Developing high throughput measurement of thiopurine in DNA by mass spectrometry

通过质谱法开发 DNA 中硫嘌呤的高通量测量

• 批准号: 9909135
• 负责人: W. Andy Tao
• 金额: $ 30万
• 依托单位: TYMORA ANALYTICAL OPERATIONS, LLC
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-02 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9909135
• 关键词: 6-Mercaptopurine; Acute Lymphocytic Leukemia; Adopted; Adverse reactions; Antimetabolites; Autoimmune Diseases; Biological; Cell Death; Chemicals; Child; Childhood Leukemia; Clinical; DNA; DNA biosynthesis; Data; Detection; Disease; Dose; Dose-Limiting; Erythrocytes; Funding; Genetic; Genetic Variation; Genotype; Hematologic Neoplasms; Hematopoietic; Inflammatory Bowel Diseases; Inherited; Knockout Mice; Lead; Leukocytes; Mass Spectrum Analysis; Measurement; Measures; Methods; Mismatch Repair; Modernization; Monitor; Morbidity - disease rate; Non-Malignant; Nucleotides; Patients; Pharmaceutical Preparations; Pharmacogenetics; Pharmacology; Phase; Phenotype; Positioning Attribute; Reaction; Reagent; Regimen; Reporting; Reproducibility; Research Personnel; Risk; Sampling; Sensitivity and Specificity; Small Business Technology Transfer Research; Solid; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stable Isotope Labeling; Sulfhydryl Compounds; TPMT gene; Techniques; Testing; Therapeutic Index; Thioguanine; Thiopurine Methyltransferase Deficiency; Toxic effect; Treatment Efficacy; Treatment Failure; Validation; Variant; White Blood Cell Count procedure; Yang; analog; analytical tool; antitumor drug; base; cohort; cytotoxicity; dose individualization; improved; individual patient; individualized medicine; ionization; leukemia/lymphoma; mortality; mouse model; novel; nucleobase; outcome prediction; patient population; personalized medicine; precision medicine; prototype; response; thiopurine; treatment response

PROJECT SUMMARY Thiopurines such as mercaptopurine are S-substituted antimetabolites used as functional analogs to natural nucleobase precursors. They are highly effective against hematologic malignancies (leukemia and lymphoma) and autoimmune diseases (inflammatory bowel diseases [IBD]), with an estimated patient population >350,000 in the US. However, the cytotoxicity of thiopurines depends on their conversion into 6-thioguanine (TG) nucleotides (TGN), which are incorporated into DNA, causing cell death by post-replicative DNA mismatch repair. They have narrow therapeutic indexes with dose-limiting hematopoietic toxicity whereas low-responders are undertreated with standard dosing. Therefore, there is enormous clinical benefit from preemptively identifying patients at risk of thiopurine toxicity and individualizing therapy to mitigate it. We propose here a high throughput method based on matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) for quantifying the pharmacological endpoint of thiopurines, TG incorporated in chromosomal DNA (DNA-TG) of white blood cells, and examine its specificity and accuracy in standard and clinical samples. To test this strategy, we propose three specific aims: Aim #1. Chemical derivatization, isolation and detection of 6-thioguanine (6-TG) by MALDI-MS. Aim #2. Examination of linear range, sensitivity, specificity and reproducibility of the method. Aim #3. Validation of high throughput 6-TG detection with biological samples. With the aim to individualize thiopurine therapy through reducing adverse reactions and obtaining optimum therapeutic efficacy for patients on thiopurine treatment, the high throughput technique facilitates continuous monitoring of thiopurine in DNA before and during the therapy. This highly translational project will likely have high impact on a large number of patients who are under chemo-treatment or thiopurine therapy for non-malignant conditions (e.g., IBD).

项目概要 硫代嘌呤如巯嘌呤是 S-取代的抗代谢物，用作功能类似物 天然核碱基前体。它们对血液恶性肿瘤非常有效 （白血病和淋巴瘤）和自身免疫性疾病（炎症性肠病 [IBD]）， 美国估计患者人数>350,000。然而，硫嘌呤的细胞毒性取决于 它们转化为 6-硫鸟嘌呤 (TG) 核苷酸 (TGN)，并整合到 DNA 中， 通过复制后 DNA 错配修复导致细胞死亡。它们的治疗指数较窄 具有剂量限制性造血毒性，而低反应者则未接受标准治疗 剂量。因此，预先识别有风险的患者具有巨大的临床益处。 硫嘌呤毒性和减轻毒性的个体化治疗。我们在这里建议高吞吐量 基于基质辅助激光解吸电离 (MALDI) 质谱 (MS) 的方法 定量纳入染色体 DNA 的硫嘌呤、TG 的药理学终点 (DNA-TG)白细胞，并检查其在标准和临床中的特异性和准确性 样品。为了测试这一策略，我们提出了三个具体目标： 目标#1。化学衍生化， 通过 MALDI-MS 分离和检测 6-硫鸟嘌呤 (6-TG)。目标#2。线性范围检查， 该方法的灵敏度、特异性和重现性。目标#3。高通量 6-TG 的验证 用生物样品进行检测。旨在通过减少硫嘌呤治疗的个体化 硫嘌呤治疗患者的不良反应并获得最佳治疗效果， 高通量技术有助于在检测前和检测前连续监测 DNA 中的硫嘌呤 治疗期间。这个高度转化的项目可能会对大量的人产生重大影响 正在接受化疗或硫嘌呤治疗的非恶性疾病的患者（例如， 炎症性肠病）。