Name: 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
Uploaded: 2017-10-15T13:00:00
Description: 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

. 和电视得到国立卫生研究院 ( R01 AI111965 ) 国家反应和传染性疾病研究所 ( NIAID ) 的支持。内容完全是作者的责任, 并不一定代表国家过敏和传染性疾病研究所或国立卫生研究院的官方观点。MC 是澳大利亚澳洲的主要研究员。. 是澳大利亚国家卫生与医学研究委员会 (澳洲) 高级研究员, 而电视是澳大利亚澳洲行业职业发展2级研究员。杨永强 S 是一个任命的年轻大使2017为 ASM (美国微生物学协会) 并且由澳洲研究生奖支持。 这项工作的一部分是在 12th Australiasian 会议上囊性纤维化在墨尔本 (5-8th 2017年8月)。

对道德的认可是由蒙纳士大学人类研究伦理委员会 (MUHREC) 获得的. 
 1. 分析的应用: 患者样本收集 <ol> <li> 记录患者服用标准剂量的150毫克 ivacaftor 或 ivacaftor 125 毫克/lumacaftor 200 毫克的时间。
	<li> 记下病人血样采集的确切时间. 
	注: 我们建议在24小时的课程中收集4-5 样本。如果只有一个样品的集合是可能的, 被表明的时间点是 2.5-4 h 岗位 ivacaftor 或 ivacaftor-lumacaftor 组合药...

<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>

如前所述, 我们的小组首次开发和验证了高效液相色谱和 LC-MS 法快速检测和定量 ivacaftor 及其主要代谢物羟甲基 M1 (活性) 和 M6 (不活泼);lumacaftor 在 CF 患者血浆和痰液中的浓度为14。本组报告的检测方法成功地用于定量测定 CF 患者的血浆和痰中 LUMA、IVA-M1、IVA-M6 的浓度, 采用150毫克/q12 h (拉丁语术语: quaque12 小时;每12小时一次) 或200毫克/q12 h LUMA-125 毫克/q12 h 综合体组合<sup cla...

囊性纤维化 (CF) 是一种常见的遗传性疾病, 涉及的外分泌粘液腺体的肺, 肝, 胰, 和肠道导致渐进性多衰竭, 如肺功能减退和胰不足1, 2,3。Ivacaftor (美国食品和药物管理局) 和欧洲药物管理局 (EMA) 批准囊性纤维化反式膜电导调节剂 (CFTR) 增药物, 其临床疗效显著G551D-CFTR 的小子集的 cf 患者肺功能改善的临床研究 [甘氨酸 (G) 在位置551中被天门冬氨酸 (D)] 义突变 (CF 种群的 4-5%)4,5。这种口服药物增加了 CFTR 通道开放, 从而增加了氯离子流和作用于主要缺陷, 导致的临床表现 CF4,6。不幸的是, 在更常见的纯合 F508del 突变的患者中, CFTR 单药的单一性是不有效的 [在帧删除的错误, 导致在位置508的苯丙氨酸 (F) 的损失, 这在50%CF 填充7,8。
最近, FDA 批准了与 CFTR 校正药物 lumacaftor 的结合。巧妙的策略, 结合 CFTR 校正器 (lumacaftor, LUMA), 这将 F508del-CFTR 到细胞表面的调制器 (伊娃) 加强 CFTR 通道活动, 有效地扩大治疗窗口的大多数 CF 人口5.关于这些药物是否能履行他们的诺言的问题仍然存在, 因为一些相互矛盾的报告已经出现, 对他们的临床功效产生了怀疑9,10。此外, 肺功能的改善只是适度的 (2.6-4% 的 ivacaftor-lumacaftor 组合) 相比, 成功地在 G551D 突变 (10.6-12.5%)8患者单药单一治疗。潜在的拮抗药物-药物相互作用可能限制 ivacaftor-lumacaftor 结合的临床疗效的 LUMA, 来自其不太理想的药代动力学属性7,11。在细胞色素 P450 酶 (CYP) 的作用下, 主要对活性代谢产物羟甲基--IVA-M1、M1) 和非活动形态 (IVA-M6, M6)7,12进行了广泛的代谢。CYP3A4 诱导 LUMA, 另一方面, 没有广泛的代谢和主要排泄不变的粪便11。由于 CYP3A4 诱导诱导细胞色素代谢, ivacaftor (CYP3A4 基质) 浓度可降低。此外, 两个 LUMA 都是非常疏水性分子和99% 绑定到血浆蛋白, 这极大地限制了自由 (有效) 药物浓度1,13。
总的来说, 这些因素可能聚集在一起, 限制了 ivacaftor-lumacaftor 结合的临床疗效。目前尚不清楚 ivacaftor-lumacaftor 组合的剂量方案是否达到最佳血浆浓度, 或者治疗阈值是否保持在8。目前, 关于 ivacaftor 或 ivacaftor lumacaftor 的药代动力学参数如峰值和稳态等离子体浓度的信息缺乏。鉴于 ivacaftor 和 lumacaftor 的新陈代谢, 对接触反应关系的监测是实现 ivacaftor 或 ivacaftor-lumacaftor 疗法最佳剂量方案的必要条件。我们的小组最近出版了第一个 HPLC/LC-MS 方法, 以监测的接触-反应关系的 LUMA 和14。目前还没有其他方法来测量 ivacaftor、其代谢物和 lumacaftor 的浓度。为了让更大的患者集体的高通量分析, 并大大减少分析时间, 我们组通过使用较小孔径的反相色谱柱和梯度溶剂系统优化了报告的方法,降低成本和运行时间。

<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

囊性纤维化膜传导调节剂 (CFTR) 的缺陷是囊肿纤维化 (CF) 的病因, 是一种具有生命危险的肺部表现的疾病。Ivacaftor (Ivacaftor) 和 lumacaftor (LUMA) 结合是两种新的突破性药物, 直接调节有缺陷的 CFTR 蛋白的活性和贩运。然而, 对药代动力学/药效学参数和 ivacaftor 和 lumacaftor 的药理研究仍然缺乏认识。高效液相色谱-MS 技术同时分析接受标准 ivacaftor 或 ivacaftor-lumacaftor 结合的 ivacaftor、羟甲基 ivacaftor、ivacaftor 羧酸和 lumacaftor 在生物体液中的浓度治疗之前, 我们的小组和部分验证的 FDA 标准。然而, 为了允许高通量分析更多的患者样本, 我们组通过使用较小孔径的反相色谱柱 (2.6 µm、C8 100 Å; 50 x 2.1 mm) 和梯度溶剂系统对报告的方法进行了优化 (0-1 分钟: 40% B;1-2 分钟: 40-70% B;2-2. 7 分钟: 举行在 70% B;2.7-2.8 分: 70-90% B;2.8-4.0 分钟: 90% B 洗涤;4.0-4.1 分: 90-40% B;4.1-6.0 分钟: 举行在 40% B) 而不是一个等洗脱。本研究的目的是将每样样品的高效液相色谱-MS 分析时间从 ~ 15 分钟减少到每样6分钟, 这对于分析大量患者样本是必不可少的。这种权宜之计的方法将有相当大的效用, 研究的接触反应的关系, 这些突破 CF 药物。

我们最近报告了一种方法, 部分验证了 FDA 的标准, 在三重四极 LC MS 和 HPLC 检测系统, 使用 C8 柱 (5 µm, 3.9 mm x 50 mm 身份) 与流动相组成的 100% ACN 和0.1% 甲酸在水中 (40:60, v/v) 的流速为1毫升/分钟。在人类血浆中, 所有代谢物、ivacaftor、Iva-M1、Iva-M6 和 lumacaftor14的浓度范围从0.01 到10µg/毫升都观察到峰值的线性相关性。此方法已经过优化, 使 LC 保留时间大大减少, 因此, 该检测的完整运?...

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 和 Ivacaftor-lumacaftor 结合是两个新的 CF 药物。然而, 在他们的 PK/PD 和药理学方面仍然缺乏了解。本文提出了一种优化的 HPLC-MS 技术, 同时分析 ivacaftor 及其主要代谢产物, 以及 lumacaftor。

经优化的 LC/ms 方法对囊性纤维化患者 Ivacaftor 及其主要代谢物和 Lumacaftor 的高通量分析

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

Research

JoVE Journal

Medicine

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

在免疫抑制剂他克莫司对干血斑使用LC-MS / MS定量

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

科研

医学

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

在耐药性颞叶癫痫患者卵圆孔电极记录的网络分析

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

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

毛喉素诱导在肠组织体溶胀：一个<em&gt;体外</em&gt;检测方法的评估囊性纤维化患者对药物的反应

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

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

优化分析<em&gt;在体内</em&gt;和<em&gt;体外</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

猪生物医学模型生物库组织样品的取样策略与处理

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

在非洲裔美国人中收集头发皮质醇分析样本

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

Generation of Human Nasal Epithelial Cell Spheroids for Individualized Cystic Fibrosis Transmembrane Conductance Regulator Study

人鼻上皮细胞球体的生成与个体化囊性纤维膜电导调节的研究

While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

使用电子地图的光映射数据的高通量分析

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

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

经过验证的LC-MS/MS面板，用于在小头发样本中量化11种耐药结核病药物

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

研究辅助供氧对囊性纤维化气道微生物组影响的模型的设计与开发

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