Analysis of mono-, di-, and triphosphates of thioguanosine and methylthioinosine in children with acute lymphoblastic leukemia by LC-MS/MS.

Mercaptopurine (6-MP) is an indispensable, first-line, drug in the treatment of pediatric acute lymphoblastic leukemia (ALL). However, 6-MP has several intrinsic drawbacks, such as large individual variability in the drug response, undesirable adverse reactions, and drug resistance in patients with release ALL, which requires therapeutic drug monitoring (TDM). Several studies analyzed the total concentration of thiopurine nucleotides in red blood cells (RBCs) after hydrolysis, and two studies detected them separately and accurately by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we developed a rapid and robust LC-MS/MS method for simultaneous quantitation of mono-, di-, and triphosphates of thioguanosine and methylthioinosine. Not only EDTA and DTT were added, but also EHT1864, a new Rac family small GTPases inhibitor, was innovatively added to ensure the stability of the analytes. Commercial availability and relatively low cost compound methotrexate-D3 was selected as internal standards. The linearity, accuracy, precision, recovery, matrix effect and stability of the method were all in line with the guidelines. This method provide an accurate and robust new solution for the determination of 6 metabolites of MP in RBCs from ALL patients with maintenance therapy.

Simultaneous quantification of eleven thiopurine nucleotides by liquid chromatography-tandem mass spectrometry.

The prodrugs azathioprine and 6-mercaptopurine, which are well-established anticancer and immunosuppressive agents, are extensively metabolized by activating and inactivating enzymes. Whereas the 6-thioguanine nucleotides (TGN) are currently being considered as major active metabolites, methylthioinosine nucleotides seem to contribute to the cytotoxic effect as well. Thiopurine-related adverse drug reactions and thiopurine failure are frequent. Thus, therapeutic monitoring of TGN and methylthioinosine derivatives has been suggested to improve thiopurine therapy, however with limited success. To elucidate systematically underlying molecular mechanisms as potential explanation for interindividual variability of thiopurine response, we developed a novel highly specific and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of eleven mono-, di-, and triphosphates of thioguanosine, methylthioinosine, methylthioguanosine, and thioinosine. Using stable isotope-labeled analogues as internal standards obtained by chemical synthesis, an intra- and interassay variability below 8% and an accuracy of 92% to 107% were achieved in spiked quality control samples with known standards. All eleven metabolites could be determined in red blood cells from patients with inflammatory bowel diseases and long-term azathioprine therapy. Thus, our novel method opens a new avenue for the understanding of the thiopurine metabolism by quantitation of all important thiopurine nucleotide metabolites in one run.

Methotrexate increases red blood cell concentrations of 6-methylmercaptopurine ribonucleotide in rats in vivo.

PURPOSE: To elucidate the effect of methotrexate (MTX) on 6-mercaptopurine (6-MP) metabolism in rats. METHODS: Fourteen rats were given 6-MP 20 mg/kg daily for 7 days. Seven of the rats were also given MTX 20 mg/kg on days 5 and 7. Blood samples were obtained from all rats on days 0.5 and 8, and red blood cell (RBC) lysates were analysed for thiopurine methyltransferase (TPMT) activity and the concentration of methylated 6-MP metabolites [methyl mercaptopurine ribonucleotides (MMPRP)] and 6-thioguanine nucleotides (6-TGN). RESULTS: The concentration of MMPRP increased 2.4 times from day 5 to day 8 in RBCs from rats given MTX in addition to 6-MP, as against 1.2 times in rats given 6-MP alone (P = 0.003). 6-TGN levels increased and TPMT activity decreased from day 5 to day 8, with no difference between the 6-MP and the 6-MP plus MTX groups. CONCLUSIONS: Single bolus doses of MTX increase the concentration of MMPRP in rats given daily s.c. doses of 6-MP, with no effect on 6-TGN concentration or TPMT activity.

Chromatographic analysis of methylmercaptopurine riboside in human plasma and urine.

An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method for the determination of methylmercaptopurine riboside (MMPR) in human plasma and urine is reported. Plasma samples were prepared for analysis by addition of internal standard (6-dimethylaminopurine 9-riboside) followed by extraction using disposable C18 cartridges. Urine samples were filtered through a 0.22-micron membrane prior to HPLC separation. The column effluent was monitored at 289 nm and quantitation performed using peak heights. The linear range for MMPR determination was from 10 to 500 ng/ml in plasma and from 0.25 to 50 micrograms/ml in urine. The reported method is convenient, sensitive, and reproducible, illustrating its usefulness for application in pharmacokinetic studies.

Persistently decreased hepatic levels of 5'-deoxy-5'-methylthioadenosine during regeneration of and chemical carcinogenesis in rat liver.

The hepatic levels of 5'-deoxy-5'-methylthioadenosine (MTA) were measured in the livers of adult male Sprague-Dawley rats a) killed at various times during the liver regeneration process, b) killed at times after partial hepatectomy when the liver mass had already been completely restored (hereafter called post-regeneration livers), or c) continuously fed 3'-methyl-4-dimethyl-aminoazobenzene (CAS: 55-80-1) up to the full development of hepatoma and killed at regular intervals during hepatocarcinogenesis. Hepatic MTA levels were always significantly decreased, although to different degrees in both in vivo models of hepatic growth and at all times during the investigation. Astonishingly, the MTA levels were also significantly decreased in the post-regeneration livers, in which there was also a significant increase in the activity of adenosylmethionine decarboxylase (S-adenosyl-L-methionine decarboxylase; EC 4.1.1.50) with normal levels of activity of ornithine decarboxylase (EC 4.1.1.17). These results demonstrate that a) the MTA content is always decreased in rat liver whenever this organ is involved in a proliferative process (whether controlled or uncontrolled); b) the decrease in hepatic MTA content is a biochemical feature necessary for, but by no means by itself sufficient for, hepatocyte proliferation to occur, since this decrease remains long after complete restoration of the liver mass; and c) the return of the hepatocytes to the normal biochemical program after restoration of the liver mass is not complete, even though these cells become quiescent, because there are still some biochemical abnormalities in the post-regeneration livers.

A rapid and sensitive high-performance liquid chromatographic assay for 6-mercaptopurine metabolites in red blood cells.

A highly sensitive and rapid assay for the detection of 6-mercaptopurine metabolites in the red blood cells of leukemic patients receiving the drug has been developed. The method employs a batch-chromatographic procedure using a mercurial cellulose resin to selectively absorb thiol compounds combined with separation by high-performance liquid chromatography using a Partisil-SAX column and uv detection. This method permits detection of 6-thioinosine monophosphate, 6-thiouric acid, and 6-thioguanosine mono-, di-, and triphosphates in patient samples with a sensitivity of 5-10 pmol. No 6-thioinosine di- or triphosphates were detected in patient samples. The results of our study indicate that 6-thioguanosine triphosphate is the major metabolite of 6-mercaptopurine retained by red blood cells after oral or iv administration of the drug.

Archaebacterial tRNA contains 1-methylinosine at residue 57 in T psi C-loop.

1-Methylinosine was isolated from unfractionated tRNAs of Sulfolobus acidocaldarius and Halobacterium volcani. The structure of 1-methylinosine was characterized by ultraviolet absorption and mass spectral analysis. 1-Methylinosine found in these archaebacterial tRNAs was located at residue 57 in T psi C-loop, indicating that the presence of 1-methylinosine is characteristic of archaebacteria.