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).
Public Health Relevance Statement: PROJECT NARRATIVE Despite wide use as anti-tumor drugs in patients with hematologic malignancies and as immunosuppressive drugs in inflammatory bowel diseases, thiopurines have a very narrow therapeutic index with dose-limiting hematopoietic toxicity that cases extensive morbidity and possible mortality. Building upon compelling data from our preliminary studies, we propose to develop a novel mass spectrometry-based high throughput method to accurately and rapidly measure thiopurines incorporated into DNA to preemptively identify patients at risk of toxicity and individualize therapy to mitigate it.
Project Terms: 6-Mercaptopurine; Acute Lymphocytic Leukemia; Adopted; Adverse reactions; analog; analytical tool; Antimetabolites; antitumor drug; Autoimmune Diseases; base; Biological; Cell Death; Chemicals; Child; Childhood Leukemia; Clinical; cohort; cytotoxicity; Data; Detection; Disease; DNA; DNA biosynthesis; Dose; dose individualization; Dose-Limiting; Erythrocytes; Funding; Genetic; Genetic Variation; Genotype; Hematologic Neoplasms; Hematopoietic; improved; individual patient; individualized medicine; Inflammatory Bowel Diseases; Inherited; ionization; Knockout Mice; Lead; leukemia/lymphoma; Leukocytes; Mass Spectrum Analysis; Measurement; Measures; Methods; Mismatch Repair; Modernization; Monitor; Morbidity - disease rate; mortality; mouse model; Non-Malignant; novel; nucleobase; Nucleotides; outcome prediction; patient population; Patients; personalized medicine; Pharmaceutical Preparations; Pharmacogenetics; Pharmacology; Phase; Phenotype; Positioning Attribute; precision medicine; prototype; Reaction; Reagent; Regimen; Reporting; Reproducibility; Research Personnel; response; Risk; Sampling; Sensitivity and Specificity; Small Business Technology Transfer Research; Solid; Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stable Isotope Labeling; Sulfhydryl Compounds; Techniques; Testing; Therapeutic Index; Thioguanine; thiopurine; Thiopurine Methyltransferase Deficiency; Toxic effect; TPMT gene; Treatment Efficacy; Treatment Failure; treatment response; Validation; Variant; White Blood Cell Count procedure; Yang
