JP5722782B2 - ナノエマルジョン治療用組成物及びその使用方法 - Google Patents
ナノエマルジョン治療用組成物及びその使用方法 Download PDFInfo
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- JP5722782B2 JP5722782B2 JP2011529276A JP2011529276A JP5722782B2 JP 5722782 B2 JP5722782 B2 JP 5722782B2 JP 2011529276 A JP2011529276 A JP 2011529276A JP 2011529276 A JP2011529276 A JP 2011529276A JP 5722782 B2 JP5722782 B2 JP 5722782B2
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Description
本願は、その開示全体が参照によって本明細書に組み込まれる、2008年9月26日出願の米国仮出願第60/100,559号の優先権を主張するものである。
本発明の理解を容易にするために、いくつかの用語及び句を、以下に定義する。
嚢胞性線維症(CF)は、電解質のバランスに関与するCFTRと呼ばれる欠陥膜貫通タンパク質により重篤な肺損傷を引き起こす、生命を危うくする障害である。濃い粘液は、肺の管(tube)、管(duct)及び通路の栓子を形成する。この環境は、バイオフィルムコミュニティーを確立して呼吸器感染症をもたらす日和見性細菌にとって理想的である。全身投与された抗生物質は、悪化の頻度及び重症度を低減することができるが、細菌が気道及び肺から完全に根絶することは決してない。噴霧器で投与する抗生物質が使用されるが、様々な耐性種の耐性の出現及び/又はコロニー形成は大きな懸案事項である。嚢胞性線維症(CF)は、黄色ブドウ球菌及びインフルエンザ菌などの病原菌種、並びに緑膿菌、アクロモバクター・キシロスオキシダンス(Achromobacter xylosoxidans)、ステノトロホモナス・マルトフィリア、ラルストニア種、パンドレア(Pandoraea)種及びバークホルデリア・セパシアコンプレックス(Bcc)種などのいくつかの日和見種のコロニー形成のための環境を提供する、先天性の呼吸器防御機構の機能障害をもたらす(例えば、LiPuma et al., (2009) Antimicrob Agents Chemother 53, 249-255参照)。Bccは、少なくとも17の系統的に関連している腐生グラム陰性桿菌の群を含み、その大部分はバイオフィルムを形成し得る(例えば、Al Bakri et al., 2004, Journal of Applied Microbiology 96, 455-463;Eberl and Tummler, 2004, International Journal of Medical Microbiology 294, 123-131;LiPuma et al., (2009) Antimicrob Agents Chemother 53, 249-255;並びにTomlin et al., 2004, Journal of Microbiological Methods 57, 95-106参照)。それらは特に治療困難であり、CF患者の高い罹患率及び死亡率に関連している。それらはまた、ヒト感染症において遭遇する最も抗菌耐性のある種に含まれる(例えば、LiPuma et al., 2005, Curr Opin Pulm Med 11, 528-533;LiPuma et al., (2009) Antimicrob Agents Chemother 53, 249-255参照)。感染症及び関連の炎症は、一度確立するとめったに排除されず、最終的には肺不全及び死亡に至る進行性肺疾患をもたらす(例えば、LiPuma et al., (2009) Antimicrob Agents Chemother 53, 249-255;Saiman and Seigel, 2003, Infect Control Hosp Epidemiol 24, S6-S52参照)。
ナノエマルジョン及びバークホルデリア(例えば、B.セノセパシア)抗原を含む免疫原性組成物は、マウス、ラット、ウサギ、モルモット、サル又はヒトなどの哺乳動物を免疫化してポリクローナル抗体を生成するために使用することができる。所望に応じて、バークホルデリア(例えば、B.セノセパシア)抗原は、ウシ血清アルブミン、サイログロブリン、キーホールリンペットヘモシアニン又は本明細書に記載の他の担体などの担体タンパク質にコンジュゲートしていてもよい。宿主の種に応じて様々なアジュバントを使用して、免疫学的応答を増大することができる。かかるアジュバントには、それに限定されるものではないが、フロイントアジュバント、鉱物ゲル剤(例えば、水酸化アルミニウム)、及び表面活性物質(例えば、リゾレシチン、プルロニックポリオール、ポリアニオン、ペプチド、本明細書に記載のナノエマルジョン、キーホールリンペットヘモシアニン及びジニトロフェノール)が含まれる。ヒトにおいて使用されるアジュバントの中では、BCG(カルメット・ゲラン(Calmette-Guerin)桿菌)及びコリネバクテリウム・パルバム(Corynebacterium parvum)が特に有用である。
現代の熱傷管理は、部分層(partial thickness)の熱傷に対して支持療法を提供し、局所用抗菌包帯材(dressing)を変えると共に、早期に壊死組織切除し、生存不可能な皮膚を再構築することを含む。現代の熱傷治療の目的は、表皮の再生に最適な環境を提供することである。皮膚の完全性の修復は、保存した毛包からのケラチノサイトの再生、又は非熱傷領域から回収した分層植皮の移植によって行われる。表皮再生期間中、皮膚に対するさらなる損傷を回避し、熱傷の進行を抑制し、創傷感染症などの続発性合併症を最小限に抑えることが重要である。全層熱傷焼痂の初期切除、即時植皮、及び局所用抗菌剤による熱傷の残りの開口又は部分層領域の治療が、熱傷コロニー形成及び侵襲性創傷感染症を最小限に抑える最も有効な手法である。(例えば、Bessey, Wound care. In Herndon DN, ed: Total Burn Care 3rd edition. Philadelphia, PA: Elsevier Inc., 2007, 127-135参照)。一般的な局所用抗菌剤には、スルファジアジン銀(SILVADENE)、酢酸マフェニド(SULFAMYLON)及び銀コロイドを含浸させた包帯材(ACTICOAT、SILVERLON)が含まれる。これらの薬剤のそれぞれは、焼痂に浸透する可変能力、グラム陰性及びグラム陽性菌の両方に対する不均等な効率、並びに宿主免疫細胞に対する潜在的毒性などの、潜在的な制限を有する(例えば、Steinstraesser et al., Antimicrob Agents Chemother 46(6):1837-1844, 2002参照)。
保存安定性
本発明のナノエマルジョンは、約40℃及び相対湿度約75%で、少なくとも最大約1カ月、少なくとも最大約3カ月、少なくとも最大約6カ月、少なくとも最大約12カ月、少なくとも最大約18カ月、少なくとも最大約2年、少なくとも最大約2.5年又は少なくとも最大約3年間、安定であり得る。
本発明のナノエマルジョンは、驚くべきことに該ナノエマルジョンが適用時にそれらの物理的構造を喪失しないので、適用時に安定である。本発明によるナノエマルジョンの適用後の皮膚表面の顕微鏡検査は、本発明のナノエマルジョンの物理的完全性を示している。この物理的完全性は、本発明のナノエマルジョンで観測される所望の吸収をもたらすことができる。
用語「ナノエマルジョン」は、本明細書で使用される場合、分散液又は液滴又は任意の他の脂質構造を指す。本発明で企図される一般的な脂質構造には、それに限定されるものではないが、単層、少数層及び多層脂質小胞、ミセル並びに層状の相が含まれる。
ある。
水相は、それに限定されるものではないが、水(例えば、H2O、蒸留水、水道水)及び溶液(例えばリン酸塩緩衝生理食塩水(PBS)溶液)を含む任意の種類の水相を含むことができる。特定の実施形態では、水相は、pH約4〜10、好ましくは約6〜8の水を含む。水は、脱イオン化することができる(以下「DiH2O」)。いくつかの実施形態では、水相は、リン酸塩緩衝生理食塩水(PBS)を含む。水相はさらに滅菌されており、発熱物質を含まないものであってよい。
本発明のナノエマルジョンの有機溶媒には、それに限定されるものではないが、C1〜C12アルコール、ジオール、トリオール、リン酸ジアルキル、リン酸トリ-n-ブチルなどのリン酸トリ-アルキル、その半合成誘導体及びその組合せが含まれる。本発明の一態様では、有機溶媒は、非極性溶媒、極性溶媒、プロトン性溶媒又は非プロトン性溶媒から選択されるアルコールである。
本発明のナノエマルジョン中の油は、任意の化粧上又は薬学的に許容される油であってよい。油は、揮発性又は不揮発性であってよく、動物油、植物油、天然油、合成油、炭化水素油、シリコーン油、その半合成誘導体及びその組合せから選択することができる。
本発明のナノエマルジョン中の界面活性剤又は洗浄剤は、薬学的に許容されるイオン性界面活性剤、薬学的に許容される非イオン性界面活性剤、薬学的に許容されるカチオン性界面活性剤、薬学的に許容されるアニオン性界面活性剤又は薬学的に許容される双性イオン性界面活性剤であってよい。
ングリコールモノヘキシルエーテル、ペンタエチレングリコールモノオクタデシルエーテル、ペンタエチレングリコールモノオクチルエーテル、ポリエチレングリコールジグリシジルエーテル、ポリエチレングリコールエーテルW-1、ポリオキシエチレン10トリデシルエーテル、ポリオキシエチレン100ステアレート、ポリオキシエチレン20イソヘキサデシルエーテル、ポリオキシエチレン20オレイルエーテル、ポリオキシエチレン40ステアレート、ポリオキシエチレン50ステアレート、ポリオキシエチレン8ステアレート、ポリオキシエチレンビス(イミダゾリルカルボニル)、ポリオキシエチレン25プロピレングリコールステアレート、キラヤ樹皮由来のサポニン、Span(登録商標)20、Span(登録商標)40、Span(登録商標)60、Span(登録商標)65、Span(登録商標)80、Span(登録商標)85、Tergitol、Type 15-S-12、Tergitol、Type 15-S-30、Tergitol、Type 15-S-5、Tergitol、Type 15-S-7、Tergitol、Type 15-S-9、Tergitol、Type NP-10、Tergitol、Type NP-4、Tergitol、Type NP-40、Tergitol、Type NP-7、Tergitol、Type NP-9、Tergitol、Tergitol、Type TMN-10、Tergitol、Type TMN-6、テトラデシル-β-D-マルトシド、テトラエチレングリコールモノデシルエーテル、テトラエチレングリコールモノドデシルエーテル、テトラエチレングリコールモノテトラデシルエーテル、トリエチレングリコールモノデシルエーテル、トリエチレングリコールモノドデシルエーテル、トリエチレングリコールモノヘキサデシルエーテル、トリエチレングリコールモノオクチルエーテル、トリエチレングリコールモノテトラデシルエーテル、Triton CF-21、Triton CF-32、Triton DF-12、Triton DF-16、Triton GR-5M、Triton QS- 15、Triton QS-44、Triton X-100、Triton X-102、Triton X-15、Triton X-151、Triton X-200、Triton X-207、Triton(登録商標)X-114、Triton(登録商標)X-165、Triton(登録商標)X-305、Triton(登録商標)X-405、Triton(登録商標)X-45、Triton(登録商標)X-705-70、TWEEN(登録商標)20、TWEEN(登録商標)21、TWEEN(登録商標)40、TWEEN(登録商標)60、TWEEN(登録商標)61、TWEEN(登録商標)65、TWEEN(登録商標)80、TWEEN(登録商標)81、TWEEN(登録商標)85、Tyloxapol、n-ウンデシルβ-D-グルコピラノシド、その半合成誘導体又はその組合せが含まれる。
ルアンモニウムクロライド(100%C16)、アルキルジメチルベンジルアンモニウムクロライド(41%C14、28%C12)、アルキルジメチルベンジルアンモニウムクロライド(47%C12、18%C14)、アルキルジメチルベンジルアンモニウムクロライド(55%C16、20%C14)、アルキルジメチルベンジルアンモニウムクロライド(58%C14、28%C16)、アルキルジメチルベンジルアンモニウムクロライド(60%C14、25%C12)、アルキルジメチルベンジルアンモニウムクロライド(61%C11、23%C14)、アルキルジメチルベンジルアンモニウムクロライド(61%C12、23%C14)、アルキルジメチルベンジルアンモニウムクロライド(65%C12、25%C14)、アルキルジメチルベンジルアンモニウムクロライド(67%C12、24%C14)、アルキルジメチルベンジルアンモニウムクロライド(67%C12、25%C14)、アルキルジメチルベンジルアンモニウムクロライド(90%C14、5%C12)、アルキルジメチルベンジルアンモニウムクロライド(93%C14、4%C12)、アルキルジメチルベンジルアンモニウムクロライド(95%C16、5%C18)、アルキルジデシルジメチルアンモニウムクロライド、アルキルジメチルベンジルアンモニウムクロライド(C12〜16)、アルキルジメチルベンジルアンモニウムクロライド(C12〜18)、ジアルキルジメチルベンジルアンモニウムクロライド、アルキルジメチルジメチルベンジル(dimethybenzyl)アンモニウムクロライド、アルキルジメチルエチルアンモニウムブロミド(90%C14、5%C16、5%C12)、アルキルジメチルエチルアンモニウムブロミド(大豆油の脂肪酸に見られるような混合アルキル基及びアルケニル基)、アルキルジメチルエチルベンジルアンモニウムクロライド、アルキルジメチルエチルベンジルアンモニウムクロライド(60%C14)、アルキルジメチルイソプロピルベンジルアンモニウムクロライド(50%C12、30%C14、17%C16、3%C18)、アルキルトリメチルアンモニウムクロライド(58%C18、40%C16、1%C14、1%C12)、アルキルトリメチルアンモニウムクロライド(90%C18、10%C16)、アルキルジメチル(エチルベンジル)アンモニウムクロライド(C12〜18)、ジ-(C8〜10)-アルキルジメチルアンモニウムクロライド、ジアルキルジメチルアンモニウムクロライド、ジアルキルメチルベンジルアンモニウムクロライド、ジデシルジメチルアンモニウムクロライド、ジイソデシルジメチルアンモニウムクロライド、ジオクチルジメチルアンモニウムクロライド、ドデシルビス(2-ヒドロキシエチル)オクチル水素アンモニウムクロライド、ドデシルジメチルベンジルアンモニウムクロライド、ドデシルカルバモイルメチルジメチル(dinethyl)ベンジルアンモニウムクロライド、ヘプタデシルヒドロキシエチルイミダゾリニウムクロライド、ヘキサヒドロ-1,3,5-トリス(2-ヒドロキシエチル)-s-トリアジン、ミリスタルコニウムクロライド(及び)Quat RNIUM 14、N,N-ジメチル-2-ヒドロキシプロピルアンモニウムクロライドポリマー、n-テトラデシルジメチルベンジルアンモニウムクロライド一水和物、オクチルデシルジメチルアンモニウムクロライド、オクチルドデシルジメチルアンモニウムクロライド、オクチルフェノキシエトキシエチル(Octyphenoxyethoxyethyl)ジメチルベンジルアンモニウムクロライド、オキシジエチレンビス(アルキルジメチルアンモニウムクロライド)、トリメトキシシリル(Trimethoxysily)プロピルジメチルオクタデシルアンモニウムクロライド、トリメトキシシリル四級(quat)、トリメチルドデシルベンジルアンモニウムクロライドなどのカチオン性ハロゲン含有化合物、その半合成誘導体及びその組合せが含まれる。
本発明のナノエマルジョンにおける使用に適した追加化合物には、それに限定されるものではないが、有機リン酸塩系溶媒、増量剤、着色剤、薬学的に許容される賦形剤、保存剤、pH調節剤、バッファー、キレート剤などの一つ又は複数の溶媒が含まれる。追加化合物は、予め乳化したナノエマルジョンに混合することができ、又は追加化合物は、乳化されるべき元の混合物に添加することができる。これらの実施形態のいくつかにおいて、一つ又は複数のさらなる化合物は、その使用の直前に、既存のナノエマルジョン組成物に混合される。
本発明のさらなる一実施形態では、ナノエマルジョンは、抗生物質又は緩和剤(熱傷治療のためなど)などの追加の活性剤を含む。別の薬剤を添加することによって、ナノエマルジョンの治療有効性を強化することができる。ナノエマルジョンは、それ自体抗菌活性を有しており、治療有効性を得るための別の活性剤と組み合わせる必要はない。細菌感染症を治療するのに適した任意の抗菌(又は抗生物質)薬剤を、本発明の局所用ナノエマルジョンに組み込むことができる。
本発明のナノエマルジョンは、任意の従来の薬学的投与方法を使用して、治療有効量のナノエマルジョン及びそれを必要としているヒト対象に投与するのに適した薬学的に許容される賦形剤を含む医薬組成物に製剤化することができる。かかる賦形剤は、当技術分野で周知である。
いくつかの例示的なナノエマルジョンを以下に記載するが、本発明の方法は、かかるナノエマルジョンの使用に限定されない。それぞれの成分及び量は、他のナノエマルジョンの調製において本明細書に記載の通り、変わり得る。(「CPC」は塩化セチルピリジニウムを指し、これはナノエマルジョンに存在するカチオン性界面活性剤である)。組成物は別段の記載がない限りw/w%である。
本発明のナノエマルジョンは、従来のエマルジョン形成技術を使用して形成することができる。例えば、米国特許出願第2004/0043041号参照。また、それら全てが参照によって組み込まれる米国特許第6,015,832号、第6,506,803号、第6,559,189号、第6,635,676号及び米国特許出願第20040043041号を参照。さらに、エマルジョンの製造方法は、米国特許第5,103,497号及び第4,895,452号(参照によって本明細書に組み込まれる)に記載されている。例示的な方法では、相対的に高いせん断力の下(例えば、高動水力及び機械力を使用して)で油を水相と混合して、約1000nm未満の平均直径を有する油液滴を含むナノエマルジョンを得る。本発明のいくつかの実施形態は、エタノールなどのアルコールを含む油相を有するナノエマルジョンを使用する。油相及び水相は、フレンチプレス又は高せん断ミキサー(例えばAdmix、Inc.、Manchester、N.H.製の、FDA承認の、例えば高せん断ミキサーが利用可能である)などの、エマルジョンを形成するのに十分なせん断力をもたらすことができる任意の装置を使用してブレンドすることができる。かかるエマルジョンの製造方法は、それら全体が参照によって本明細書に組み込まれる米国特許第5,103,497号及び第4,895,452号に記載されている。
本願を通して、より詳細に説明する通り、本発明は、嚢胞性線維症(CF)を有する対象における呼吸器感染症を治療且つ/又は予防する方法を対象とする。一般に、該方法は、ナノエマルジョンを対象に投与するステップを含み、該ナノエマルジョンは、(i)水、(ii)少なくとも一つの有機溶媒、(iii)少なくとも一つの界面活性剤、及び(iv)少なくとも一つの油を含み、約1000nm未満の平均直径を有する液滴を含む。本発明の一実施形態では、対象は、スタフィロコッカス属の種、ヘモフィルス属の種、シュードモナス属の種、バークホルデリア属の種、アシネトバクター属の種、ステノトロホモナス属の種、エシェリキア属の種、クレブシエラ属の種及びプロテウス属の種からなる群から選択される一つ又は複数の細菌種による感染症に罹患しやすいか、又はそれを有する。ナノエマルジョンは、任意の薬学的に許容される手段を使用して、有用な一投与方法である吸入により送達することができる。
本発明は、免疫応答(例えば、先天性及び/又は適応性免疫応答(例えば、バークホルデリア属の細菌種(例えば、B.セノセパシア、B.マルチボランス等)に対する宿主免疫の発生のため))の誘発のための、免疫原性ナノエマルジョン組成物及びその使用方法を提供する。本発明の組成物及び方法は、中でも、臨床(例えば、治療用及び予防用医薬品(例えば、ワクチン接種))及び研究適用における使用が見出される。
本発明のナノエマルジョン組成物を試験して、組成物を対象に肺内投与することにより、任意の組織学的な変化及び/又は病変が誘発されるかどうかを決定した。
ナノエマルジョン(W805EC)を単独で、若しくはEDTAと組み合わせて含む組成物、及び/又は高張食塩水が存在することにより、呼吸器で発見される細菌について、増殖を減衰させる、且つ/又は死滅させることができるかどうかを決定した。
単独又は20mMのEDTAとあわせた20%のナノエマルジョン(NE)による、10分でのPBS中のセパシア菌の死滅(図1参照);
単独又は20mMのEDTAと合わせた20%のNEによる、20分以内のPBS中のセパシア菌の死滅(図2参照);
単独又は20mMのEDTAと合わせた20%のNEによる、40分以内のPBS中のセパシア菌の死滅(図3参照);
単独又は20mMのEDTAと合わせた20%のNEによる、40分以内又は60分以内のPBS中のセパシア菌の死滅(図4参照);
NE単独及びNEとEDTAによる、15分及び30分での高張食塩水(6%NaCl)中のセパシア菌の死滅(図5参照);
7%NaCl中のセパシア菌及び緑膿菌の15分以内の死滅(図6参照);及び
7%NaCl中のセパシア菌及び緑膿菌(混合培養物)の15分以内の死滅(図7参照)。
材料と方法
細菌株及び培養条件。バークホルデリア分離株75株、並びに緑膿菌、アクロモバクター・キシロスオキシダンス、ステノトロホモナス・マルトフィリア、アシネトバクター種、パンドレア種(P.アピスタ(P. apista)、P.プノメヌサ(P. pnomenusa)、P.プルモニコーラ(P. pulmonicola)、P.ノリンベルゲンシス(P. norimburgensis)、及びP.スプトラム(P. sputorum))、及びラルストニア種(R.マンニトリリティカ(R. mannitolilytica)及びR.ピッケティ(R. pickettii))を含めた他のCF関連種に属する分離株75株を含めた150株の分離株を分析した。臨床分離株145株を、Burkholderia cepacia Research Laboratory and Repository(BcRLR、University of Michigan、Ann Arbor、MI)から入手した。これらは、1997年9月〜2007年10月に142個体から回収され、米国内のCF治療センター62ヶ所から分析のためにBcRLRに付託された。残りの5株は、環境分離株であるB.マルチボランス、ATCC17616(American Type Culture Collection、Manassas、Virginia)、P.ノリンベルゲンシス、LMG 18379T(BCCM/LMG Bacteria Collection、Laboratorium voor Micrbiologie Gent、Universiteit Gent、Ghent、Belgium)及びB.ピロシニア(B. pyrrocinia)、HI3642(BcRLR collection)、並びに臨床型の株であるB.セノセパシア、LMG 16656T(aka J2315)及びP.プルモニコーラ、LMG 18106Tを含んだ。臨床分離株147株のうち134株(91%)は、CFの人間から回収され;そのうち114株(78%)が痰培養からのものであり、残りは咽頭スワブからのもの(n=17)、気管内吸引/気管吸引からのもの(n=5)、血液からのもの(n=5)、気管支洗浄からのもの(n=3)、並びに、上顎洞、腹膜腔、及び喉頭蓋それぞれからのものであった。分離株150株のうち49株(33%)が、付託された微生物研究所で行われた感受性試験に基づいて、多剤耐性であると定義された(抗生物質の二つ又は三つのクラス:カルバペネムを含むラクタム系、アミノグリコシド系、及びキノロン系の試験した全ての薬物に対して耐性;例えば、Taccetti et al., Eur J Epidemiol. 1999 Jan;15(l):85-8を参照されたい);そのうち20株(41%)が汎耐性であった。残りの分離株のうち72株(48%)は、少なくとも一つの抗生物質に対して感受性であり、29株(19%)の分離株については感受性試験の結果が入手できなかった。全ての分離株が、BcRLRにおいて、記載の通り表現型アッセイ及び遺伝子型アッセイを使用した多相分析によって種のレベルまで同定された(例えば、Reik et al., J Clin Microbiol. 2005 Jun;43(6):2926-8を参照されたい)。例外として、アシネトバクターについては、決定的な種特異的アッセイを欠いたために属のレベルまでしか同定されなかった。全ての分離株は、以前に記載された通り、BOX AlRプライマーを使用した反復性の遺伝子外エレメントPCR(rep-PCR)分類にも供して(例えば、Coenye et al., J Clin Microbiol. 2002 Sep;40(9):3300-7を参照されたい)、試験パネルに含まれる分離株150株全てが遺伝子型的に明らかに異なることを確実にした。細菌を、脱脂乳中又は15%のグリセロールを含んだルリア・ベルターニ(LB)培地中、-8O℃で貯蔵し、一晩の凍結貯蔵物からミューラー・ヒントン(MH)寒天上に回収した。
浮遊性細菌の感受性。P4075ECが不透明な白色であるために、標準のCLSI承認マイクロタイター段階希釈法を、細菌の生存可能性の指標としてレサズリンを加えることを含むように改変した。P4075ECを15.6〜2000μg/mlの濃度範囲で試験した。MICを、青色から淡紅色への色の変化を生じなかったP4075ECの最低濃度として定義した(図8参照)。この視覚的な屈曲点と蛍光定量的分析の比較により、63%のMICウェルが無処理の対照のウェルの代謝活性の≦1%を有し;91%が対照のウェルと比較して≦5%の代謝活性を有し、96%が対照のウェルと比較して≦10%の代謝活性を有した。MICの結果を図11に示す。全ての株が、試験した濃度のP4075ECによって阻害された。150株のパネル全体に対するMIC50は31.2μg/mlであり、MIC90は125μg/mlであった。38株(25%)が、試験した最低濃度のP4075ECによって阻害され(15.6μg/ml)、単一の株のみが、阻害のためにそれぞれ250μg/ml及び500μg/mlの濃度を必要とした。P4075ECは、バークホルデリア株(MIC5062.5μg/ml;MIC90125μg/ml)よりも非バークホルデリア株に対してわずかに活性であった(MIC5031.2μg/ml;MIC9062.5μg/ml)(図9参照)。活性は、試験した10株のバークホルデリア種にわたって同程度であった。多剤耐性バークホルデリア株に対するP4075ECの活性は、一つ又は複数の抗生物質に対して感受性の株と比較して差異は見られなかった。
ナノエマルジョンP4075EC、7%の生理食塩水及び20mMのEDTAを含む組成物を、バイオフィルム(個々又は混合)中に存在する細菌を死滅させるその能力について試験した。
薬物の供給源。W205EC、ロット×1151、濃度5000μg/ml(貯蔵濃度)は、Patheon、Mississaauga、Ontario、Canadaで製造された臨床試験材料(NB-001)のプール試料であった。P4075EC、ロット×l138、及びW205GBA2、ロット×1103は、NanoBio Corporationにおいて製造された。比較化合物であるアジスロマイシン、アンピシリン、セフェピム及びクリンダマイシンは、The United States Pharmacopeiaから購入した;カタログ番号は、それぞれ、1046056、1033000、1097636、1136002であった。テトラサイクリンは、Fluka Biochemikaから購入し、カタログ番号は87128であった。エマルジョンは、水と不混和性の油相を水相と混合することによって作製した。基剤は、以下の表5に示し、それは未希釈のエマルジョンを表しており、それを所望の%にさらに希釈した。組成物は、特に断りのない限り、w/w%である。
薬物及び分離株の供給源。12種の比較化合物、クリンダマイシン(USP#1136002)、ドキシサイクリン(USP#1226003)、エリスロマイシン(Sigma#E0774)、レボフロキサシン(Sigma#28266)、酢酸マフェニド(USP#1373008)、ムピロシン(Sigma#M7694)、シルバデン(Silvadene)(USP#61260)、バンコマイシン(Sigma#V1764)、オキサシリン(Sigma#28221)、スルファメトキサゾール(USP#1631001)、トリメトプリム(USP#1692505)、及び硝酸銀(VWR #VW3462-0)を4種のナノエマルジョンと比較した。4種のナノエマルジョンは、(a)W805EC、(b)W205ECEDL2、(c)W205GBA2ED及び(d)P4075ECであった。薬物のパネルのテンプレートを提供している表10を参照されたい(パネルの横方向に希釈が増加している)。エマルジョンは、水不混和性の油相を水相と混合することによって作製した。W205ECEDL2製剤は微小流動化した。基剤は以下の表9に示し、それは未希釈のエマルジョンを表しており、それを所望の%にさらに希釈した。特に断りのない限り、組成物はw/w%である。
MIC及びMBCについてのデータを表11及び12に示す。4種のナノエマルジョン全てが、MRSAの臨床分離株に対して効力があり、市中感染MRSA(SCCmecIV型)又は院内感染MRSA(SCCmecI〜III型)に対して示される活性と差異がなかった。さらに、公知の抗生物質のいずれに対してもナノエマルジョンの交差耐性はないように見えた。P4075EC、W205ECEDL2及びW805ECについてMIC90値は2μg/mlであった。W205GBA2EDは、4μg/mlのMIC90値を有した。MIC90値により、このMRSAコレクションが、経口用抗生物質であるドキシサイクリン(MIC90=4μg/ml)、スルファメトキサゾール(MIC90=4μg/ml)、トリメトプリム(MIC9O=0.5μg/ml)、及びバンコマイシン(MIC90=1μg/ml)に対して感受性であることが示された。分離株はおおむね、経口用抗生物質であるレボフロキサシン(MIC90=32μg/ml)、クリンダマイシン(MIC90=>64μg/ml)及びエリスロマイシン(MIC90=>128μg/ml)に対して耐性であった。熱傷の創傷感染症の治療に使用される局所用抗生物質であるシルバデン及び硝酸銀は、それぞれ、32μg/ml及び0.00063%のMIC90値を有した。酢酸マフェニド(Sulfamylon)は、熱傷の創傷感染症に対する別の局所的治療法であるが、MRSAに対して一様に不活性であった(MIC90=>128μg/ml)。ムピロシンは、皮膚及び軟組織の感染症に対する局所的治療法であり、MRSAの保因を根絶するために同様に使用される;分離株の31%がこの抗生物質に対して耐性であった。
W805EC、W205ECEDL2、W205GBA2ED及びP4075ECは、MIC90値が2μg/ml又は4μg/mlであり、MRSA臨床分離株に対して同等に有効であると思われた。さらに、MBC90/MIC90比に基づいて、4種のナノエマルジョンは殺菌性であった。予想されたように、MRSAは多剤耐性であり、MRSA感染症を治療するための新規薬剤の必要性が強調された。本発明によるナノエマルジョンは緑膿菌及びバークホルデリア種などの重大なグラム陰性病原体に対する活性も有するので(LiPuma et al., "In vitro activities of a novel nanoemulsions against Burkholderia and other multidrug-resistant cystic fibrosis-associated bacterial species," Antimicrob. Agents Chemother., 53:249-255 (2009))、ナノエマルジョンは嚢胞性線維症患者の吸入治療/維持に有用である。さらに、ナノエマルジョンは、本明細書に記載の病原因子の感染を予防/治療するための熱傷創傷の治療において有用である。
CF患者から単離した多剤耐性のグラム陰性細菌分離株を本実施例において試験した。MIC及びMBCを、CLSIガイドライン及び標準的な方法であるM7-A7及びM100-S17を使用して決定した。属に応じて、1mM又は5mMの、NE活性の透過性増強剤であるEDTAの存在下でMICを行った。ステノトロホモナス及びアシネトバクターは、ナノエマルジョン中に1mMのEDTAを有した。シュードモナス及びバークホルデリアは、ナノエマルジョン中に5mMのEDTAを有した。これは、標準のMIC及びMBCの決定、並びにチェッカーボード相乗効果研究の間の場合であり、時間-死滅試験については以下に記載する。NE(P4075EC)は高い濃度において不透明であるので、代謝活性に応答して発色変化をもたらす酸化還元指示薬であるalamar blueの添加を使用してNE(P4075EC)のMICを決定した。
薬物A及び薬物Bの両方についてこの比を算出する。分数を合計する。合計を以下の範囲と比較する。
不関:2種の薬物についてのFICの合計 >0.5〜≦4;
拮抗的:2種の薬物についてのFICの合計 >4
図19Aにおいて、横行Dは、組み合わせる2種の薬物のフランキング濃度を選択するために使用する予め決定したMICを示す。図19Bにおいて、X軸及びY軸上の水平のスラッシュ記号は、各薬物単独の典型的なMIC濃度を示す。
分離株46株を評価し、その全てはUniversity of Maryland-Baltimore School of Dentistryから入手した。受け取った分離株は以下の通りであった:シュードモナス20株、バークホルデリア11株、アシネトバクター10株及びステノトロホモナス5株。緑膿菌分離株は、多くのCF患者において実証されている定義済みのリピドA修飾を有した。ステノトロホモナスの追加の分離株10株を第2の供給源から受け取った(Eurofins、Virginia)。これらの10株は一覧の最後にあり、試料を取り出した個体の年齢を伴う分離株である。分離株を以下の表14に要約する。
薬物濃度の調製を以下の表17及び18に記載する。特に、表17は、96ウェルの薬物パネルプレートの調製に関する詳細を提供し、表18は、96ウェルのチェッカーボード相乗効果プレートの調製に関する詳細を提供する。
96ウェルマイクロタイタープレートにおいて、播種されたウェルを35℃で20時間インキュベートした後に観察した。各横行には、横行それぞれに左から右に高濃度から低濃度までの範囲で薬物が存在し、各ウェルは左隣のウェルの1/2の濃度である。一連で最初の透明のウェルをMICとみなした。これは、培地を濁らせる細菌の増殖を阻害した薬物の濃度である。ナノエマルジョンには本質的に濁度があるので、ナノエマルジョンを含有する横行には、alamar blue(CellTiter Blue、Promega)を培養物の容量の20μL〜100μLの割合で加えた。プレートを1時間インキュベーターに戻し、最初の青色のウェルをMICとみなした(低濃度のナノエマルジョンで、細菌が増殖しているウェルは淡紅色に変化した)。各ウェル中の出発接種物は、一般に2〜7×l05cfu/mLであった。出発接種物に対する特定の濃度は、各試験プレートについて、マイクロタイタープレートを設置した時点で、マイクロタイタープレートに播種するのに使用する接種物をサンプリングすることによって決定した。正確な細菌濃度を使用して各試験パネルのパネル内の各薬物についてのMBCを決定した。MBCを、細菌数を99.9%減少させた薬物濃度として定義する。MICは、MICと、それに加えて左に4つのウェルから10μL/ウェルのサンプリングを始め、寒天プレート上に、プレート当たり試料一つを置くために使用する目印である。寒天プレートを35℃で24時間インキュベートし、コロニーを計数した。得られたコロニーの数値を接種物の出発濃度と比較して、どのウェル、すなわちどの薬物濃度がcfu/mLで3logの降下をもたらしたかを決定した。
各ウェル中の出発接種物は、一般に2〜7×105cfu/mLであった。出発接種物に対する特定の濃度は、各試験プレートについて、マイクロタイタープレートを設置した時点で、マイクロタイタープレートに播種するのに使用する接種物をサンプリングすることによって決定した。これらの正確な細菌濃度を使用して各試験パネルのパネル内の各薬物についてのMBCを決定した。MBCを、細菌数を99.9%減少、細菌の元の濃度から3log降下させた薬物濃度として定義する。MICは、MICと、それに加えて右に縦行11までの全ウェルから10μL/ウェルのサンプリングを始め、寒天プレート上に、プレート当たり試料一つを置くために使用する目印である。寒天プレートを35℃で24時間インキュベートし、コロニーを計数した。得られたコロニーの数値を接種物の出発濃度と比較して、どのウェル、すなわちどの薬物濃度がcfu/mLで3logの降下をもたらしたかを決定した。
バークホルデリアNB 29 @ O.D. 0.14〜0.20の細菌懸濁液を用意した。T0、無処理の対照のために、生理食塩水チューブの中に生理食塩水500μL及び細菌懸濁液500μLを混合し、次いで、5mMのCaCl2を含んだTA(トリプトン-アゾレクチン)培地と1対1で混合して希釈し、下記の通りプレーティングした。処理チューブ中、細菌懸濁液を処理混合物(CPCが64μg/mLの2×P4075EC及び10mMのEDTA)に1対1で加え、10分後、20分後及び30分後の各時点で400μLを抜き取り、5mMのCaCl2を含んだTA培地400μLと混合した。次に、100μLを抜き取って、生理食塩水中、10-8まで段階的に1/10希釈した。次いで、その一連の各希釈チューブから100μLを抜き取り、3連でプレーティングした。これらのプレートを、35℃で一晩インキュベートし、計数して各時点におけるcfu/mLを決定した。その時点で、無処理の試料又は処理した細菌を同時に加えてTA溶液を中和し、それぞれの時点でグルタルアルデヒド113μLに試料450μLを加えた。
走査型電子顕微鏡(SEM)用に、Sorensonバッファー中グルタルアルデヒド10%水溶液113μL、pH7.4を細菌懸濁液450μLと混合し、ナノエマルジョンに曝露させた。混合物をボルテックスにかけ、4℃で少なくとも18時間置いた。表19は、試料を走査型電子顕微鏡用に固定し、染色する手順を提供する。
a.MIC及びMBCのデータ
P4075ECについてのMIC90/MBC90値は、緑膿菌に対して8/64μg/ml、B.セノセパシアに対して64/>514μg/ml、A.バウマンニに対して8/64μg/ml、及びS.マルトフィリアに対して8/32μg/mlであった。コリスチンは、緑膿菌、B.セノセパシア、A.バウマンニ及びS.マルトフィリアに対して、それぞれ、2/8、>32/>32、1/>16及び>32/>32のMIC90/MBC90値を有した。セフェピム、イミペネム、レボフロキサシン及びトブラマイシンは、全ての株に対して、それぞれ、≧32/>32μg/ml、≧32/>32μg/ml、16/16μg/ml及び>32/>32μg/mlのMIC90/MBC90値を有した。
10株のバークホルデリア、ステノトロホモナス及び10株のアシネトバクターを試験して、P4075EC+EDTAを、慢性肺感染症を治療するためにCF患者の肺に使用される二つの伝統的な抗微生物剤である、コリスチン又はトブラマイシンのいずれかと組み合わせたときのMICの変化を決定した。コリスチンと組み合わせたP4075EC+EDTAは、ステノトロホモナス株に関して90%(FICに関して)及び70% (FBCに関して)相乗的であるが、トブラマイシンと組み合わせたときは、FICで20%相乗的、FBCで0%相乗的なだけであり、不関であることがわかった。アシネトバクター株に対しては、コリスチンと組み合わせたP4075EC+EDTAは、FICで20%相乗的、FBCで0%相乗的なだけであり、同様にトブラマイシンと組み合わせたとき、FICで10%相乗的、FBCで10%相乗的なだけであり、不関であることがわかった。バークホルデリア株に対しては、コリスチンと組み合わせたP4075EC+EDTAは、FICで30%相乗的、FBCで10%相乗的なだけであり、不関であるが、トブラマイシンと組み合わせたとき、FICで50%相乗的、FBCで20%相乗的であることがわかった。
時間-死滅は、全体で、無処理の開始時から30分時点までで、cfu/mlで4.44logの減少という結果であった。10分時点それぞれは、以下の通り、1〜2logの減少を有した:無処理時点から10分時点までは1.40logの減少、10分時点から20分時点までは、1.91logの減少、及び20分時点から30分時点までは1.13logの減少であった。図20A〜20Dを参照されたい。
本実施例は、試験したP4075ECなどのナノエマルジョンが、バークホルデリア及びステノトロホモナスのコリスチン耐性分離株を含めた、多剤耐性株に対して有効であることを実証している。記載のシュードモナス種におけるリピドA修飾体はいずれもP4075ECによってMIC/MBCに影響がなかった。2種の主要な抗生物質であるコリスチン及びトブラマイシンによる拮抗の証拠は観察されず、ステノトロホモナスの場合、相乗効果が明らかであった。このことは、CF患者の治療について、その治療プログラムを複雑にしている、ナノエマルジョンを使用するために通常の抗生物質レジメンを一時停止する必要がないはずなので、有用である。SEM画像は、この場合では、丈夫な外膜を有するという評判を持つグラム陰性細菌の、接触機構での死滅を実証している。
材料と方法
試薬。特に断りがなければ、全ての試薬は、Sigma-Aldrich Corp.(St. Louis、MO)から購入した。
ナノエマルジョンで処置した動物は、生理食塩水で処置した対照と比較して、皮膚組織のグラム当たりの細菌CFUの平均値(6.5×104対7.9×107、p=0.07)及び中央値(0対4.4×106、p<0.05)の数値が減少した(図21参照)。W205GBA2EDで処置した動物対W205GBA2EDプラセボで処理した動物について、皮膚の細菌数に同様の減少が見られた(平均値:6.5×104対5.5×106、p=0.02)。臨床組織試料に対して定量的な創傷培養を行う場合、一般に、組織グラム当たり、生物がCFUで1×105超増殖することを陽性の結果とみなす(例えば、Neal et al., J Burn Care Rehabil 2:35-39, 1981;Taddonio et al., Burns Incl Therm Inj 14(3): 180-184, 1988; Uppal et al., Burns 33(4):460-463, 2007を参照されたい)。これらの基準を使用して、対照群の32体の動物のうち29体が定量的な創傷培養の陽性の証拠を示し、ナノエマルジョン群の23体の動物のうち3体のみがこのレベルの創傷感染症の証明を実証した(91%対13%、p<0.0001、以下の表24を参照されたい)。Sulfamylonで処理した動物でも、生理食塩水対照と比較して、創傷の細菌レベルの中央値の有意な減少が実証された(3×104対4.4×106、p<0.05)。ナノエマルジョン又はSulfamylonを用いた処置によって、生理食塩で処置した動物と比較して、熱傷創傷から培養したシュードモナスのレベルに同様の減少が生じた。しかし、プラセボ群とSulfamylon群の間に統計的有意差はなかったのに対して、W205GBA2EDプラセボと比較したW205GBA2ED群については差異があった。
中間層の熱傷をもたらす熱湯損傷により、偽の損傷動物と比較して、損傷の32時間後に得た皮膚のホモジネート中の皮膚のIL-Iβ及びサイトカイン誘導性好中球化学誘引物質-3(CINC-3)のレベルに差異が生じた(図22A及びE参照)。熱傷の16時間後及び24時間後にW205GBA2EDを用いて処置すると、細菌感染の非存在下で、皮膚のこれらの2種の炎症性メディエーターのレベルが減少して基線(偽)まで戻った。細菌の創傷感染を発生させなかった実験では、熱傷を受けた皮膚においてラットCXCケモカインCINC-3が上昇したにもかかわらず、ミエロペルオキシダーゼアッセイによって測定された好中球分画における差異は観察されなかった。これらの三つの群全て(偽、熱傷、及び熱傷+W205GBA2ED)でCINC-1について差異が見られたが(p=0.04、ANOVA)、群間比較の値は統計的有意に達しなかった。
ナノエマルジョンで処置した群からの皮膚のホモジネートは、IL-Iβ及びIL-6のレベルは、生理食塩水で処置した動物において測定されたレベルと比較して大幅に縮小した(図23A及びB参照)。動物の二つの実験群間で、TNF-αレベルに統計的有意差は見られなかった(図22C及びD参照)。感染した熱傷創傷を、Sulfamylonを用いて処置すると、対照と比較して、測定された炎症性サイトカイン(IL-1β、IL-6、TNF-α、CINC-1又はCINC-3)の皮膚レベルにどんな有意な変化ももたらされなかった。W205GBA2ED又はSulfamylonいずれかを用いた治療によって、感染した熱傷創傷において損傷の32時間後に見られたミエロペルオキシダーゼのレベルが減少した。したがって、一部の実施形態では、ナノエマルジョン及び/又は抗微生物剤を用いた処置により、中間層熱傷創傷への好中球分画が減少する。
熱傷創傷は、著しいレベルの毛細管漏出に関連する。これは血管内容積の枯渇及び静脈内への大量の晶質液投与の必要性につながる。ナノエマルジョンを用いた処置により、炎症が減少すると同時に毛細管漏出が減少するかどうかを評価するために、エバンスブルーアッセイを利用して血管透過性を測定した。血流から浸出し皮膚組織内に浸出するこの色素の量を定量的に測定することにより、ナノエマルジョンで処置した動物が、生理食塩水で処置した対照よりも少ない、熱傷後の毛細管漏出及び組織水腫の証拠を有したことが明らかになった(図26参照)。
熱損傷の後、毛包細胞において皮膚のアポトーシスが起こる。蛍光標識TUNELアッセイを使用すると、生理食塩水で処置した熱傷創傷は、緑色に見える強いFITC-TUNEL陽性細胞の証拠を示した(図27参照)。DAPI核染色により、青色に染色された細胞を持つ、冠状又は矢状の薄片に切った毛包を同定することが可能になる。FITC-TUNEL陽性細胞は緑色に見え、アポトーシス細胞を表している。合成した画像では、アポトーシス毛包細胞は、熱傷+生理食塩水動物からのスライドガラスにおいて明らかであり、これらの変化は、熱傷+W205GBA2EDで処理した動物では縮小する。対象の毛包部内のTUNEL陽性細胞のピクセルを計数することにより、局所用ナノエマルジョンを用いた処置による毛包細胞のアポトーシスの減少を定量化することが可能になる(図28参照)。生理食塩水で処置した対照動物では、偽の熱傷動物と比較してTUNEL陽性細胞の量が増加した。W205GBA2ED処置及びプラセボ処置ではどちらも、熱損傷の12時間後に回収した組織において、中間層熱傷損傷後の毛包細胞のアポトーシスの減少がもたらされた。この差異は、熱傷の24時間後に試料採取された皮膚においては明らかでなかった。したがって、一部の実施形態では、本発明は、熱傷後の早期の毛包におけるアポトーシスによる細胞死を減少させる方法であって、熱傷後の早期にナノエマルジョン(例えば、W205GBA2ED)を熱傷創傷に投与することを含む方法を提供する。
材料と方法
B.セノセパシア株K56-2の貯蔵維持及び培養。バークホルデリア・セノセパシア株K56-2は、Dr. Pam Sokol(University of Calgary)から豊富に提供された。K56-2は、臨床分離株であり、バークホルデリアの分子微生物学及びゲノム研究に使用されている(例えば、Goldberg, 2007 Burkholderia Molecular Microbiology and Genomics. Gent, Belgium: Taylor & Francisを参照されたい)。これは伝染性且つ悪性のB.セノセパシアETl 2系統の代表である(例えば、Johnson et al., 1994, J Clin Microbiol 32, 924-930; Saijan et al., 2008, Infect Immun 76, 5447-5455を参照されたい)。株間の中和アッセイのために、バークホルデリア・マルチボランス、ATCC 17616を使用した。K56-2株及びATCC 17616株をどちらも、15%グリセロールを含んだルリア・ベルターニ培地中、-80℃で貯蔵し、凍結貯蔵物からブレインハートインフュージョン寒天上に回収し、37℃で一晩置いた。
免疫反応性OMPタンパク質を、銀染色(図29B参照)及びウェスタンブロット(図29C及びD参照)によって同定した。主要な反応性バンドは、どちらの場合でも62KDa、45KDa、17KDa、及び10KDaにおいて実証された。しかし、バンドの強度は、PBS調製物中OMPを用いて免疫化したマウスからの等価の血清希釈物を用いて探索したブロット(図29D参照)と比較して、NEベースのワクチンを用いて免疫化したマウスからの血清のほうがずっと高かった(図29C参照)。
OMPを用いた鼻部ワクチンによって誘導されたOMPに対する体液性免疫応答を、CD-1マウスにおいてin vivoで特徴付けた。OMP調製物+NE(OMP-NE)を5μg又は15μgのいずれかを用いて鼻腔内ワクチン接種することにより、最初のワクチン接種後6週間の時点で、それぞれ、2.8×105及び5.1×105の、高いOMP特異的IgG抗体の血清力価がもたらされた(図30A参照)。アジュバントなしのOMPは免疫原性であり、最初のi.n.免疫化の6週間後、1.9×104及び3.8×104の血清抗OMPIgG力価をもたらした。しかし、アジュバントとしてNEを含んだ処置群は、追加免疫後の全ての時点でアジュバントなしの群よりも有意に高度に応答した(13〜30倍)(p<0.05)。さらに、PBS中OMPを用いて免疫化したマウスは、2回目のワクチン接種の後に有意な追加免疫の効果を実証しなかった(図30A参照)。
血清IgGサブクラスの分析を行って、細胞性応答のヘルパーT細胞型のバイアスを決定した。IgGサブクラスの分布パターンは、OMP-NEを用いてi.n.免疫化することにより、Th1型IgG2bサブクラス抗体対Th2型IgG1サブクラス抗体のレベルが上昇した(p=0.048)(図31A参照)。それに対し、PBS中OMPを用いた免疫化では、同様のレベルのIgG1抗体及びIgG2b抗体が生じた(図31A参照)。
LPSが本質的にバークホルデリア種のOMP調製物に関連していることが以前実証されている(例えば、Bertot et al., 2007, Infect Immun 75, 2740-2752を参照されたい)。免疫応答の特異性を調査し、B.セノセパシアのOMP調製物中のエンドトキシンの、可能性のある免疫を増強する役割を評価するために、OMP-NEを用いて免疫化したマウスからの血清を、粗製OMP、エンドトキシン枯渇OMP、又はLPSのいずれかを用いたELISAを使用して分析した。IgGの反応性は、エンドトキシン枯渇OMP調製物及び粗製OMP調製物に対して最も高かった。統計分析により、LPSに対するエンドトキシン枯渇OMP(OD405=2.37±0.11(p=0.001))及びLPSに対する粗製OMP(OD405=1.49±0.14(p=0.01))において有意に高い応答性が示された。しかし、血清は、有意なレベルのIgG抗LPS抗体も含有した(OD405=0.59±0.05)。
B.セノセパシア中和抗体を、コロニー減少アッセイ使用して決定した。鼻腔内に5μg又は15μgのOMP-NEをワクチン接種したマウスは、ワクチン接種していないマウスからの血清と比較して、それぞれ、92.5%又は98.3%のB.セノセパシアコロニーを阻害した血清中和抗体を産生した(図33参照)。アジュバントを欠くOMPを用いてワクチン接種したマウスも同様に、33.3%及び46.7%(それぞれ、5μgのOMP-PBS及び15μgのOMP-PBSについて)のcfuの減少をもたらし、比較的高レベルの阻害を実証した。統計分析により、B.セノセパシアの増殖が、OMP調製物単独で免疫化されたものからの血清と比較して、OMP-NEから導かれる血清によって有意に阻害されたことが示された(図33参照)。
鼻腔内免疫化の保護作用を、肺感染症モデルにおいて、in vivoでさらに試験した。OMP-NE又はアジュバントなしのOMPを用いて鼻腔内免疫化したマウスにおいて、気管内接種した後6日間、肺組織からのB.セノセパシアのクリアランスを評価した。15μgのOMP-NEをワクチン接種することにより、ワクチン接種していないマウスと比較して有意に高い肺のクリアランス速度がもたらされた(p=9.2×10-3)(図34A参照)。6日目の肺の平均細菌負荷量は、肺当たり1.28×106±3.36×106cfuであったワクチン接種してないマウスと比較して、15μgのOMP-NEを用いてワクチン接種したマウスでは、22.5±26.2cfuであり、細菌負荷量が5log超減少したことを示している。
B.セノセパシアの感染により、OMP-NE又はアジュバントなしのOMPを用いて免疫化したマウスよりも、ワクチン接種していないマウスにおいて、6日目までに体温調節が大幅に失われる。B.セノセパシアを感染させた6日後の平均体温は、15μgのOMP-NEを用いてワクチン接種したマウス(36.8℃±0.31、p=0.008)、5μgのOMP-NEを用いてワクチン接種したマウス(37.1℃±0.42、p=0.008)、又は5μgのアジュバントなしのOMPを用いてワクチン接種したマウス(36.8℃±0.61、p=0.01)と比較して、ワクチン接種していないマウス(35.4℃±0.7)では有意に低下した。
Claims (10)
- 対象における肺疾患又は呼吸器感染症の治療又は予防に用いるためのナノエマルジョンであって、
(a)治療又は予防がナノエマルジョンを対象に肺送達により投与するステップを含み;
(b)対象が、一種又は複数種の微生物による感染症に罹患しやすいか、又はその感染症を有し;
(c)ナノエマルジョンが、
(i)水、
(ii)約0.001%〜約10%の、塩化セチルピリジニウム、Poloxamer 407、Tween 20、ポリソルベート80、アルキルジメチルベンジルアンモニウムクロライド、又は塩化ベンザルコニウムである少なくとも一種の界面活性剤、
(iii)約0.1%〜約50%の少なくとも一種の有機溶媒、及び
(iv)約1%〜約80%の少なくとも一種の油
を含み;
(d)ナノエマルジョンが約1000nm未満の平均直径を有する液滴を含む;
前記ナノエマルジョン。 - 肺疾患が急性肺胞疾患、急性気管支炎、急性肺損傷(ALI)、急性呼吸促迫症候群(ARDS)、アレルギー感染症、石綿肺症、喘息、細菌性肺炎、ベリリウム肺症、気管支炎、気管支拡張症、綿肺症、慢性閉塞性肺疾患(COPD)、炭坑夫塵肺症(CWP)、嚢胞性線維症(CF)、肺気腫放射線肺炎、好酸球性肺炎、真菌性肺炎、特発性肺線維症(ILD)、コラーゲン血管障害に関連するILD、間質性肺疾患、人工呼吸器誘発肺損傷、胎便吸引症候群(MAS)、マイコバクテリア肺炎、院内肺炎、閉塞性呼吸器疾患、中耳炎、肺炎、ニューモシスチス・カリニ(Pneumocystis carinii)肺炎、塵肺症、ニューモシスチス・カリニ(Pneyumocystis carinii)、肺浮腫、インフルエンザなどの別の障害の結果であるか又はそれに続発する肺炎症、呼吸器感染症、呼吸器合胞体ウイルス(RSV)、呼吸促迫症候群(RDS)、喫煙に起因する炎症に関連する呼吸器感染症、関節リウマチ、鼻炎/副鼻腔炎、サルコイドーシス、敗血症、珪肺症、煙吸引、全身性エリテマトーデス、全身性硬化症、結核、上気道疾患、人工呼吸器関連肺炎、及びウイルス肺炎からなる群から選択される、請求項1に記載のナノエマルジョン。
- 微生物が細菌、グラム陽性菌種、グラム陰性菌種、古細菌、真菌、カビ、酵母、原生動物、ウイルス、マイコプラズマ、皮膚糸状菌、プリオン、寄生生物、又はそれらの任意の組み合わせである、請求項1又は2に記載のナノエマルジョン。
- 呼吸器感染症又は肺疾患が微生物のバイオフィルム及び/又は嚢胞性線維症の痰と関連している、請求項1から3のいずれか一項に記載のナノエマルジョン。
- 治療または予防が、ナノエマルジョンの投与の前、最中又は後のいずれかに、一種又は複数種の抗微生物剤を投与するステップをさらに含み、
ここで、
(a)ナノエマルジョン及び少なくとも一種の抗微生物剤の投与が、部分阻害濃度(FIC)指数、部分殺菌濃度(FBC)指数又はその組合せによって定義されるように相乗的であり;
(b)ナノエマルジョンが、抗微生物剤といかなる拮抗作用も示さず;
(c)抗微生物剤が抗生物質、抗体、抗菌酵素、ペプチド、及びランチオン(lanthione)含有分子からなる群から選択され;又は
(d)(a)、(b)、及び/又は(c)の任意の組み合わせ;
である、請求項1から4のいずれか一項に記載のナノエマルジョン。 - (a)ナノエマルジョンが、最小の毒性若しくは副作用を示すか、又は毒性若しくは副作用を全く示さず;
(b)ナノエマルジョンの最小阻害濃度(MIC)、最小殺菌濃度(MBC)又はその組合せが、ナノエマルジョンの静菌活性又は殺菌活性を示し;
(c)一種又は複数種の微生物種が、一種又は複数種の抗生物質に対して耐性を示し;
(d)微生物種がメチシリン耐性黄色ブドウ球菌(MRSA)であり;
(e)微生物種がバンコマイシンに耐性であり;
(f)微生物種がフルオロキノロンに耐性であり;
(g)微生物種がコリスチンに耐性であり;
(h)微生物種が多剤耐性であり;
(i)微生物種がバークホルデリア(Burkholderia)属の薬剤耐性菌種であり;
(j)微生物種がシュードモナス(Pseudomonas)属の薬剤耐性菌種であり;
(k)ナノエマルジョンが微生物耐性を示さず;又は
(l)それらの任意の組み合わせ;
である、請求項1から5のいずれか一項に記載のナノエマルジョン。 - (a)キレート剤;
(b)少なくとも一種の保存剤;
(c)少なくとも一種のpH調節剤;
(d)少なくとも一種のバッファー;
(e)少なくとも一種の抗生物質製剤;又は
(f)それらの任意の組合せ;
をさらに含む、請求項1から6のいずれか一項に記載のナノエマルジョン。 - ナノエマルジョンが対象において全身的に吸収されないか、又はごくわずかなナノエマルジョンが対象において全身的に吸収される、請求項1から7のいずれか一項に記載のナノエマルジョン。
- 対象が嚢胞性線維症(CF)を有する、請求項1から8のいずれか一項に記載のナノエマルジョン。
- ワクチンに用いるための、請求項1から9のいずれか一項に記載のナノエマルジョン。
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PCT/US2009/058449 WO2010036938A2 (en) | 2008-09-26 | 2009-09-25 | Nanoemulsion therapeutic compositions and methods of using the same |
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EP (1) | EP2349209A2 (ja) |
JP (1) | JP5722782B2 (ja) |
CN (1) | CN102223876A (ja) |
AU (1) | AU2009296458A1 (ja) |
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WO (2) | WO2010036945A2 (ja) |
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2009
- 2009-09-25 CA CA2738245A patent/CA2738245A1/en not_active Abandoned
- 2009-09-25 US US12/567,470 patent/US20100092526A1/en not_active Abandoned
- 2009-09-25 JP JP2011529276A patent/JP5722782B2/ja active Active
- 2009-09-25 EP EP09793025A patent/EP2349209A2/en not_active Withdrawn
- 2009-09-25 AU AU2009296458A patent/AU2009296458A1/en not_active Abandoned
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WO2010036945A3 (en) | 2010-05-20 |
US20160158179A1 (en) | 2016-06-09 |
US20150216826A1 (en) | 2015-08-06 |
WO2010036945A2 (en) | 2010-04-01 |
US8962026B2 (en) | 2015-02-24 |
US20100092526A1 (en) | 2010-04-15 |
US9259407B2 (en) | 2016-02-16 |
EP2349209A2 (en) | 2011-08-03 |
CA2738245A1 (en) | 2010-04-01 |
AU2009296458A1 (en) | 2010-04-01 |
WO2010036938A3 (en) | 2010-06-10 |
JP2012504134A (ja) | 2012-02-16 |
WO2010036938A2 (en) | 2010-04-01 |
US20100203139A1 (en) | 2010-08-12 |
CN102223876A (zh) | 2011-10-19 |
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