6-TGN Concentrations Research Articles

MONITORED HIGH-DOSE AZATHIOPRINE TREATMENT REDUCES ACUTE REJECTION EPISODES AFTER RENAL TRANSPLANTATION

Azathioprine (AZA) is widely used in organ transplantation. Common practice is to adjust dose according to body weight only, despite documented pharmacokinetic variability. The purpose of this study was to investigate whether high-dose AZA treatment monitored by 6-thioguanine nucleotides (6-TGN) levels reduces the incidence of rejection episodes in renal transplantation without a corresponding increase in myelotoxicity. Patients receiving cyclosporine, steroids, and AZA were randomized into either the low-dose AZA group (3 mg/kg on day 0, then 2 mg/kg/day the first week and 1 mg/kg/day thereafter) or the high-dose AZA group. In the latter, AZA was started at 5 mg/kg/day and then adjusted to keep 6-TGN concentrations (measured twice weekly) between 100 and 200 pmol/8 x 10(8) RBCs. A total of 360 transplant recipients were included in the final analysis. The cumulative incidence of first rejection episodes was reduced by 21%, from 62.8% in the low-dose group to 49.4% in the high-dose group (difference: 13.3%; 95% confidence interval: 3.2-23.5). Similar results were found in subgroups according to HLA-DR match. The 6-TGN concentration was significantly higher in the high-dose AZA group during the first month, and the reduction in rejection episodes was achieved in the same period. A larger proportion of patients in the high-dose group had nadir white blood cell count below 2.0 x 10(9) leukocytes/L (13.3% vs. 4.4%; difference: 8.9%; confidence interval: 3.1-14.7). High-dose AZA therapy in a triple-drug regimen, monitored by 6-TGN, will keep myelotoxicity within acceptable limits with the benefit of a reduction in acute rejection episodes.

Optimisation of Azathioprine Immunosuppression After Organ Transplantation by Pharmacological Measurements

Azathioprine undergoes extensive metabolism in vivo. Most of its immunosuppressive and myelotoxic effects are exerted by the intracellular metabolites 6-thioguanine nucleotides (6-TGN). There is large individual variability in thiopurine pharmacokinetics. When transplant recipients are started on the standard azathioprine dosage, low and probably subtherapeutic 6-TGN concentrations [<100 pmol/8 x 10(8) red blood cells (RBC)] are measured in the majority of patients with normal kidney function. When renal function is severely impaired, 6-TGN concentrations rise 8- to 10-fold or higher. Due to genetic polymorphism, the activity of the enzyme thiopurine methyltransferase (TPMT) is intermediate to undetectable in approximately 11% of the population. With low TPMT activity, transmethylation is reduced and more intermediate metabolites are left for alternative pathways such as 6-TGN formation. High 6-TGN concentrations are associated with increased frequency and severity of leucopenia. It has been suggested that active monitoring of azathioprine to keep 6-TGN concentrations between 100 and 200 pmol/8 x 10(8) RBC may contribute to more effective treatment by reducing the incidence of rejection episodes and leucopenia. Such monitoring is currently being evaluated in a controlled, prospective study of renal allograft recipients.

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

To elucidate the effect of methotrexate (MTX) on 6-mercaptopurine (6-MP) metabolism in rats. 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). 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. 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.

Possibilities for therapeutic drug monitoring of azathioprine: 6-thioguanine nucleotide concentrations and thiopurine methyltransferase activity in red blood cells.

The objectives of this study were to establish monitoring of azathioprine (AZA) treatment in renal allograft recipients by red blood cell (RBC) 6-thioguanine nucleotide (6-TGN) measurements and to characterize the variability of RBC thiopurine methyltransferase (TPMT) activity and the effects on 6-TGN levels and the incidence of rejection episodes. In 82 renal allograft recipients, the effect of standard AZA dosage (3 mg/kg tapered to 1 mg/kg) was compared with higher dosages (3 mg/kg for several days) under 6-TGN monitoring. The authors measured TPMT in these patients and in a group not receiving AZA. The authors did not find an inverse correlation between RBC TPMT activity and 6-TGN concentrations, and baseline TPMT activity did not predict the incidence of rejection episodes The slight increase in RBC TPMT activity after transplant was associated with the use of furosemide rather than AZA; in the five patients receiving furosemide for less than 10 days, TPMT activity declined. The higher AZA dosage in the 6-TGN monitored group was not sufficient to increase RBC 6-TGN to target levels (100 to 200 pmol/8 x 10(8) RBC); median 6-TGN levels were similar in the two groups, as was the incidence of rejection episodes. Based on these findings, the authors suggest that higher dosages be studied in conjunction with 6-TGN monitoring, to explore the possibilities for therapeutic improvements.

Intracellular thiopurine nucleotides and azathioprine myelotoxicity in organ transplant patients.

Despite widespread use of azathioprine in organ transplant recipients, the mechanism of its myelotoxicity remains unclear. The aim of this study was to assess the importance of thiopurine metabolites on bone marrow toxicity. We investigated the relationship between intracellular concentrations of 6-thioguanine (6-TGN), 6-mercaptopurine (6-MPN) and 6-thioxanthine (6 TXN) nucleotides and the absolute count of white or red cells in forty-seven lung or heart/lung transplant patients after oral administration of azathioprine. No significant correlation between red cell concentrations of 6-TGN or total thiopurine metabolites and white or red cell counts was found, with no difference between the sexes. Likewise, high 6-TGN levels were not related to bone marrow depletion. These results suggest that red blood cell 6-TGN alone do not predict the haematopoietic toxicity of azathioprine.

MONITORING OF AZATHIOPRINE TREATMENT BY DETERMINATION OF 6-THIOGUANINE NUCLEOTIDE CONCENTRATIONS IN ERYTHROCYTES1

Thioguanine nucleotides (6-TGN) are intracellular metabolites that may contribute to the antiproliferative effects of AZA. The objectives of our study were to describe the variability of 6-TGN concentrations during AZA therapy and to investigate possible correlations between 6-TGN levels and subsequent myelosuppression. We measured 6-TGN concentrations in RBC of 65 renal transplant recipients from day 0 until 11-64 days after transplantation. High 6-TGN concentrations were observed in relation to elevated S-creatinine. In 15 patients, 6-TGN concentrations above 200 pmol/8 x 10(8) RBCs were measured (high 6-TGN group: mean maximal 6-TGN = 552 pmol/8 x 10(8) RBCs, SE = 91). In the remaining 50 patients, mean maximal 6-TGN was 82 pmol/8 x 10(8) RBCs, SE = 6.1 (low t-TGN group). In the former group, mean S-creatinine measured on the day of maximal 6-TGN was 466 mumol/L (SE = 62.3), while in the latter it was 190 (SE = 14.7). In the high 6-TGN group, we observed a lower mean nadir neutrophil count than in the low 6-TGN group (3.4 vs. 5.1 x 10(9) neutrophils/L). The nadir neutrophil count occurred, on the average, 12.7 days after maximal 6-TGN in the high 6-TGN group, with no such delay in the low 6-TGN group. This study demonstrates for the first time that 6-TGN in RBCs may rise to very high levels during impaired renal function. Furthermore, the results support the hypothesis that myelosuppressive side effects of AZA therapy correlate with 6-TGN concentrations. Renal transplant recipients may benefit from the monitoring of AZA through RBC 6-TGN measurements.

MONITORING OF AZATHIOPRINE TREATMENT BY DETERMINATION OF 6-THIOGUANINE NUCLEOTIDE CONCENTRATIONS IN ERYTHROCYTES1

Thioguanine nucleotides (6-TGN) are intracellular metabolites that may contribute to the antiproliferative effects of AZA. The objectives of our study were to describe the variability of 6-TGN concentrations during AZA therapy and to investigate possible correlations between 6-TGN levels and subsequent myelosuppression. We measured 6-TGN concentrations in RBC of 65 renal transplant recipients from day 0 until 11–64 days after transplantation. High 6-TGN concentrations were observed in relation to elevated S-creatinine. In 15 patients, 6-TGN concentrations above 200 pmol/8×108 RBCs were measured (high 6-TGN group: mean maximal 6-TGN = 552 pmol/8×108 RBCs, SE = 91). In the remaining 50 patients, mean maximal 6-TGN was 82 pmol/8×108 RBCs, SE = 6.1 (low 6-TGN group). In the former group, mean S-creatinine measured on the day of maximal 6-TGN was 466 μmol/L (SE = 62.3), while in the latter it was 190 (SE = 14.7). In the high 6-TGN group, we observed a lower mean nadir neutrophil count than in the low 6-TGN group (3.4 vs. 5.1 × 109 neutrophils/L). The nadir neutrophil count occurred, on the average, 12.7 days after maximal 6-TGN in the high 6-TGN group, with no such delay in the low 6-TGN group.

Kinetics of Mercaptopurine and Thioguanine Nucleotides in Renal Transplant Recipients During Azathioprine Treatment

The purpose of this study was to examine the pharmacokinetics of mercaptopurine (6-MP) and thioguanine nucleotides (6-TGN) during azathioprine treatment. Plasma profiles and urinary excretion of 6-MP and 6-TGN concentrations in red blood cells (RBCs) were measured repeatedly during the first 3 weeks following transplantation in 10 adults, who had received kidney grafts from living related donors. Mean maximal 6-MP plasma concentration (Cmax) was 340 nmol/L (SD = 290), mean time to Cmax (Tmax) was 2 h (SD = 1.8), and mean area under the plasma concentration-time curve (AUC) was 930 nmol/L/h (SD = 770). The mean fraction of azathioprine dose excreted as 6-MP in urine was 1.32% (SD = 1.11). Up to eightfold variability of Cmax and AUC was observed from day to day within each patient. The correlation between 6-MP AUC and amount excreted in the urine was weak (r = 0.37, 95% CI from 0.02 to 0.64). In this group of patients the observed 6-TGN levels in RBCs were low; maxima during the observation period ranged from undetectable to 250 pmol/8 x 10(8) RBCs. In individual patients, 6-TGN levels were relatively stable throughout the dosing interval ("within-dose-interval-CV" < 19%), even when sharp and high 6-MP peaks in plasma were observed.

Is 6-thioguanine more appropriate than 6-mercaptopurine for children with acute lymphoblastic leukaemia?

The cytotoxic activity of 6-mercaptopurine (6-MP) is affected by thiopurine methyltransferase (TPMT), a genetically regulated and variable intracellular enzyme. 6-Thioguanine (6-TG), a closely related thiopurine, is less affected by that enzyme and so it may be a more reliable drug-at least for patients with constitutionally high TPMT activity. We attempted to assess its suitability as an alternative by comparing the pharmacokinetics of both drugs in a small group of children with lymphoblastic leukaemia (ALL). Patients were included who were in their second or subsequent remission, who would otherwise have received 6-MP, and on whom pharmacokinetic data concerning 6-MP metabolism had been collected in a previous remission. Plasma 6-TG concentrations were assayed following an oral dose of 40 mg m-2, and the accumulation and fluctuation of intracellular (erythrocyte, RBC) 6-TG nucleotides (6-TGNs) were measured at regular intervals during daily oral therapy. Seven children were studied. Plasma 6-TG concentrations were low and cleared within 6 h of oral dosing. At 7 days, 6-TGN concentrations ranged from 959 to 2361 pmol 8 x 10(-8) RBCs, in all cases significantly higher (P = 0.002) than those produced by the same patients on 6-MP. After a total therapy time of 35 patient months, a modest rise of alanine aminotransferase was seen on one occasion, otherwise no toxicity apart from myelosuppression was encountered. In the context used, 6-TG appears well tolerated and produces higher concentrations of intracellular cytotoxic metabolites than 6-MP. For children constitutionally 'resistant' to the traditional drug, if not all, it may be a preferable alternative.

Genetic variation in response to 6-mercaptopurine for childhood acute lymphoblastic leukaemia

6-mercaptopurine (6-MP) can be inactivated by S-methylation, which is catalysed by thiopurine methyltransferase (TPMT). An alternative metabolic route leads to the formation of cytotoxic 6-thioguanine nucleotides (6-TGN). To investigate whether these two pathways compete with each other to affect the therapeutic response to 6-MP, 6-TGN concentrations and TPMT enzymatic activity were measured in erythrocytes (RBC) from 95 children on long-term 6-MP therapy for lymphoblastic leukaemia (ALL). RBC TPMT activities were also measured in 130 control children and 104 long-term survivors of ALL no longer on treatment. The 95 children on 6-MP showed wide interindividual differences in RBC 6-TGN concentrations at the full protocol dose of 75 mg/m2, and RBC 6-TGN concentrations correlated negatively with RBC TPMT activity. Children with 6-TGN concentrations below the group median had higher TPMT activities and a higher subsequent relapse rate. 50 of the 104 long-term survivors had been treated with "gentle" low-dose protocols, and this subgroup contained an excess of children with lower TPMT activities compared with normal controls. These results indicate that genetically determined TPMT activity may be a substantial regulator of the cytotoxic effect of 6-MP, an effect which in turn could be important in influencing the outcome of therapy for childhood ALL.

Variable mercaptopurine metabolism and treatment outcome in childhood lymphoblastic leukemia.

Intracellular thioguanine nucleotides (6-TGN) are the major cytotoxic metabolites of mercaptopurine (6-MP). Red blood cell (RBC) 6-TGN concentrations were measured in a group of 120 consecutive children with lymphoblastic leukemia (ALL) to assess interpatient variability and its clinical importance. Assays were performed after at least 2 months 6-MP maintanance chemotherapy and a minimum 7 days unattenuated protocol dose of 75 mg/m2. Observed 6-TGN concentrations ranged from 126 to 832 pmol/8 x 10(8) RBCs (median, 275). There was a correlation between 6-TGN and neutropenia 14 days postassay (rs = .51; P less than .0005), and an inverse correlation between 6-TGN and the length of time uninterrupted full protocol dose was tolerated without neutropenia (rs = -.3; P less than .01). After a median follow-up of 49 months, 19 children had relapsed, of whom 17 (89%) had 6-TGN concentrations below the group median (log-rank chi 2 = 11.9; P less than .001). Multivariate analysis using Cox's proportional hazards regression showed the 6-TGN effect on disease control to be independent of diagnostic WBC count, sex, age, immunological cell type, French-American-British (FAB) type, variation in other antineoplastic therapy, and duration of remission at the time of 6-TGN assay. Children with ALL taking the same dose of 6-MP show great variability in its measurable cytotoxic effect, and this variability is apparently important in predicting treatment outcome.

Pharmacogenetics of acute azathioprine toxicity: Relationship to thiopurine methyltransferase genetic polymorphism

Azathioprine therapy can cause acute myelosuppression. Toxicity is in part caused by the incorporation of azathioprine-derived 6-thioguanine nucleotides (6-TGN) into deoxyribonucleic acid (DNA). The enzyme thiopurine methyltransferase (TPMT) plays an important role in azathioprine catabolism. TPMT activity is controlled by a common genetic polymorphism, and one in 300 subjects has very low enzyme activity. Azathioprine was withdrawn in five study patients because of acute myelosuppression. The duration of azathioprine treatment was 21 to 70 days (median, 28), and the daily oral dose was 1.0 to 2.5 mg/kg. Sixteen control patients who had been taking oral azathioprine (1.1 to 2.0 mg/kg daily for more than 6 months) with no history of myelosuppression were studied. All subjects had normal liver and kidney function. When compared with the control group, the five patients with myelosuppression had very low TPMT activities and abnormally high 6-TGN concentrations. Inherited low TPMT activity appears to be a major risk factor for acute azathioprine-induced myelosuppression.

Metabolism of azathioprine to 6-thioguanine nucleotides in patients with pemphigus vulgaris.

Azathioprine metabolism, to red cell 6-thioguanine nucleotides (6TGN), was studied in four patients with pemphigus vulgaris. Throughout treatment blood samples were taken for red cell 6TGN assay and differential white cell counts. Metabolite steady-state occurred in three patients in 2, 2 and 4 months respectively. In the fourth patient red cell 6TGN concentrations increased slowly over 3 years. There was a significant negative correlation between 6TGN concentrations and the white blood cell count (rs = -0.92, P less than 0.0005) in this patient.

Thiopurine pharmacogenetics in leukemia: correlation of erythrocyte thiopurine methyltransferase activity and 6-thioguanine nucleotide concentrations.

Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine (6-MP) and azathioprine. Human erythrocyte (RBC) TPMT activity is controlled by a common genetic polymorphism. On a genetic basis approximately one in every 300 subjects lacks TPMT activity, and 11% of subjects have intermediate activities. 6-Thioguanine nucleotides (6-TGN) are major metabolites of 6-MP and azathioprine in humans. RBC 6-TGN concentrations are correlated directly with risk for the development of leukopenia in patients treated with thiopurine drugs. Our studies were performed to determine whether there was a relationship between genetically controlled levels of RBC TPMT activity and RBC concentrations of 6-TGN. We found a significant negative correlation between RBC TPMT activity and 6-TGN concentrations in blood samples from 40 children with acute lymphoblastic leukemia receiving long-term therapy with 6-MP (rs = -0.474; P less than 0.005). In addition, RBC TPMT activities were significantly higher in blood samples from these patients than in blood samples from adult control subjects (P less than 0.0001) or children with acute lymphoblastic leukemia who were in remission but were not receiving drug therapy (P less than 0.0001). Finally, three adult patients were studied who developed very high RBC 6-TGN concentrations and thiopurine-induced leukopenia. Two of the three patients had no detectable RBC TPMT activity--presumably on a genetic basis. These results indicate that low TPMT activity may be a risk factor for the occurrence of elevated 6-TGN concentrations and for the development of severe leukopenia in patients treated with thiopurine drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

Assay of 6-thioinosinic acid and 6-thioguanine nucleotides, active metabolites of 6-mercaptopurine, in human red blood cells

A highly sensitive reversed-phase high-performance liquid chromatographic assay, with ultraviolet detection, for 6-thioinosinic acid and the 6-thioguanine nucleotides (6TGNs) was developed. The 6TGNs are major red blood cell metabolites of the immunosuppressive agent azathioprine and the cytotoxic drugs 6-thioguanine and 6-mercaptopurine. The assay is based on the specific extraction, via phenyl mercury adduct formation, of the thiopurine released on acid hydrolysis of the thionucleotide metabolite. Red blood cell 6TGN concentrations in eighteen leukaemic children receiving chronic 6-mercaptopurine chemotherapy were measured and compared to a previously published spectrophotofluorometric assay. Linear regression analysis gave r = 0.991; P < 0.001; y = 40 + 0.94 x.

Childhood lymphoblastic leukaemia: sex difference in 6-mercaptopurine utilization.

Twelve boys and 10 girls on similar long term remission maintenance treatment for lymphoblastic leukaemia had 79 random assays of their red cell 6 thioguanine nucleotide ( 6TGN ) concentrations performed as an index of cytotoxic activity generated by oral 6-mercaptopurine ( 6MP ). Correlation between the dose of 6MP and 6TGN was statistically significant in the girls (r = 0.58, P less than 0.001) but not in the boys (r = 0.15). Additionally, as a group the boys tolerated more 6MP (P less than 0.05), despite similar prescribing criteria, but this did not result in a higher mean 6TGN concentration or increased myelotoxicity. It appears that girls develop 6MP cytotoxicity at lower doses and more predictably than boys. If so, this may be relevant to the as yet unexplained but marked sex difference in prognosis apparent in some studies.