Measurement of the intracellular active metabolites of thiopurine drugs to evaluate the enzymatic activity of nudix hydrolase 15 in human blood samples

Abstract

Thiopurine is metabolized to 6-thio-(deoxy) guanosine triphosphate (6-thio-(d) GTP), which is then incorporated into DNA or RNA and causes cytotoxicity. Nudix hydrolase 15 (NUDT15) reduces the cytotoxic effects of thiopurine by converting 6-thio-(d) GTP to 6-thio-(d) guanosine monophosphate (6-thio-(d) GMP). NUDT15 polymorphisms like the Arg139Cys variant are strongly linked to thiopurine-induced severe leukocytopenia and alopecia. Therefore, measurement of NUDT15 enzymatic activity in individual patients can help predict thiopurine tolerability and adjust the dosage. We aimed to develop a quantitative assay for NUDT15 enzymatic activity in human blood samples. Blood samples were collected from donors whose NUDT15 genetic status was determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the 6-thio-GTP metabolic activity in cell extracts. Because 6-thio-guanosine diphosphate (6-thio-GDP) and 6-thio-GMP were generated upon incubation of 6-thio-GTP with human blood cell extracts, the method detecting 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP was validated. All three metabolites were linearly detected, and the lower limit of quantification (LLOQ) of 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 5 μM, 1 μM, and 2 μM, respectively. Matrix effects of human blood cell extracts to detect 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 99.0 %, 100.5 %, and 101.4 %, respectively, relative to the signals in the absence of blood cell extracts. The accuracy and precision of the method and the stability of the samples were also assessed. Using this established method, the genotype-dependent differences in NUDT15 activities were successfully determined using cell extracts derived from human blood cells with NUDT15 wild-type (WT) or Arg139Cys variant and 6-thio-GTP (100 μM) as a substrate (18.1, 14.9, and 6.43 μM/h/106 cells for WT, Arg139Cys heterozygous, and homozygous variant, respectively). We developed a method for quantifying intracellular NUDT15 activity in peripheral blood mononuclear cells (PBMCs), which we defined as the conversion of 6-thio-GTP to 6-thio-GMP. Although PBMCs preparation takes some time, its reproducibility in experiments makes it a promising candidate for clinical application. This method can tell the difference between WT and Arg139Cys homozygous blood samples. Even in patients with WT NUDT15, WT samples showed variations in NUDT15 activity, which may correlate with variations in thiopurine dosage.