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. Measure ment of the low concentrations of mono-, di-and triphosphate thioguanine nucleotides in red blood cells (detection limit 20 pmol/109 cells) is possible after extraction with methanol and methylene chloride, followed by oxidation of thioguanine nucleotides with permanganate and fluorimetric detection.
Thiopurine methyltransferase (TPMT) is an important enzyme in the metabolism of 6-mercaptoptirine (6MP), which is used in the treat men t of acute lymphoblastic leukemia (ALL). TPMT catalyzes the formation of methylthioi nosine monophosphate (MetlMP), which is cytotoxic for cultured cell lines, and it plays a role in detoxification of 6MP, Population studies show a genetic polymorphism for TPMT with both high and low activity alleles. About 1 of 300 subjects is homozygous for the low activity. The function TPMT plays in detoxification or therapeutic efficacy of 6MP in vivo is not clear. In this article the genetic polymorphism of TPMT is reviewed and the contribution of TPMT to the cytotoxic action, or detoxification, of 6MP in children with ALL is discussed. Induction of TPMT activity has been described during the treatment for ALL. We performed a pilot study on the influence of higlvdose 6MP infusions (1300 m g/m 2 in 24 h) on TPMT activity of peripheral blood mononuclear cells (pMNC) of eleven patients with ALL. The TPMT activities were in, or, above the normal range. There was no statistically significant difference between the TPMT activities before and after the 6MP infusions. MetlMP levels in pMNC increased during successive courses. This might be explained by TPMT induction, but other explanations are plausible as well. Twenty five percent of the TPMT assays failed, because less than the necessary 5*i0f' pMNC could be isolated from the blood of leukopenic patients. Red blood cells can not be used for TPMT measurements, since transfusions are frequently required during the treatment with 6MP infusions. Therefore, the influence of high-dose 6MP infusions on TPMT activity can only be investigated further when a TPMT assay which requires less pMNC has been developed.
6MP is extensively metabolized in patients with NHL treated with HD-6MP. Thiopurine methylation, at the levels of nucleotide, 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.
']a r is e n R o n n e y A. De Ahreu,*f HenkJ. Blom* Jos P.M. Bokkerinkf andj. M. Frans Trijbels* ♦ L a b o r a t o r y o f P e d i a t r i c s , a n d t C e n t e r f o r P e d ia t r ic : O n c o l o g y S.E. N e t h e r l a n d s , U n i v e r s i t y H o s p i t a l S t . R a d b o u d , P.O. Box 9101» 6500 HB N ijm e g e n , T h e N e t h e r l a n d s A BSTRA CT. 6 -mercaptopurine (6MP) cytotoxicity is caused by thioguanine and methylthioinosine nucleo tides. Thiopurine méthylation occurs to a large extent in vivo and in vitro. In this reaction, S-adenosyl-Lmethionine (AdoMet), produced from methionine and ATP, is converted into S-adenusy l-L-htunocysreine (AdoHcy) which, in turn, is hydrolyzed into homocysteine. Remethylation of homocysteine into methionine is inhibited by methotrexate (MTX). In cultured lymphoblasts, AdoMenAdoHcy ratio and DNA méthylation decrease after incubation with 6MP. The aim of the present study was to investigate the influence of high-do.se 6MP on the méthylation capacity in children with acute lymphoblastic leukemia. Five patients received 4 courses with high-dose intravenous MTX (5 g • m in 24 hr) immediately followed by high-dose 6MP (1300 mg • m~2 in 24 hr). Five control patients received high-dose MTX and oral 6MP (25 mg • m~2 daily for 8 weeks). Leucovorin rescue was started at .36 hr in both groups.In the intravenous 6MP group, 6-methylmercaptopurine, its riboside, and 6-methylmercapto-8-hydroxypurine were detectable in plasma in concentrations of 0.3-2.6 |jlM (6MP steady state levels: 11.6 (xM). In red blood cells, mean methylthioinosine nucleotide levels were one third of those of ATP (13,1 nmol/10"). AdoHcy levels (10 pmol/108) remained constant in both groups and AdoMet was not detectable (<20 pmol/10,s). In both groups, plasma homocysteine increased and methionine decreased following administration of MTX. The delay in the recovery of methionine in the intravenous 6MP group after MTX infusion is probably the result-of an increased demand on methyl groups during 6MP infusion. BIOCHKM PHARMACOL 51;9:1165-1171, KEY W ORDS. 6 -mercaptopurine; methotrexate; acute lymphoblastic leukemia; méthylation § is used in the treatment of ALL. It has no intrinsic cytotoxic activity, but is converted into active metabolites intracellularly (Scheme 1). 6MP tlMP which, itself, can be conve MetlMP. Both pathways ferase (EC 2.1.1.67) (TPMT). The TPMT activity is con trolled by a genetic polymorphism and the activity in RBC correlates with that in lymphoblasts, lymphocytes, plate lets, liver, and ki •' V ** % oi in cytotoxicity in vitro, ei ther by incorporation of thioguanine nucleotides into DNA and RNA [1,2] or by inhibition of purine de novo synthesis by MetlMP [2,3]. 6MP is methylated into MeMP and 6MP-riboside into MeMPR (Scheme 1). The thiopurine meththe frequency distribution is trimodal with subjects displaying high activity, 11.1% intermediate activ ity, and 1 out of 300 subjects having undeteetahle TPMT . Thiopurine méthylation requires AdoMet as onor [9]. AdoMet is the universa...
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