C.W. Keuzenkamp-Jansen

Determination of extracellular and intracellular thiopurines and methylthiopurines by high-performance liquid chromatography

The thiopurine antimetabolites 6-thioguanine and 6-mercaptopurine are important chemotherapeutic drugs in the treatment of childhood acute lymphoblastic leukaemia. Measurement of metabolites of these thiopurines is important because correlations exist between levels of these metabolites and the prognosis in childhood acute lymphoblastic leukaemia. The reversed-phase method for the determination of extracellular thiopurine nucleosides and bases was previously developed and has been modified such that methylthiopurine nucleosides, bases, thioxanthine and thiouric acid can be measured also. The anion-exchange method enables the determination of intracellular mono-, di- and triphosphate (methyl)thiopurine nucleotides in one run. Extraction on ice with perchloric acid and dipotassium hydrogenphosphate results in good recoveries for (methyl)thiopurine nucleotides in lymphoblasts and peripheral mononuclear cells and for methylthioinosine nucleotides in red blood cells. Measurement of the low concentrations of mono-, di- and triphosphate thioguanine nucleotides in red blood cells (detection limit 20 pmol/10(9) cells) is possible after extraction with methanol and methylene chloride, followed by oxidation of thioguanine nucleotides with permanganate and fluorimetric detection.

Metabolism of intravenously administered high-dose 6-mercaptopurine with and without allopurinol treatment in patients with non-Hodgkin lymphoma.

Purpose: We investigated the metabolism of high dose 6 mer-captopurine (HD-6MP) infusions and its influenee on the metabolism by allopurinol, an inhibitor of xanthinc oxidase, the enzyme that catabolizes 6MP into thioxanthine and thiourie acid. Patients and Methods: Nine patients (aged 2− 11 years) with non-Hodgkin lymphoma (NHL) were treated with HD-6MP (1300 mg/m2.24 h) within a therapeutic window after diagnosis. Four patients received oral allopurinol (200 mg/m2.day) to prevent urate nephropathy, and five did not. Plasma and RBC were isolated before and 4, 20, 24, 28, and 48 h after the start of the infusion. All measurements were performed with HPLC. Results: Considerable variations were found in the plasma levels of 6MP. thioxanthine. and thiourie acid and of RBC-MeTIN levels. 6MP-ribosidc was not detectable, and MeMP and MeMPR levels were <1.3 M in the plasma. In general, 6MP, thioxanthine, and McMP levels in plasma were higher, and thiourie acid plasma levels and RBC-MeTIN levels were lower in the patients treated with allopurinol compared to those who did not receive allopurinol. Conclusions: 6MP is extensively metabolized in patients with NHL treated with HD-6MP. Thiopurine mcthylation, at the levels of nuclcotide. nucleoside. and base, is an important metabolic pathway after HD-6MP. Co-administration of allopurinol can result in both a decreased catabolism and anabolism of 6MP compared to treatment with HD-6MP alone. This observation may have consequences for the therapeutic efficacy and toxic effects of 6MP in combination with allopurinol.

Thiopurine Treatment in Childhood Leukemia

The thiopurine antimetabolites 6-mercaptopurine (6MP) and 6-thioguanine (TG) are important chemotherapeutic drugs in the treatment of acute lymphobastic leukemia. Both drugs are initially converted by the purine enzyme hypoxanthine guanine phos-phoribosyl transferase (HGPRT) into 6-thio-inosine monophoshate (TIMP) and 6-thio-guanosine monophosphate (TGMP), respectively. Further conversion of TIMP to TGMP via 6-thio-xanthine monophosphate (TXMP) is catalyzed by two enzymes, IMP-dehydro-genase (IMPDH) and GMP-synthetase, respectively. IMPDH is the rate-limiting enzyme in these two enzymatic steps (1). Subsequently, TGMP is converted by consecutive steps to the 6-thio-guanosine triphosphates: 6-thio-GTP (TGTP) and 6-thio-deoxy-GTP (TdGTP), which are incorporated into RNA and DNA, respectively.

Intracellular Pharmacology and Biochemistry of Methotrexate and 6-Mercaptopurine in Childhood Acute Lymphoblastic Leukemia

After treatment with methotrexate (MTX), 6-mercaptopurine (6MP) demonstrated a synergistic effect and an enhanced incorporation into DNA and RNA in human lymphoblastic cell lines1.