Liu Lu, Detering Jan-Christoph, Milde Till, Haefeli Walter Emil, Witt Olaf, Burhenne Jürgen
Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Im Neuenheimer Feld 410, Heidelberg, Germany.
Department of Pediatric Oncology, Hematology, Immunology and Pulmonology, University of Heidelberg, Im Neuenheimer Feld 430, Heidelberg, Germany; Clinical Cooperation Unit Pediatric Oncology (G340), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Aug 1;964:212-21. doi: 10.1016/j.jchromb.2014.02.014. Epub 2014 Feb 18.
Vorinostat (suberoylanilide hydroxamic acid) is the first approved histone deacetylase (HDAC) inhibitor for the treatment of cutaneous T-cell lymphoma after progressive disease following two systemic therapies. Intracellular access of vorinostat is essential to exert its epigenetic effects. Therefore, we studied the relationship between vorinostat extracellular (plasma) and intracellular (peripheral blood mononuclear cells, PBMCs) concentration and assessed its concentration-effect relationship by HDAC activity testing. Assays were developed and validated for the low nanomolar quantification of vorinostat and two inactive metabolites in human plasma and PBMCs. For the vorinostat extraction from plasma and PBMCs solid-phase extraction and liquid-liquid extraction methods were applied. Extraction recoveries ranged from 88.6% to 114.4% for all analytes and extraction methods. Extracts were chromatographed on a Phenomenex Luna column isocratically (plasma) or by gradient (PBMCs) consisting of acidic ammonium acetate, acetonitrile, and methanol. The analytes were quantified using deuterated internal standards and positive electrospray tandem mass spectrometry (multiple reaction monitoring) with lower limits of quantification of 11.0 ng/mL (plasma) and 0.1 ng/3 × 10(6) cells (PBMCs). The calibrated ranges were linear for vorinostat in plasma 11.0-1100 (11,000) ng/mL (metabolites) and PBMCs 0.1-10.0 ng/3 × 10(6) cells with correlation coefficients >0.99, an overall accuracy varying between -6.7% and +3.8% in plasma, -8.1% and -1.5% in PBMCs, and an overall precision ranging from 3.2% to 6.1% in plasma and 0.8% to 4.0% in PBMCs (SD batch-to-batch). The application to blood samples from healthy volunteers incubated with vorinostat revealed accumulation of vorinostat in PBMCs, effective intracellular HDAC inhibition at therapeutic vorinostat concentrations and a direct vorinostat concentration dependency to HDAC inhibition.
伏立诺他（辛二酰苯胺异羟肟酸）是首个获批用于治疗经两种全身治疗后病情进展的皮肤T细胞淋巴瘤的组蛋白脱乙酰酶（HDAC）抑制剂。伏立诺他的细胞内摄取对于发挥其表观遗传效应至关重要。因此，我们研究了伏立诺他细胞外（血浆）和细胞内（外周血单核细胞，PBMC）浓度之间的关系，并通过HDAC活性检测评估了其浓度-效应关系。开发并验证了用于低纳摩尔定量人血浆和PBMC中伏立诺他及两种无活性代谢物的分析方法。对于从血浆和PBMC中提取伏立诺他，应用了固相萃取和液-液萃取方法。所有分析物和提取方法的提取回收率在88.6%至114.4%之间。提取物在Phenomenex Luna柱上进行等度（血浆）或梯度（PBMC）色谱分析，流动相由酸性醋酸铵、乙腈和甲醇组成。使用氘代内标和正电喷雾串联质谱（多反应监测）对分析物进行定量，血浆的定量下限为11.0 ng/mL，PBMC为0.1 ng/3×10⁶个细胞。伏立诺他在血浆中的校准范围为11.0 - 1100（11,000）ng/mL（代谢物），在PBMC中为0.1 - 10.0 ng/3×10⁶个细胞，相关系数>0.99，血浆中的总体准确度在-6.7%至+3.8%之间，PBMC中为-8.1%至-1.5%，血浆中的总体精密度在3.2%至6.1%之间，PBMC中为0.8%至4.0%（批次间标准差）。对用伏立诺他孵育的健康志愿者血样的应用显示，伏立诺他在PBMC中蓄积，在治疗性伏立诺他浓度下有效抑制细胞内HDAC，且HDAC抑制与伏立诺他浓度直接相关。
