J. l. と t. v. は、国立研究所のアレルギーおよび感染症 (NIAID) の健康の国民の協会 (R01 AI111965) によってサポートされます。内容は著者の責任と国立研究所のアレルギーと感染症や健康の国民の協会の公式の見解を必ずしも表さない。三菱商事は、オーストラリア NHMRC 首席研究員です。J. l. はオーストラリア国立保健医療研究評議会 (NHMRC) 主任研究員、および t. v. は、オーストラリア NHMRC 産業キャリア開発レベル 2 研究員。E.K.S は、ASM (微生物学のためのアメリカの社会) の任命された若者大使 2017年でオーストラリアの大学院賞を受賞しています。 この作業の一部は、Australiasian 会議メルボルンに嚢胞性線維症 (2017 年 8 月の 5-8日) 12thで発表されました。

倫理の承認からモナッシュ大学人間研究倫理委員会 (MUHREC) が得られた。
 1 アッセイのアプリケーション: 患者サンプル コレクション <ol><li>とき患者と 150 mg ivacaftor または ivacaftor 125 mg/lumacaftor の標準的な量の時間を記録 200 mg。</li>。
	<li>注下患者の血液サンプルを収集するとき正確な時刻です 。
	注: 24 時間コース上 4-5 の試料を収集をお勧めします。示された時間ポイントは 2.5-4 h ポスト ivacaftor または ivacaftor lumacaftor の組み合わせ投与、定常状態で C の 最大 濃度は後達成される場合のみ 1 つのサンプルのコレクションは、&#62; 連続投与後 5 日</li>。
	<li>市販の未処理青いチューブの血液の 4 ~ 5 mL を収集します</li>。
	<li>全血のコレクションの後許可を室温で邪魔されずに残して凝固血 。
	注: これは通常 15-30 分かかる</li>
	<li>は、冷却遠心機で 4 ° C で 10 分間 1,000-2,000 × g で遠心分離によって血腫を除去します。プラズマとして結果の上澄みを指定します</li>。
	<li>遠心分離後すぐにパスツール ピペットを使用して清潔なポリプロピレン チューブに上清 (プラズマ) を転送します。プラズマ管を患者 ID を (例えば 患者 1)、(例えば、ivacaftor lumacaftor の組み合わせ)、投与薬と患者のサンプルの時間のラベルは収集した (例えば、2.5 時間後の投与量).</li>
	<li>の処理中に 2-8 ° C で維持するサンプル。処理が完了すると、ストアでサンプル &#8211; 20 ° C</li>
	<li>必要に応じて、出荷サンプル分析研究所に承認されたクーリエ 。
	注: サンプルをドライアイスに到着まで発送して保管 &#8211; 解析までの 80 ° C.</li>
</ol> 2。準備および発生したサンプルの処理と基準 注: ivacaftor/lumacaftor 療法を健康なドナーの世間知らずからプラズマがオーストラリア赤十字社から得られました。整合性を確保して収集と処理中にすべてサンプル/基準 2-8 ° C でおきます。処理が完了すると、ストアでサンプル &#8211; 20 ° C
<ol><li>IVA、IVA-カルボン酸、ヒドロキシメチル-IVA、社内基準への参照となる輝度を量り</li>。
	<li>LC MS グレードのメタノール 100 μ g/mL、10 μ g/mL の各試料 (IVA、M1、M6 および LUMA) の 2 つの独立した在庫ソリューションを準備します 。
	注: 化合物は、ベンチトップで 10 日後切れます</li>。
	<li>たて準備各解析実行前に液体クロマトグラフィー-質量校正標準器校正基準の濃度希釈原液以下の濃度を達成するためにひと血漿に: 0.01 0.025 0.05、0.1、0.2、0.5、1.0、2.5、5.0、10.0 μ g/mL。これらのサンプルを基準と呼びます</li>。
	<li>蛋白質の沈殿物、アセトニ トリル (ACN、LC MS グレード) の 0.1% ギ酸 (FA) を準備し、必要になるまで冷蔵庫にしておきます</li>。
</ol> 3。発生したサンプルおよび標準の前処理 <ol><li>部屋の温度平衡に両方の患者と空白の血漿サンプルを許可します</li>。
	<li>15 の血漿を混合する渦サンプル毎秒です</li>。
	<li>1.5 mL ポリプロピレン微量遠心チューブに基準のいずれかの空白プラズマまたは分析のための患者のサンプルの 100 μ 因数を転送します</li>。
	<li>は、標準的な管のそれぞれに内部標準 IVA を追加 (例えば 0.01、0.025、0.05、0.1、0.2、0.5、1.0、2.5、5.0、および潜在的な濃度範囲をカバーする 10.0 μ G/ml)、蓋を閉めるし、2-3 s. 注内部標準の渦は、のみ空白のプラズマに追加と内部標準は患者のサンプルに追加されません</li>。
	<li>3.4 の手順を繰り返します内部の標準 M1、M6、ルミナンス。</li>。
	<li>スピン ・ ダウン各チューブに簡単に蓋の上に水滴がないことを確認します</li>。
	<li>0.1% のミックスの追加 200 μ L を血しょう蛋白質を沈殿させた各チューブに ACN で FA</li>。
	<li>渦約 15 のために精力的に混合 s.</li>
	<li>冷蔵庫に 10 分放置する管を許可します</li>。
	<li>可能であれば 4 ° C で 10 分間、10,000 × g で遠心分離機します</li>。
	<li>1.5 mL の HPLC バイアルに 13 mm のシリンジ フィルター通過の上清 200 μ L 因数をフィルター処理します</li>。
	<li>高速液体クロマトグラフィー質量分析用 LC MS バイアルに上澄みの転送 100 μ L.</li>
</ol> 4。高速液体クロマトグラフィー質量分析 注: トリプル四重極質量分析計 (表 1) と相まって LC MS システム上の高速液体クロマトグラフィー-質量分析を行った.
<ol><li>に LC MS システム上自動サンプラでサンプルとトレイを配置します</li>。
	<li>ガード列に列し LC MS システムに接続します</li>。
	<li>移動相の両方の本を添付 (ボトル a: 100 %acn; ボトル水の b: 0.1% ギ酸) LC MS システムの平衡と</li>。
	<li>次パラメーターを組み込む LC MS プロトコル。
	<ol><li>2:1 の比率で質量分析計に入る前に移動相の流れを分割 (廃棄物: MS 入口).</li>
		<li>グラジエント移動相に水の 100 %acn と 0.1% ギ酸を使用して 0.5 mL/min の流量での実行 (開始点、40: 60 で v/v)。移動相の容積 % 分析を通じて変更されるに注意してください</li>。
		<li>肯定的なエレクトロ スプレー イオン化モードで質量を操作します</li>。
		<li>LC MS 設定は次のとおりであることを確認: イオン スプレー電圧 4.5 kV、衝突エネルギー 295.9 V、噴霧ガス: 3 L/分で窒素; 衝突ガス: アルゴン; 乾燥ガス 20 L/分の流量、電圧 Q3 のレンズ:-22 V、溶媒の温度熱ブロック 250 ° C400 の温度 ° C</li>
		<li>サンプルあたり注入 10 μ L のボリューム</li>。
		<li>監視 (MRM) 複数の反応を利用して検出検体。M/z 392.49 のイオンの遷移を監視 &#8594;、393 m/z 408.49 &#8594; 409、m/z 422.47 &#8594; 423 と m/z 452.40 &#8594; ルミナンス、イヴァ M6、イヴァ M1 IVA 453 それぞれ 。
		注: 新しく最適化されたメソッドがまだされて完全に FDA の基準に従って検証される</li>。
	</ol></li></ol> 5。較正曲線 <ol><li>構築 LC MS 内部標準へ 4 つの analytes のそれぞれのピーク面積比との校正の標準的な公称濃度との関係を使用して各解析実行前に検LC MS 設定プログラム内 ivacaftor (IVA M1、イヴァ M6 またはルミナンス). 
	注: 検量線の建設のための正確な手順は使用されている LC MS 機器のモデルによって異なります。この情報機器マニュアルにあります</li>。
	<li>によると濃度の逆数 1/C 2 を重み付けによる線形最小二乗回帰分析を実行します</li>。
</ol>

<ol>
	<li>Schneider, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-drug+plasma+protein+binding+interactions+of+ivacaftor.">Drug-drug plasma protein binding interactions of ivacaftor.</a> J Mol Recognit. 28 (6), 339-348 (2015).</li><li>Solomon, M., Leatte, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Treatments for Cystic fibrosis. , (2009).</li><li>O'Sullivan, B. P., Flume, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+approach+to+lung+disease+in+patients+with+cystic+fibrosis.">The clinical approach to lung disease in patients with cystic fibrosis.</a> Semin Respir Crit Care Med. 30 (5), 505-513 (2009).</li><li>Ramsey, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+CFTR+potentiator+in+patients+with+cystic+fibrosis+and+the+G551D+mutation.">A CFTR potentiator in patients with cystic fibrosis and the G551D mutation.</a> N Engl J Med. 365 (18), 1663-1672 (2011).</li><li>Wainwright, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumacaftor-Ivacaftor+in+Patients+with+Cystic+Fibrosis+Homozygous+for+Phe508del+CFTR.">Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR.</a> N Engl J Med. 373 (3), 220-231 (2015).</li><li>Hadida, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide+(VX-770,+ivacaftor),+a+potent+and+orally+bioavailable+CFTR+potentiator.">Discovery of N-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide (VX-770, ivacaftor), a potent and orally bioavailable CFTR potentiator.</a> J Med Chem. 57 (23), 9776-9795 (2014).</li><li>FDA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> FDA Advisory Commitee Briefing Material VERTEX-FDA Pulmonary-Allergy drugs advisory commitee. 98 , (2015).</li><li>Holmes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=False+dawn+for+cystic+fibrosis+disease+modifiers?.">False dawn for cystic fibrosis disease modifiers?.</a> Nat Rev Drug Discov. 13 (10), 713-714 (2014).</li><li>Veit, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+gating+potentiators,+including+VX-770,+diminish+DeltaF508-CFTR+functional+expression.">Some gating potentiators, including VX-770, diminish DeltaF508-CFTR functional expression.</a> Sci Transl Med. 6 (246), 246ra297 (2014).</li><li>Cholon, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potentiator+ivacaftor+abrogates+pharmacological+correction+of+DeltaF508+CFTR+in+cystic+fibrosis.">Potentiator ivacaftor abrogates pharmacological correction of DeltaF508 CFTR in cystic fibrosis.</a> Sci Transl Med. 6 (246), 246ra297 (2014).</li><li>EMA, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Assessment report ORKAMBI (ivacaftor/lumacaftor) European medicines agency. , (2015).</li><li>VERTEX. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Vertex prescribing infomation. , (2015).</li><li>Matthes, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+free+drug+concentration+prevents+inhibition+of+F508del+CFTR+functional+expression+by+the+potentiator+VX-770+(ivacaftor).">Low free drug concentration prevents inhibition of F508del CFTR functional expression by the potentiator VX-770 (ivacaftor).</a> Br J Pharmacol. 173 (3), 459-470 (2016).</li><li>Schneider, E. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+HPLC+and+LC-MS/MS+methods+for+the+analysis+of+ivacaftor,+its+major+metabolites+and+lumacaftor+in+plasma+and+sputum+of+cystic+fibrosis+patients+treated+with+ORKAMBI+or+KALYDECO.">Development of HPLC and LC-MS/MS methods for the analysis of ivacaftor, its major metabolites and lumacaftor in plasma and sputum of cystic fibrosis patients treated with ORKAMBI or KALYDECO.</a> J Chrom B Analyt Technol Biomed Life Sci. 1038, 57-62 (2016).</li><li>Su, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+LC-MS/MS+method+for+the+quantitation+of+cabozantinib+in+rat+plasma:+application+to+a+pharmacokinetic+study.">An LC-MS/MS method for the quantitation of cabozantinib in rat plasma: application to a pharmacokinetic study.</a> J Chromatogr B Analyt Technol Biomed Life Sci. 985, 119-123 (2015).</li></ol>

著者が明らかに何もありません。

既報のように、私たちのグループは、初めて開発および HPLC や LCMS 迅速検出と ivacaftor とヒドロキシメチル IVA M1 (アクティブ) とイヴァ カルボン酸 M6 (非アクティブ); その主要代謝物の定量法を検証血漿および CF の患者の14の喀痰中 lumacaftor。以前私たちのグループによって報告されたアッセイ ルミナンス、IVA、イヴァ-M1 とプラズマで IVA M6 の濃度とイヴァ 150 mg/q12 h と定常?...

嚢胞性線維症 (CF) は肺、肝臓、膵臓、肺機能、膵機能不全1,の減少などの進行性多臓器不全を引き起こす腸の分泌粘液腺を含む一般的な遺伝的疾患2,3。Ivacaftor (IVA) は最初の食品医薬品局 (FDA 米国) と欧州医薬品庁 (EMA) 明らかな臨床的有効性の重要な生産と嚢胞性線維症膜コンダクタンス レギュレータ (CFTR) 増強剤を含む薬を承認ベアリング G551D CFTR CF の患者の小さいサブセットでプラセボに対する肺機能の改善 [グリシン (G) 551 の位置にはアスパラギン酸 (D) 置き換え] ミスセンス変異 (CF 人口の 4 〜 5%)4,5。これは経口投与薬剤増加 CFTR チャネルを開く、塩化物イオン流を増加させると CF4,6の臨床症状につながる主要な欠陥に作用します。残念ながら、イヴァ単独療法は [フレーム位置 508 にフェニルアラニン (F) の損失の結果 CFTR 遺伝子の削除] でより一般的なホモ F508del 変異を有する患者における効果的なの 〜 50% に見られる変性 CFTR の結果CF 人口7,8。
最近、FDA は薬 lumacaftor CFTR 補正とイヴァを組み合わせることの承認を付与します。CFTR チャネル活性を増強する変調器 (IVA) を用いた細胞表面に F508del CFTR を救助する「CFTR 補正 (lumacaftor、LUMA) を組み合わせた巧妙な戦略効果的に CF 人口5のほとんどに治療ウィンドウを表示.競合レポートの数の臨床9,10時にその疑いが浮上しているようにこれらの薬が彼らの約束を果たすかどうか以上の質問が残っています。さらに、肺の機能の改善だけささやかな (2.6 4 の % であった ivacaftor lumacaftor 組み合わせ) 軸受 G551D 突然変異 (10.6 12.5%)8患者の IVA 単独で達成した成功と比較して。IVA とルミナンス ivacaftor lumacaftor 併用の臨床的有効性が制限される可能性がの潜在的な敵対的薬物相互作用は、その理想的な未満の薬物動態的特性7,11から来る。IVA は活性代謝物ヒドロキシメチル-イヴァ (IVA M1, M1) と不活性形 IVA-カルボン酸 (IVA M6 M6)7,12主にチトクローム p450 (CYP) によって代謝されます。CYP3A4 誘導ルミナンス、その一方で、広範囲で代謝されてし、は主としてそのまま糞便11に排泄されます。CYP3A4 誘導剤がチトクローム代謝を誘導する、ivacaftor (CYP3A4 基質) 濃度を減らすことができます。また、イヴァとルミナンスの両方は非常に疎水性の分子、~ 99% にバインドされる血漿蛋白 (アクティブ) 遊離型薬物濃度1,13を大幅に制限します。
総称して、これらの要因は、ivacaftor lumacaftor 併用の臨床的有効性を制限する一緒に来るかもしれません。Ivacaftor lumacaftor の組み合わせで現在服用下で最適な血中濃度を達成するかどうかまたはかどうか治療のしきい値は8を維持されますが不明です。現在、ピークといった薬物動態学的パラメーターに関する情報の少なさと ivacaftor または ivacaftor lumacaftor の定常状態血漿濃度があります。Ivacaftor と lumacaftor の指摘の代謝を考えると、すると、ivacaftor または ivacaftor lumacaftor 療法の最適用量レジメンを達成するために必要な暴露-反応関係の監視です。当社グループは最近、イヴァとルミナンス14の量反応関係の監視のための最初の高速液体クロマトグラフィー/液体クロマトグラフィー-質量方法を公開しました。これまで ivacaftor、その代謝産物および lumacaftor 濃度測定の代替技術は報告されていません。大規模患者集団の高スループット解析を許可し、分析時間を大幅に削減する、当社グループが小さい細孔サイズ逆相クロマトグラフィー コラムおよび勾配溶剤系を使用して報告された方法を最適化します。コストと実行時間を削減します。

<table border="0" cellpadding="0" cellspacing="0" width="915">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:480px;">IVA (Cat#S114)&#160;</td>
			<td style="width:229px;">SelleckChem (USA).&#160;</td>
			<td style="width:143px;"></td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LUMA (Cat#S1565)&#160;</td>
			<td>SelleckChem (USA).&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IVA-carboxylate (Cat# 510242247CS)&#160;</td>
			<td>Clearsynth (Canada).&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hydroxymethyl-IVA (Cat# 510240849CS)&#160;</td>
			<td>Clearsynth (Canada).&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol (MeOH, LC-MS grade),&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acetonitrile (ACN, LC-MS grade)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">formic acid (FA)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">triple-quadrupole Shimadzu 8030 LC-MS&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenomenex Kinetex (2.6 &#181;m C8 100 &#197;; 50 &#215; 2.1mm)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(KrudKatcher Ultrea HPLC In-Line Filter 0.5 m Depth Filter x 0.004inID).&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL polypropylene microcentrifuge tube (VWR).&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eppendorf Centrifuge 5430</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">13-mm syringe filter (0.45 &#181;m nylon, GRACE, USA)&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">[Phenomenex VEREX, 9 mm, PP, 300 &#181;L, PTFE/Silicone septa].&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

optimized lc-ms/ms method for the high-throughput analysis of clinical samples of ivacaftor, its major metabolites, and lumacaftor in biological fluids of cystic fibrosis patients

嚢胞性線維症膜コンダクタンス制御因子 (CFTR) の欠陥は、嚢胞性線維症 (CF)、生命にかかわる肺症状を病気の原因です。Ivacaftor (IVA) と ivacaftor lumacaftor (輝度) の組み合わせは、直接活動と欠陥 CFTR 蛋白質の輸送を調節する 2 つの新しい CF の画期的な薬です。ただし、薬物動態/薬力学パラメーターに関する理解の不足、ivacaftor と lumacaftor の薬理学はまだです。高速液体クロマトグラフィー MS ivacaftor、ヒドロキシメチル ivacaftor、ivacaftor-カルボン酸、および lumacaftor 標準の ivacaftor または ivacaftor lumacaftor の組み合わせを受けている患者の体液中の濃度の同時分析法治療は、以前されて私たちのグループによって開発され、FDA の基準を部分的に検証されます。ただし、患者サンプルの大きい数の高スループット解析をできるように、私たちのグループが小さい細孔サイズ逆相クロマトグラフィー コラムを使用して報告されたメソッドに最適化 (2.6 μ m、C8 100 Å; 50 x 2.1 mm) と溶媒混合系のグラデーション (0 1 分: 40 %b;1-2 分: 40-70 %b;2 2.7 分: 70 %b にて開催2.7 2.8 分: 70-90 %b;2.8-4.0 分: 90 %b 洗濯;4.0 4.1 分: 90-40 %b;4.1 6.0 分: 40 %b で開催) ではなくイソクラティック溶出。本研究の目的は、大量の患者のサンプルの分析のために不可欠であるサンプルあたり 6 分のみ 〜 15 分から大幅にサンプルあたりの高速液体クロマトグラフィー-質量分析時間を減らすことでした。これらの画期的な CF 薬物の量反応関係を研究のためかなりユーティリティのこの便宜的なメソッドになります。

我々 は最近の C8 列 (5 μ m、内径 3.9 × 50 mm) を用いた 100% 水 (40: 60 の ACN と 0.1% ギ酸から成る移動相をトリプル四重極液体クロマトグラフィー-質量と高速液体クロマトグラフィー検出器システム、FDA の基準を部分的に検証メソッドに報告しています。、v/v) 1 mL/min の流速で。すべての代謝物、ivacaftor、Iva M1、Iva-M6、lumacaftor14のひと血漿は 0.01 から 10 μ g/mL までの濃度範囲でピ...

Watch this Scientific Journal Video about Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cys

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Ivacaftor と ivacaftor lumacaftor の組み合わせは、2 つの新しい CF 薬です。ただし、まだ自分の PK/PD と薬理学の理解の不足があります。Ivacaftor 主要な代謝物と lumacaftor の同時解析のために最適化された HPLC MS 技術を提案します。

Ivacaftor、主要な代謝産物および嚢胞性線維症患者の体液中の Lumacaftor の臨床サンプルの高スループット分析のための最適化された LC-MS/MS 法

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

optimized-lc-msms-method-for-high-throughput-analysis-clinical

Research

JoVE Journal

Medicine

8.2K ビュー.  Monash University. Ivacaftor と ivacaftor lumacaftor の組み合わせは、2 つの新しい CF 薬です。ただし、まだ自分の PK/PD と薬理学の理解の不足があります。Ivacaftor 主要な代謝物と lumacaftor の同時解析のために最適化された HPLC MS 技術を提案します。

ビデオ: Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

LC-MS / MSを用いて乾燥血液スポット上で免疫抑制タクロリムスの定量化

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

医学

Network Analysis of Foramen Ovale Electrode Recordings in Drug-resistant Temporal Lobe Epilepsy Patients

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control seizures. However, a significant minority of patients continues to exhibit post-operative seizures, even in those cases in which the suspected source of seizures has been correctly localized and resected. The protocol presented here combines a clinical procedure routinely employed during the pre-operative evaluation of temporal lobe epilepsy (TLE) patients with a novel technique for network analysis. The method allows for the evaluation of the temporal evolution of mesial network parameters. The bilateral insertion of foramen ovale electrodes (FOE) into the ambient cistern simultaneously records electrocortical activity at several mesial areas in the temporal lobe. Furthermore, network methodology applied to the recorded time series tracks the temporal evolution of the mesial networks both interictally and during the seizures. In this way, the presented protocol offers a unique way to visualize and quantify measures that considers the relationships between several mesial areas instead of a single area.

This protocol describes a procedure to track the evolution of mesial network measures in temporal lobe epilepsy (TLE) patients. It is based on the combination of intracranial recordings with a novel numerical technique for data analysis. Specifically, we present a protocol for network analyses of foramen ovale recordings.

薬剤耐性側頭葉てんかん患者における孔開存電極レコーディングのネットワーク解析

Approximately 30% of epilepsy patients are refractory to antiepileptic drugs. In these cases, surgery is the only alternative to eliminate/control ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

フォルスコリン誘発性腸オルガノイドに腫れ：<em&gt;インビトロ</em&gt;アッセイ嚢胞性線維症患者における薬物反応を評価するための

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

の最適化分析<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

サンプリング戦略とブタの生物医学的モデルからバイオバンク組織サンプルの処理

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Collecting Hair Samples for Hair Cortisol Analysis in African Americans

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, including humans. The advantage of hair cortisol concentration is that it reflects a retrospective representation of hypothalamic-pituitary-adrenal (HPA) axis function over time, much like hemoglobin A1C represents glycemic control. However, obtaining hair samples can be challenging, due to the cultural beliefs and hair care practices of minority participants. For example, African Americans may be reluctant to provide samples. Additionally, few researchers are trained to collect hair samples from African Americans. The purpose of this paper is to present a culturally informed protocol to help researchers obtain hair samples from African Americans. To illustrate the representative results of this protocol implementation, de-identified data from African Americans that participated in a community-based study on chronic stress are provided. Hair practice preferences are assessed. The participants are made comfortable by showing pictures of hair samples prior to cutting their hair. The single strain twist and gently pull method is used to collect approximately 30 - 50 strands of hair from the posterior vertex region of the scalp. This protocol will significantly improve collection of hair samples from African Americans.

Hair cortisol concentration analysis provides an alternative to traditional measures of cortisol; however, to collect hair samples from African Americans, scientists need to be culturally informed and competent. The purpose of this protocol is to demonstrate a culturally informed technique to collect hair samples for cortisol analysis from African Americans.

髪コルチゾールにおけるアフリカ系アメリカ人の毛のサンプルの収集

The hormone cortisol is typically assessed in saliva, serum, or urine samples. More recently, cortisol has been successfully extracted from hair, ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

エレクトロマップを用いた光マッピングデータのハイスループット解析

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

A High-Throughput In Situ Method for Estimation of Hepatocyte Nuclear Ploidy in Mice

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as cholangiocytes, myofibroblasts, progenitors and inflammatory cells also indicate chronic liver damage, tissue remodeling and disease progression. In this protocol, we describe a simple high-throughput approach for calculating changes in the cellular composition of the liver that are associated with injury, chronic disease and cancer. We show how information extracted from two-dimensional (2D) tissue sections can be used to quantify and calibrate hepatocyte nuclear ploidy within a sample and enable the user to locate specific ploidy subsets within the liver in situ. Our method requires access to fixed/frozen liver material, basic immunocytochemistry reagents and any standard high-content imaging platform. It serves as a powerful alternative to standard flow cytometry techniques, which require disruption of freshly collected tissue, loss of spatial information and potential disaggregation bias.

We present a robust, cost-effective, and flexible method for measuring changes in hepatocyte number and nuclear ploidy within fixed/cryopreserved tissue samples that does not require flow cytometry. Our approach provides a powerful sample-wide signature of liver cytology ideal for tracking the progression of liver injury and disease.

マウスにおける肝細胞核プロイドの推定に対するSitu法におけるハイスループット

When the liver is injured, hepatocyte numbers decrease, while cell size, nuclear size and ploidy increase. The expansion of non-parenchymal cells such as ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

小さな毛髪サンプルで11の薬剤耐性結核薬を定量化するための検証済みのLC-MS/MSパネル

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

嚢胞性線維症気道マイクロバイオームに対する酸素付加の効果を研究するモデルの設計と開発

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...