mixing the two formulations (A and B) just prior to spray drying.
[0281]
Formulation A: 0.6 g BGG (Calbiochem, San Diego, CA), 0.42 g lactose (Sigma Chemical, St. Louis, MO), and 25 mg Pluronic F-68, NF grade (BASF, Parsippany, New York) 21 g of 0.2% saline solution was used to dissolve the state).
[0282]
Formulation B: A phospholipid-stabilized fluorocarbon-in-water emulsion was prepared as follows. The Phospholipid (0.3 g EPC-100-3 (Lipoid KG, Ludwigshafen, Germany), Ultra-Turax Mixer (T-25 type) in 33 g hot deionized water (T = 50-60 ° C.) Was homogenized at 8000 rpm for 2 to 5 minutes (T = 60-70 ° C.) 35 g of F-decalin (Air Products, Allentown, Pa.) Were added dropwise during mixing. After that, the emulsion was mixed for at least 4 minutes The resulting crude emulsion was then passed through a high pressure homogenizer (Avestin, Ottawa, Canada) in 5 passes at 124,106 kPa (18,000 psi).
[0283]
Formulations A and B were combined and fed to the spray dryer under the conditions described above. A free flowing white powder was collected with a cyclone separator. The hollow porous particles had a volume-weighted mean aerodynamic diameter of 1.27 ± 1.42 μm as determined by time-of-flight analysis (Aerosizer, Amherst Process Instruments, Amherst, Mass.) .
[0284]
XXII Andersen CASK for bovine gamma globulin MDI formulation Impactor result
The inhalability of a dose measuring inhaler (MDI) formulated with porous particles of BGG, prepared according to Example XXI, was evaluated using an Andersen Cascade Impactor. 83 mg of hollow porous BGG particles were weighed into a 10 ml aluminum can and dried in a vacuum oven under a stream of nitrogen at 40 ° C. for 3 to 4 hours. The can was crimp sealed using a DF 31/50 act 50 l valve (Vallois, Greenwich, Conn., USA) and filled with 9.64 g of HFA-I 34a (Dupont, Wilmington, Del.) Propellant by over pressure through the stem .
[0285]
By operation of the device, a fine particle fraction of 61% and a fine dose of 68 μg were observed (FIG. 15). This example illustrates that relatively large bioactive agents such as BGG can be formulated and effectively delivered from MDI.
[0286]
Those skilled in the art will further appreciate that the present invention can be embodied in other specific forms without departing from its spirit or central attributes. It should be understood that other variations are considered to be within the scope of the present invention in that the foregoing description of the present invention discloses only its exemplary aspects. Thus, the present invention is not limited to the specific embodiments detailed herein. Rather, reference should be made to the appended claims as an indication of the scope and content of the present invention.
Brief Description of the Drawings
[Fig. 1]
FIG. 1 is a diagrammatic representation of the level of functional HA peptide (residues 110-120 of influenza virus hemagglutinin) according to the formulation in the microstructure according to the invention.
[Fig. 2]
FIG. 2 illustrates the fact that antigens formulated in microstructure do not require intracellular processing to activate T cells.
[Fig. 3]
FIG. 3 compares plasma concentrations of HA microparticles delivered using nasally administered microparticles and intravenous injection.
[Fig. 4]
FIG. 4 represents a calibration curve for human IgG formulated in microparticles as described in this application, with selected controls.
FIG. 5A
FIG. 5A graphically illustrates the release kinetics for IgG formulated microparticulates and HA peptide formulated microparticulates.
FIG. 5B
FIG. 5B graphically illustrates the release kinetics for IgG formulated microparticulates and HA peptide formulated microparticulates.
FIG. 6A
FIG. 6A shows the persistence of IgG in plasma following intratracheal and nasal administration using formulated microparticulates.
FIG. 6B
FIG. 6B shows the persistence of IgG in plasma following intratracheal and nasal administration using formulated microparticulates.
[FIG. 7A]
FIG. 7A shows systemic and local antibody responses to intratracheally administered IgG as microparticulates formulated in accordance with the present invention.
[FIG. 7B]
FIG. 7B shows systemic and local antibody responses to intratracheally administered IgG as microparticulates formulated in accordance with the present invention.
[Fig. 8]
FIG. 8 graphically illustrates the levels of cytokines indicative of T cell responses following intratracheal administration of IgG microparticulates to mice.
[Fig. 9]
FIG. 9 shows murine antibody responses to intranasally administered IgG microparticulates.
FIG. 10A
FIG. 10A shows antibody titers in mice 7 and 14 days after intraperitoneal injection of IgG microparticulates, respectively.
[FIG. 10B]
FIG. 10B shows antibody titers in mice 7 and 14 days after intraperitoneal injection of IgG microparticulates, respectively.
FIG. 11A
FIG. 11A describes virus of microparticulate formulation, T cell response to viral control, and infection titer of both formulated and unformulated virus, respectively.
[FIG. 11B]
FIG. 11B illustrates virus of microparticulate formulation, T cell response to viral control, and infection titer of both formulated and unformulated virus, respectively.
[FIG. 11C]
FIG. 11C describes virus of microparticulate formulation, T cell response to viral control, and infection titer of both formulated and unformulated virus, respectively.
[Fig. 12]
FIG. 12 shows antibody responses of mice to microparticulate-formed viable and killed influenza virus 7 and 14 days after intranasal administration.
FIG. 13A
FIG. 13A shows murine levels of viral microparticulate or viable virus or factor indicating T cell response following intranasal inoculation of killed virus or dead virus.
[FIG. 13B]
FIG. 13B shows murine levels of viral microparticulate or viable virus or factor indicating T cell response following intranasal inoculation of killed virus or dead virus.
[FIG. 13C]
FIG. 13C shows murine levels of viral microparticulate or viable virus or factor indicating T cell response following intranasal inoculation of killed virus or dead virus.
FIG. 14A
FIG. 14A illustrates viral shedding and weight change in mice inoculated intranasally with microparticles containing both surviving and dead virus, respectively.
[FIG. 14B]
FIG. 14B illustrates viral shedding and weight change in mice inoculated intranasally with microparticles containing both surviving and dead virus, respectively.
[Fig. 15]
FIG. 15 shows the results of an in vitro Andersen Cascade Impactor study showing efficient delivery of formulated microspheres containing bovine gamma globulin from a dose metered inhaler.

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