JP2016196495A - 抗菌剤と分散剤または接着阻害剤組成物 - Google Patents
抗菌剤と分散剤または接着阻害剤組成物 Download PDFInfo
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- JP2016196495A JP2016196495A JP2016129761A JP2016129761A JP2016196495A JP 2016196495 A JP2016196495 A JP 2016196495A JP 2016129761 A JP2016129761 A JP 2016129761A JP 2016129761 A JP2016129761 A JP 2016129761A JP 2016196495 A JP2016196495 A JP 2016196495A
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Abstract
Description
本発明の第一の態様によると、抗菌剤と、分散剤または接着阻害剤、特に粘液溶解性分散剤または粘液溶解性接着阻害剤である第二の薬剤とを含有してなる製造品が提供される。
製造品
本発明の第一の態様によると、抗菌剤と、分散剤または接着阻害剤である第二薬剤とを含有してなる製造品が提供される。
驚くべきことに、抗菌剤と分散剤または接着阻害剤の抗菌作用は概して組み合わせることにより相乗的に増加する。
FIC=MICA[併用]/MICA[単独]+MICB[併用]/MICB[単独]
「抗菌剤」なる用語は、細菌源に由来しうる抗菌剤を指すために用いられる。抗菌剤は殺菌性および/または静菌性であってよい。
第二薬剤は、分散剤および接着防止剤から選択されうる。特に、第二薬剤は、粘液溶解性分散剤および粘液溶解性接着防止剤から選択される。第二薬剤は、細菌コロニーの形成を阻害する任意の薬剤、特にバイオフィルム形成を阻害する任意の薬剤であってよい。例として、第二薬剤は、細菌の接着、疎水性または粘液産生を阻害しうる。
上述の活性作用物質は、自由なまたは固定された組み合わせで投与することができる。自由な組み合わせは、全ての活性作用物質を自由な組み合わせで含有する、組み合わせパッケージとして提供され得る。固定された組み合わせは、錠剤またはカプセルであることが多い。
一実施形態によると、本発明の製造品の抗菌剤はペプチドを含まない。好適には、本発明の製造品はペプチドを含まない。
本発明は、細菌感染を治療または予防する方法を提供する。細菌感染は、典型的には、播種性感染、または特に肺などの粘膜豊富な環境におけるもの、例えば、CFに罹患している患者の肺または細菌関連の慢性閉塞性肺疾患(COPD)であってよい。本発明の方法は、本発明に係る製造品をその環境に投与する工程を含む。この方法は生体内または生体外であってよい。
・抗菌剤と、
・分散剤または接着阻害剤、好ましくはシステアミンである第二薬剤と
を投与する工程を含む。
材料と方法
1.1 菌株
緑膿菌ATCC27853およびバークホルデリア・セパシアNCTC10743を本研究に用いた。表1および表2に示す、緑膿菌の他の菌株を使用した。
この研究において試験された抗菌剤はシステアミン(NM001)、トブラマイシン、シプロフロキサシン、コリスチンおよびゲンタマイシンとした。これら作用物質は、Sigma−Aldrich(ギリンガム、英国)から入手し、ストック溶液を、14〜18MΩcmの純水中20mg/mlに調製した(Purite HP40水精製システム、Oxon、UK)。溶解した後、調製物を0.22μmのフィルター(Millipore、ウォトフォード、イングランド)を用いて濾過滅菌し、−20℃で保存した。
細菌接種材料は、CLSI M26−A法において記載されている0.5マクファーランド濁度標準にて標準化した、ミューラーヒントン(MH)培養液において活性に増殖している培養物の希釈法によって得た。
細菌接種材料と、構造的にシステアミンと関連する化合物を含む抗菌剤の両方をプレートに同時に添加した。プレートを37℃で24時間にわたりインキュベートし、光学密度をマイクロタイタープレート読み取り機(BioTek Powerwave XS、ウィヌースキ、米国)にて、625nmで読み取った。細菌増殖の完全な阻害を示す抗微生物の最低濃度として、MICを得た。
システアミンに化学構造が関連する多数の化合物:シスタミン(塩酸塩)、タウリンおよび2,3−ジメルカプトコハク酸の抗菌活性を調べた。化学的にシステアミンに関連する他の化合物も知られているが、有毒であるのでこのような性質の薬に実用性はない。
シスタミン:
タウリン:
2,3−ジメルカプトコハク酸
高イオン濃度の存在下でのシステアミン(Lynovex(登録商標))の抗菌活性を測定するために、上記(1.4)と同様の方法を用いた。システアミンの抗菌活性(MIC100)を、通常条件(CLSI M7−A7法に記載されるとおり)下と、150mM 塩化ナトリウム(NaCl)、1.7mM 塩化カルシウム(CaCl2)、1mg/ml DNAまたは1%(w/v)ブタ胃ムチンの存在下とで、96ウェルプレートにて3通り比較した。24時間にわたって細菌増殖を追跡し、ビオテックマイクロタイタープレート読み取り機を用いて625nmの吸光度を読み取った。
抗バイオフィルム活性は、マイクロ流体BioFlux800フローセルシステム(Fluxion、サウスサンフランシスコ、米国)を用いて評価した。48ウェルBioFluxプレートのマイクロ流体チャネルに、ミューラーヒントン培養物中の緑膿菌PAO1の0.5マクファーランド同等接種物を接種した。この微生物細胞を37℃で約30分かけて接着させ、微小コロニーを形成させた。粘液溶解薬は、ミューラーヒントン培養液中1mg/mlに調製し、37℃で16時間、0.5Dyn/cm2の流量(53μl/hに相当)でマイクロ流体を流した。顕微鏡観察を行い、倒立AXIOVERT40顕微鏡を用いて100倍の倍率(Carl Zeiss、ウェリンガーデンシティー、英国)の写真を記録した。
FICは、抗菌剤の組み合わせが相乗的、相加的、拮抗的、または中立的であるかどうかを示す相互作用係数に対応する。FICは、以下のように、組み合わされた作用物質の活性(作用物質A+作用物質BのMIC)を、単独の作用物質の活性(作用物質Aまたは作用物質BのMIC)と比較することによって決定される(Singhら、2000年)。
FIC=MICA[併用]/MICA[単独]+MICB[併用]/MICB[単独]
PAEは、処置後、マイクロタイタープレートにおいて3通り、90μlの滅菌MH培養液に10μlのバイオフィルム培養物を移すことにより決定した。細菌増殖は、37℃で24時間バイオテックプレート読み取り機にて追跡した。バイオフィルムの処理後の微生物の代謝活性は、PAE決定のためと同様の方法を用い、蛍光細胞生存率指標であるレサズリン(アラマーブルー、Serotec)を10%(v/v)添加して決定した。このレサズリンは蛍光の増加により微生物の代謝活性の増大が検出される。蛍光は、バイオテックシナジーHTマイクロプレート読み取り機(BioTek、ウィヌースキ、米国)を用いて530/590nmで37℃、24時間にわたって測定した。
システアミンの粘液溶解活性は、処理後にムチン溶液の粘度を測定することにより、N−アセチルシステイン(Mucomyst(登録商標))、DNアーゼI(ドルナーゼアルファ(登録商標))、アルギン酸リアーゼおよびシステアミン塩酸塩などの他の粘液溶解剤と比較した。ブタ胃のムチン(Sigma−Aldrich、ギリンガム、英国)は、滅菌精製水にて20%(w/v)に調製した。粘液溶解剤は、20%(w/v)のムチン溶液中10mg/mlに調製した。ムチンの速度は粘液溶解剤に約5分曝露させた後に測定した。
2.1 細菌コロニー形成の予防と治療
抗菌剤の濃度範囲は0〜10μg/mlであった。
トブラマイシンおよびシプロフロキサシンのMICは、単独投与と比較して、システアミンと共に投与すると大幅に低減した。この効果は、CF肺に見られた環境を模倣した環境において、現れた。
Claims (1)
- 抗菌剤と、分散剤または接着阻害剤である第二薬剤とを含有してなる製造品。
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