The impact of high-dose methotrexate on intracellular 6-mercaptopurine disposition during interval therapy of childhood acute lymphoblastic leukemia.

PURPOSE: Low-dose methotrexate (MTX) therapy is the cornerstone treatment of acute lymphoblastic leukemia (ALL) and may enhance the activation of 6-mercaptopurine (6-MP) to 6-thioguanine nucleotides (6-TGN). Yet, data have established that high-dose MTX (HDMTX) hampers the accumulation of 6-TGN in red blood cells (RBC) and lymphoblasts. METHODS: To clarify the pharmacokinetic interactions between these two antimetabolites, we serially measured RBC 6-TGN and MTX polyglutamates (MTXPG) levels following repeated courses of HDMTX (5 g/m(2) over 24 h) with daily oral 6-MP (25 mg/m(2)) during interval therapy in 20 children with ALL. RESULTS: HDMTX produced a rapid reduction in RBC 6-TGN 24 h after the start of MTX, and this effect was sustained at least by the third day (median decrease -21%; P < 0.001). However, a return to pre-infusion of 6-TGN levels was observed by the time of the following HDMTX course 14 days later (P < 0.001). RBC MTX polyglutamates accumulation followed Michaelis-Menten kinetics but was not associated with the change in pre-infusion 6-TGN levels which remained stable during the interval period. CONCLUSION: HDMTX does not appear to enhance 6-MP activation to 6-TGN. Moreover, given that the deleterious effect of HDMTX on intracellular 6-MP disposition has been shown to be several folds greater in lymphoblasts than in RBC. Our data warrant additional studies elucidating the optimal MTX dose synergizing with 6-MP.

Should TPMT genotype and activity be used to monitor 6-mercaptopurine treatment in children with acute lymphoblastic leukaemia?

BACKGROUND AND OBJECTIVE: The activity of thiopurine S-methyltransferase (TPMT), a key enzyme in the metabolism of purine analogues, displays wide inter-subject variability partly due to a genetic polymorphism. Previous studies have suggested adjusting purine analogues dosing according to TPMT activity but measurements are costly and time-consuming. It is still unclear, especially under treatment, whether the simpler TPMT genotyping reliably predicts enzyme activity. Our aim was to study the possible correlation of TPMT genotype with phenotype. METHODS: We determined the genotypic status and TMPT activity, at diagnosis and after 6 months of maintenance therapy, of 118 children with acute lymphoblastic leukaemia (ALL). RESULTS AND DISCUSSION: Eighty-nine per cent of the children had a homozygous wild-type genotype (group 1), 11% had one or two mutant allele(s) (group 2). At both time points, TPMT activity (U/mL peripheral red blood cell) was significantly higher in group 1 than in group 2 (P < 0.001) but inter-group levels overlapped considerably. There was considerable heterogeneity in the percentage increase in TPMT activity after therapy, and little correlation between metabolites ratio [6-methylmercaptopurine derivative/6-thioguanine nucleotides (6-TGN)] and TPMT activity at the end of 6 months' maintenance treatment. These results show that TPMT activity cannot be used as an accurate tool for 6-mercaptopurine monitoring. CONCLUSION: Genotyping at diagnosis identifies patients with a homozygous mutant TPMT and may prevent severe and life-threatening toxicity. ALL treatment monitoring should preferentially be based on repeated determinations of intracellular active metabolites (6-TGN) and methylated metabolites.

Possible implication of thiopurine S-methyltransferase in occurrence of infectious episodes during maintenance therapy for childhood lymphoblastic leukemia with mercaptopurine.

6-Mercaptopurine (6-MP) is metabolized by thiopurine S-methyltransferase (TPMT), an enzyme subject to genetic polymorphism. We investigated the relationships between the TPMT locus (TPMT activity and genotype) and the pharmacological response to 6-MP during maintenance therapy of 78 children with acute lymphoblastic leukemia (ALL). For each patient, 6-MP dosage, leukocyte counts and occurrence of infectious episodes were monitored on an 8 week basis. Higher 6-MP dosage was associated with higher TPMT activity (P = 0.03) and higher average leukocyte counts (P < 0.01). Eight patients (10%) carrying a TPMT mutant genotype (one homozygous and seven heterozygous) received lower 6-MP doses (average: 48 vs 65 mg/m2/day; P = 0.02) and had on average lower leukocyte counts (2834 vs 3398 cells/mm3; P = 0.003) than patients carrying the wild-type TPMT genotype. Higher occurrence of infectious episodes graded 2 or 3 was correlated with higher 6-MP dosage (P < 0.01) but no difference was observed between TPMT mutants and TPMT wild-type patients. Patients who received 6-MP dosage above the group median (62 mg/m2/day) or having a TPMT activity above the group median (21.5 nmol/h/ml) had a higher percentage of 8 week periods with infectious episodes requiring treatment (34% vs 17% and 33% vs 19%, respectively) than those with 6-MP dose or TPMT activity below the group median (P < 0.01). In the last 25 patients enrolled in the study, steady-state erythrocyte thioguanine nucleotide (TGN) concentrations were associated with lower leukocyte counts (P= 0.01) but not with a higher occurrence of infectious episodes. In contrast, higher steady-state erythrocyte methylmercaptopurine nucleotide (MeMPN) concentrations were associated with higher 6-MP dosage (P< 0.01) and higher occurrence of infectious episodes (P < 0.001). In conclusion, during maintenance therapy of ALL, children with higher TPMT activity receive a higher 6-MP dosage and may have infectious episodes caused by metabolism of 6-MP into methylmercaptopurine nucleotides.

Thiopurine methyltransferase activity and its relationship to the occurrence of rejection episodes in paediatric renal transplant recipients treated with azathioprine.

AIMS: Azathioprine is a prodrug commonly used in combination therapy to prevent allograft rejection after renal transplantation. After conversion to 6-mercaptopurine, the drug is metabolized into 6-thioguanine nucleotides (6-TGN) and catabolized by thiopurine methyltransferase (TPMT), an enzyme under monogenic control. The aim of this study was to evaluate the inter- and intraindividual variability of red blood cell thiopurine methyltransferase and 6-TGN concentrations and their relationship to the clinical effects of azathioprine in paediatric patients. METHODS: In the present study, the inter- and intraindividual variations in red blood cell TPMT activity and 6-TGN concentrations and their relationship to the actions of azathioprine were evaluated during the first year after renal transplantation in 22 paediatric patients. RESULTS: 6-TGN concentration reached steady-state values after 6 months and correlated negatively with TPMT activity (P=0.004). Initial TPMT activity (median: 20.8 nmol h-1 ml-1, range 7.8-34.6) and 6-TGN concentration at steady-state (median: 80 pmol 8 x 10(8-1) cells, range not detected to 366) were not related to the occurrence of rejection episodes during the period of the study. In contrast, TPMT activity and the percentage difference in TPMT activity from the day of transplantation determined at month 1 were higher in the patients with rejection episodes by comparison with those that did not reject during the first 3 months or the first year following transplantation (P<0.005). CONCLUSIONS: We report a relationship between TPMT activity and occurrence of rejection in paediatric kidney transplant patients undergoing azathioprine therapy. These data suggest a link between high red blood cell TPMT activity and poor clinical outcome probably caused by rapid azathioprine catabolism.

High-performance liquid chromatographic method for quantification of busulfan in plasma after derivatization by tetrafluorothiophenol.

A high-performance liquid chromatographic (HPLC) method was developed and validated for the determination of busulfan in plasma. Busulfan was extracted in toluene, derivatized by 2,3,5,6-tetrafluorothiophenol to obtain di-TFTP-butane, the derivatization product was then re-extracted in toluene and injected into the HPLC system with ultraviolet detection (wavelength: 275 nm). Recovery from extraction was 80%, the limit of quantification was 50 ng/ml and linearity ranged from 50 to 2000 ng/ml. In addition, forty-two samples obtained from pediatric patients treated with busulfan were analyzed by the HPLC and GC-MS assays based on the same derivatization procedure. The correlation between the di-TFTP-butane concentrations was highly significant (p<0.0001), demonstrating that the two methods were in good agreement.

In-vitro cytochrome P450 dependent metabolism of oxybutynin to N-deethyloxybutynin in humans.

Oxybutynin (Ditropan), a direct antagonist of acetylcholine on muscarinic receptors, is administered in children for the treatment of vesical immaturity and is responsible for atropinic adverse effects, which are more frequent in paediatric than in adult patients. Oxybutynin is metabolized by oxidation to N-desethyloxybutynin, a stable and toxic metabolite, and previous results in vivo have suggested that cytochrome P450 2D6 (CYP2D6) may be involved in this pathway of metabolism. We used human liver microsomes genotyped as extensive metabolizers for CYP2D6 to determine the kinetic parameters of N-desethyloxybutynin formation: Km was 16.5+/-5.2 microM and Vmax was 76.8+/-3.7 mmol/mg/h. Quinidine and anti-liver kidney microsome antibody type I had no inhibitory effects on N-desethyloxybutynin formation, demonstrating that CYP2D6 has no role in oxybutynin metabolism. The effects of specific inhibitors of other cytochromes P450 were also investigated. Recombinant human CYP 3A4 but not 2B6, 2D6, 2C8 and 2E1, displayed significant N-desethylation activity. Our results demonstrate that the CYP3A subfamily, and not CYP2D6, is involved in N-desethyloxybutynin formation.

Thiopurine methyltransferase activity: new high-performance liquid chromatographic assay conditions.

This paper reports changes to our previously published high-performance liquid chromatographic method for the measurement of 6-methylmercaptopurine (6-MMP) in red blood cell lysates. The extraction procedure and chromatographic conditions have been improved and the range of the calibration curves has been modified. The recoveries of 10 and 100 ng ml(-1) 6-MMP were 99.0+/-6.0% and 96.3+/-4.0% respectively and the limit of quantification was lowered to 5 ng ml(-1). This method, which does not require radioactive S-adenosyl-L-methionine, is more sensitive, specific and reproducible and may prove useful for routine determination of thiopurine methyltransferase activity in red blood cells.

Pharmacokinetics and distribution of 6-mercaptopurine administered intravenously in children with lymphoblastic leukaemia.

OBJECTIVE: The pharmacokinetics of 6-mercaptopurine, including cerebrospinal-fluid (CSF) distribution, and the erythrocyte 6-thioguanine nucleotide concentrations were determined in children randomised to receive intravenous mercaptopurine for acute lymphoblastic leukaemia (ALL), according to the EORTC protocol ALL n.58881. RESULTS: After 1 month of oral treatment at a dose of 50 mg.m-2.day-1, the pharmacokinetic parameters were determined after the first i.v. administration of 1 g.m-2 (bolus dose of 0.2 g.m-2 followed by an 8-h infusion of 0.8 g.m-2) in 11 patients: systemic clearance was 23.02 1.h-1, volume of distribution was 0.75 l.kg-1, and elimination half-life was 1.64 h. The erythrocyte thioguanine concentrations were measured in the same 11 patients and increased significantly between the beginning and the end of infusion (10 pmol x 10(8) packed RBC) or within 24 h of infusion (223 pmol x 10(8) packed RBC). The CSF concentration was 3.78 mumol.1(-1), 1-6 h after the beginning of infusion (n = 28) and the CSF to plasma ratio was 0.15 (n = 16). In patients receiving the oral dose of 50-165 mg.m-2.day-1 of 6-mercaptopurine, CSF concentrations were below 0.18 mumol.1(-1), 1-24 h after drug intake (n = 67), and the CSF to plasma ratio was not calculated. CONCLUSION: Following the i.v. administration of 6-mercaptopurine, we observed high CSF concentrations of 6-mercaptopurine and an acute increase of erythrocyte thioguanine nucleotide concentrations. The clinical trial (EORTC protocol ALL n[symbol: see text]5881), comparing the oral and i.v. administrations of mercaptopurine, will demonstrate if the i.v. administration reduces the incidence of CNS relapses.

Thiopurine methyltransferase activity in a French population: h.p.l.c. assay conditions and effects of drugs and inhibitors.

1. Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the catabolism of thiopurine drugs, which are used to treat cancer patients and organ transplant recipients. Because TPMT activity is polymorphic and under genetic control, large interindividual variations in the immunosuppressive activity and toxicity of these drugs may, at least in part, be inherited. 2. We have developed a specific h.p.l.c. method for measuring 6-methyl mercaptopurine formed from 6-mercaptopurine (6-MP) in red blood cell lysates during the TPMT assay procedure. In blinded assays of 55 samples from adult blood donors, the results of the h.p.l.c. method correlated with those of the radiochemical reference method (r = 0.83, P < 0.001). 3. Using this h.p.l.c. assay, we tested the effect of known inhibitors of TPMT activity (syringic acid, p-anisic acid and tropolone) in vitro and showed that they were highly inhibitory. We also found that drugs often administered concomitantly with 6-MP (prednisone, prednisolone, 6-methylprednisolone, cyclophosphamide, methotrexate, and trimethoprim-sulphamethoxazole) had little or no effect on TPMT activity in vitro. 4. In a group of 300 French individuals, TMPT activity was highly variable, ranging from 4.7 to 35.3 nmol h-1 ml-1 of packed red blood cells (nmol h-1 ml-1 PRBC) with a mean value of 19.3 +/- 4.9. TMPT activity was not influenced by sex. 5. This sensitive and reproducible h.p.l.c. assay for TPMT activity in red blood cells may prove useful for prospective clinical studies designed to optimise dosage regimens of thiopurine drugs (detection limit for 6-methyl mercaptopurine is 5 ng ml-1, intra- and inter-assay variations are 6.8 and 8.2%, respectively).