Metabolites of mercaptopurine in red blood cells: a relationship between 6-thioguanine nucleotides and 6-methylmercaptopurine metabolite concentrations in children with lymphoblastic leukemia

Intracellular concentrations of 6-mercaptopurine metabolites, i.e. of 6-thioguanine nucleotides (6-TGN) and of 6-methylmercaptopurine metabolites (6-mMP) were analysed in red blood cells (RBC) of 19 children with acute lymphoblastic leukemia (ALL), the subjects of a maintenance chemotherapy of their first remission. Interpatient variations in concentrations of both metabolites were high; concentrations of 6-TGN varied from <60 to 833 pmol/8×10⁸ RBC (median value, 144) and those of 6-mMP metabolites from <150 to 19 000 pmol/8×10⁸ RBC (median value, 3250). In two patients, 6-TGN appeared at concentrations below the limits of assay sensitivity, and 6-mMP metabolites were not detected. In another child the concentrations of both metabolites were at the limit of the assay sensitivity. In three other children the concentrations of both metabolites were below the median value of the group. In the analysed group of children, significant correlations were found between the white cell count (WBC) and RBC 6-TGN (r_s=−0.72, P<0.005) as well as between the neutrophil count and RBC 6-TGN (r_s=−0.60, P<0.01). No significant correlation was found between the concentrations of 6-TGN and 6-mMP metabolites. The monitoring of concentrations of 6-TGN as well as of 6-mMP metabolites allows an early identification of patients who are at an increased risk of the disease relapse as indicated by the low levels of either 6-TGN itself or of its two metabolites.     

The role of xanthine oxidase in thiopurine metabolism: a case report

Azathioprine (AZA) is widely used in the treatment of autoimmune inflammatory diseases. AZA is normally rapidly and almost completely converted to 6-mercaptopurine (6-MP) in the liver, which is further metabolized into a variety of pharmacologic active thiopurine metabolites. 6-MP is catabolized by xanthine oxidase (XO) to the inactive metabolite 6-thiouric acid. The authors report the case of a woman with chronic autoimmune pancreatitis unable to form active thiopurine metabolites. The 55-year-old woman presented with weight loss, progressive elevation of liver transaminases, and serum amylase. She was treated with prednisolone 30 mg/day (1 mg/kg) and AZA was increased to 75 mg/day (2.5 mg/kg); this was later increased to 150 mg/day (5 mg/kg). Despite good patient compliance, the active metabolites of AZA, 6-thioguanine nucleotides (6-TGN), and 6-methylmercaptopurine ribonucleotides (6-MMPR) could not be detected in the erythrocytes (RBC). Subsequently, AZA was switched to high-dose 6-MP (2.5 mg/kg) and the XO inhibitor allopurinol was added. After 1 week, this combination led to a high 6-TGN level of 616 pmol/8 x 10(8) RBC and a 6-MMPR level of 1319 pmol/8 x 10(8) RBC. Three weeks after starting treatment, 6-TGN and 6-MMPR even reached toxic levels (1163 pmol/8 x 10(8) RBC and 10015 pmol/8 x 10(8) RBC, respectively) so that 6-MP treatment was discontinued. To elucidate this finding, 6-MP (1.7 mg/kg) was prescribed for 3 days without allopurinol. The woman was not able to form active thiopurine metabolites. According to the authors, this is the first report of a patient unable to form detectable active thiopurine metabolites on AZA and 6-MP therapy despite good patient compliance. High XO activity led to an inability to form detectable levels of active thiopurine metabolites 6-TGN and 6-MMPR. This finding emphasizes the important role of XO in the biotransformation of thiopurines.     

Thiopurine S-methyltransferase phenotype-genotype correlation in hemodialyzed patients

Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme, catalyzing S-methylation of thiopurine drugs. TPMT exhibits autosomal codominant polymorphism. Patients carrying a variant genotype have low TPMT activity, and produce elevated levels of 6-thioguanine nucleotides (6-TGN) in their red blood cells (RBC). 6-TGN accumulation may result in azathioprine (AZA)-induced bone marrow myelosuppression in the course of treatment with the drug in a standard dosage regimen in patients following renal transplantation. In the current study, TPMT activity (phenotype) and genotype were determined in dialyzed patients, qualified for renal transplantation. TPMT activity was measured in RBC after dialysis by HPLC method. Patients were genotyped for TPMT *2, *3A and *3C variant alleles using PCR-RFLP and allele-specific PCR methods. TPMT activity ranged between 12.2 and 45.5 nmol 6-mMP/g Hb/h (median value 30.6). A significant correlation between TPMTphenotype and genotype was noted: the heterozygous patients (11.5%) demonstrated significantly lower mean TPMT activity as compared to the wild homozygotes (17 +/- 3.6 vs. 32.4 +/- 4.8 nmol 6-mMP/g Hb/h, p < 0.0003). No overlap in TPMT activity values between the group of heterozygous (range 12.2-20.6) and wild-type homozygous patients (range 22.7-45.5) was noted. TPMT activity, established after hemodialysis and TPMT genotyping results seem to be convergent in dialyzed patients, so both methods can be used for the identification of patients with lower TPMT activity. Such tests could be helpful in AZA dose individualization, and thus in reducing the risk of myelosuppression during AZA therapy following renal transplantation.     

中国肾移植患者红细胞内嘌呤类药物活性代谢物6-硫鸟嘌呤核苷酸浓度监测及影响因素分析

目的：研究服用硫唑嘌呤（AZA）中国肾移植患者红细胞（RBC）内活性代谢物6-硫鸟嘌呤核苷酸（6-TGNs）分布特征及影响因素，为临床合理应用嘌呤类药物提供依据。方法：以89例中国肾移植患者为研究对象，关联分析年龄、性别、体质量、AZA剂量和TPMT活性对RBC内6-TGNs浓度的影响，并应用SPSS v20.0软件进行多元线性回归分析。结果：89例中国肾移植患者RBC内6-TGNs浓度呈非正态分布（P<0.000 1），6-TGNs浓度中位数为167.60（四分位间距，108.10~300.80） pmol/8×10⁸ RBC，个体间差异约24.3倍。关联分析显示患者年龄、性别、体质量、TPMT活性对6-TGNs浓度均无显著影响（P>0.05）；而AZA剂量与6-TGNs浓度间呈显著正相关性（r_s=0.307 1，P<0.01）。多元线性回归分析显示，RBC内6-TGNs浓度与AZA剂量间呈显著正相关（P<0.001），与TPMT活性呈显著负相关（P<0.05）。结论：AZA剂量和RBC内TPMT活性协同影响嘌呤类药物活性代谢物6-TGNs浓度，进而影响该类药物临床疗效和毒性反应。     

Pharmacokinetics of 6-thioguanine in patients with inflammatory bowel disease

6-Thioguanine (6-TG) seems to be an attractive alternative in both AZA- and 6-MP-intolerant and -resistant IBD populations. However, little is known of 6-TG pharmacokinetics, metabolite levels, and their correlation with drug efficacy and toxicity in IBD patients. This study reports the 6-TG pharmacokinetics in a population of IBD patients and the predictive value of metabolite concentrations. Red blood cell (RBC) 6-thioguanine nucleotide (6-TGN) concentrations were measured in 28 IBD patients at t = 1, 2, 4, and 8 weeks after starting 6-TG, 20 mg once daily. Outcome measures included mean 6-TGN concentrations (+/-95% confidence interval [CI95%]) and their associations with TPMT genotype, 6-TG dose, and hematological, hepatic, pancreatic, and efficacy parameters during the 8 week period. Steady-state 6-TGN concentrations were reached after 4 weeks, indicating a half-life of approximately 5 days, and measured 856 (CI95% 715-997) pmol/8 x 10 RBCs. Large interpatient variability occurred at all time-points. No correlation was found between steady-state 6-TGN concentrations and drug dose per kilogram body weight. No significant differences in 6-TGN concentrations were found between patients with adverse events and patients without any event. Also, mean 6-TGN concentrations did not differ in patients with active disease versus patients in remission. In IBD patients on 6-TG treatment, large interindividual differences in metabolite concentrations occur. In our population, we could not demonstrate a clear relationship between 6-TGN concentrations on one hand and toxicity and efficacy on the other, as exist in AZA- and 6-MP-treated patients.     

Red blood cell hypoxanthine phosphoribosyltransferase activity measured using 6-mercaptopurine as a substrate: a population study in children with acute lymphoblastic leukaemia

1. 6-Mercaptopurine (6-MP) is used in the continuing chemotherapy of childhood acute lymphoblastic leukaemia. The formation of red blood cell (RBC) 6-thioguanine nucleotide (6-TGN) active metabolites, not the dose of 6-MP, is related to cytotoxicity and prognosis. But there is an apparent sex difference in 6-MP metabolism. Boys require more 6-MP than girls to produce the same range of 6-TGN concentrations. Given the same dose, they experience fewer dose reductions because of cytotoxicity, and have a higher relapse rate. 2. The enzyme hypoxanthine phosphoribosyltransferase (HPRT) catalyses the initial activation step in the metabolism of 6-MP to 6-TGNs, a step that requires endogenous phosphoribosyl pyrophosphate (PRPP) as a cosubstrate. Both HPRT and the enzyme responsible for the formation of PRPP are X-linked. 3. RBC HPRT activity was measured in two populations, 86 control children and 63 children with acute lymphoblastic leukaemia. 6-MP was used as the substrate and the formation of the nucleotide product, 6-thioinosinic acid (TIA) was measured. RBC 6-TGN concentrations were measured in the leukaemic children at a standard dose of 6-MP. 4. There was a 1.3 to 1.7 fold range in HPRT activity when measured under optimal conditions. The leukaemic children had significantly higher HPRT activities than the controls (median difference 4.2 micromol TIA ml(-1) RBCs h(-1), 95% C.I. 3.7 to 4.7, P < 0.0001). In the leukaemic children HPRT activity (range 20.4 to 26.6 micromol TIA ml(-1) RBCs h(-1), median 23.6) was not related to the production of 6-TGNs (range 60 to 1,024 pmol 8 x 10(-8) RBCs, median 323). RBC HPRT was present at a high activity even in those children with low 6-TGN concentrations. 5. When HPRT is measured under optimal conditions it does not appear to be the metabolic step responsible for the observed sex difference in 6-MP metabolism. This may be because RBC HPRT activity is not representative of other tissues but it could equally be because other sex-linked factors are influencing substrate availability.     

Pharmacokinetics of 6-mercaptopurine in patients with inflammatory bowel disease: implications for therapy

Proper prospective pharmacokinetic studies of 6-mercaptopurine (6-MP) in inflammatory bowel disease (IBD) patients are lacking. As a result, conflicting recommendations have been made for metabolite monitoring in routine practice. The authors have evaluated 6-MP pharmacokinetics in IBD patients, including the genetic background for thiopurine methyltransferase (TPMT). Red blood cell (RBC) 6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine ribonucleotide (6-MMPR) concentrations were measured in 30 IBD patients at 1, 2, 4, and 8 weeks after starting 6-MP, 50 mg once daily. Outcome measures included mean 6-TGN and 6-MMPR concentrations (+/- 95% confidence interval, CI95%) and their associations with TPMT genotype, 6-MP dose, and hematologic, hepatic, pancreatic, and efficacy parameters during the 8-week period. Steady-state concentrations were reached after 4 weeks, indicating a half-life of approximately 5 days for both 6-TGN and 6-MMPR; the concentrations were 368 (CI95% 284-452) and 2837 (CI95% 2101-3573) pmol/8 x 10 RBCs, respectively. Large interpatient variability occurred at all time points. TPMT genotype correlated with 6-TGN concentrations (0.576, P < 0.01), and patients with mutant alleles had a relative risk (RR) of 12.0 (CI95% 1.7-92.3) of developing leukopenia. A 6-MMPR/6-TGN ratio less than 11 was associated with therapeutic efficacy. Based on this pharmacokinetic analysis, therapeutic drug monitoring is essential for rational 6-MP dosing.     

Azathioprine metabolism: pharmacokinetics of 6-mercaptopurine, 6-thiouric acid and 6-thioguanine nucleotides in renal transplant patients

Despite extensive clinical experience with azathioprine (AZA), the disposition of various AZA metabolites remains obscure. We therefore evaluated the pharmacokinetics of three AZA metabolites: 6-mercaptopurine (6-MP), the immediate metabolite; 6-thiouric acid (6-TU), the final end product; and 6-thioguanine nucleotides (TGN), the active moiety; in eight renal transplant patients after oral administration of AZA. The low peak plasma 6-MP level of 73.7 +/- 23.7 ng/mL (mean +/- SD) and the short half-life (t1/2) of 1.9 +/- 0.6 hours suggest rapid conversion of 6-MP to other metabolites. A peak plasma 6-TU concentration of 1210 +/- 785 ng/mL was observed at 3.5 +/- 0.6 hours after the AZA dose. The strong correlation between 6-TU t1/2 and serum creatinine (r = 0.98, P = .0008) supported our previous work showing that 6-TU is primarily excreted by the kidneys. The total TGN levels in red blood cells (RBCs) in each patient remained largely unchanged over 24 hours with the intraindividual coefficient of variation ranging from 4.4% to 29.8%. In comparison, the mean TGN level varied considerably between patients, and ranged from undetectable to 413 pmol per 8 X 10(8) RBCs. However, there was no apparent correlation between white cell counts on day 0 (P greater than .5), day 7 (P greater than .5), or day 14 (P greater than .5) and RBC TGN level. The persistence of TGN in body tissues thus provides a pharmacokinetic rationale for the conventional once or twice daily AZA regimen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extended thiopurine metabolite assessment during 6-thioguanine therapy for immunomodulation in Crohn's disease

The proposed metabolic advantage of 6-thioguanine (6-TG) is the direct conversion into the pharmacologically active 6-thioguaninenucleotides (6-TGN). The authors assessed metabolic characteristics of 6-TG treatment in patients with Crohn's disease (N = 7) on therapy with 20 mg 6-TG. 6-thioguanine-monophosphate (6-TGMP), 6-thioguanine-diphosphate (6-TGDP), and 6-thioguanine-triphosphate (6-TGTP) were measured by high-performance liquid chromatography analysis in erythrocytes. Thiopurine S-methyltransferase activity and total 6-TGN levels were determined by standard methods. High interindividual variance in metabolite measurements was observed. Main metabolites were 6-TGTP (median = 531 pmol/8 x 10(8) red blood cells) and 6-TGDP (median = 199 pmol/8 x 10(8) red blood cells). Traces of 6-TGMP (median = 39 pmol/8 x 10(8) red blood cells) and 6-TG (2 patients) could be detected. 6-TGN levels correlated with 6-TGTP levels (r = 0.929, P = .003) and with the sum of separate nucleotides (r = 0.929, P = .003). No correlations were established between TPMT activity (median = 13 pmol/h/10(7)) and 6-TG metabolites. The 1-step metabolism of 6-TG still leads to high interindividual variance in metabolite concentrations. Total 6-TGN level monitoring may suffice for clinical practice.     

Assessment of thiopurine methyltransferase and metabolite formation during thiopurine therapy: results from a large Swedish patient population

This study examined thiopurine methyltransferase (TPMT) and the relationship to thioguanine nucleotides (TGN) and methylthioinosine monophosphate (meTIMP) in a large Swedish patient population. The current hypothesis is that the cytotoxic effects of thiopurine drugs are mediated by the incorporation of TGN into DNA. The authors assayed the TPMT activity in red blood cells from 1151 subjects and the concentrations of TGN (n = 602) and meTIMP (n = 593) from patients treated with thiopurine drugs. The TPMT frequency distribution in both adults and children showed some differences from what had been found in unselected general populations. Children had lower median TPMT activity than adults (12.0 versus 12.9 U/mL RBC; P < 0.001). Relative differences in both TGN formation [medians: normal TPMT, 1.3; intermediate TPMT, 3.3; low TPMT, 47.9 pmol/8 x 10(8) RBC per mg azathioprine (AZA); P < 0.001] and meTIMP formation (medians: normal TPMT, 13; intermediate TPMT, 7.3; low TPMT, 0 pmol/8 x 10(8) RBC per mg AZA; P = 0.001) per 1 mg administered drug were noted among the 3 TPMT activity groups. Women formed higher concentrations of both TGN (1.5 versus 1.3 pmol/8 x 10(8) RBC per mg AZA; P = 0.01) and meTIMP (14.4 versus 10.7 pmol/8 x 10(8) RBC per mg AZA; P = 0.01) than men did. There was a significant correlation between the AZA dose and the meTIMP concentrations (r = 0.45; P < 0.001). Furthermore, dose alterations made in subjects with normal TPMT (n = 84) and intermediate TPMT (n = 22) activity resulted in more pronounced increases in TGN concentrations (170 versus 30 pmol/8 x 10(8) RBC; P < 0.001) in intermediate TPMT activity, whereas in normal TPMT activity changes in meTIMP concentrations were more pronounced (1.3 versus 0 nmol/8 x 10(8) RBC; P < 0.001). In normal TPMT activity both metabolites increased in a dose-dependent fashion, whereas in intermediate TPMT activity only TGN concentrations increased. The results of this study demonstrate the dynamic nature of thiopurine metabolism and its importance for thiopurine dosing.     

Low allopurinol doses are sufficient to optimize azathioprine therapy in inflammatory bowel disease patients with inadequate thiopurine metabolite concentrations

Purpose

Recent studies in patients with inflammatory bowel diseases (IBD) on thiopurine therapy suggest that too low 6-thioguanine nucleotide concentrations (6-TGN) and too high methylmercaptopurine nucleotide concentrations (MMPN) can be reversed by a combination therapy of allopurinol and low-dose thiopurines. To date, however, optimal dosing has not been established. The aim of this study was to evaluate the minimal allopurinol doses necessary to achieve adequate 6-TGN concentrations in combination with low-dose azathioprine.

Methods

A stepwise dose-escalation of allopurinol was performed in 11 azathioprine-pretreated IBD patients with inadequately low 6-TGN concentrations (<235 pmol/8?×?10⁸ erythrocytes) and/or elevated MMPN concentrations (>5,000 pmol/8?×?10⁸ erythrocytes) and/or elevated liver enzymes (alanine aminotransferase and/or aspartate aminotransferase levels one- to threefold the upper limit of normal). Six patients were recruited into an open study, and five were treated in the context of an individualized therapeutic approach. Adverse effects, azathioprine metabolites, liver enzymes and whole blood counts were monitored two to three times per month.

Results

Adequate 6-TGN concentrations were achieved with a combination of 25 mg allopurinol and 50 mg azathioprine in one patient and with 50 mg allopurinol and 50 mg azathioprine in nine patients. Median 6-TGN concentrations (range) were 336 (290–488) pmol/8?×?10⁸ erythrocytes after an 8-week-long intake of the final dose combination. One patient dropped out due to nausea after the first intake. MMPN concentrations and liver enzymes normalized immediately in all affected patients. All patients finishing the dose-escalation regimen tolerated the treatment without toxicity.

Conclusions

Combination therapy with only 50 mg allopurinol and 50 mg azathioprine daily is sufficient, efficacious and safe in most IBD patients with inadequate thiopurine metabolite concentrations to optimize azathioprine-based IBD therapy.     

Thiopurine metabolite monitoring in paediatric inflammatory bowel disease

BACKGROUND: Measurement of thiopurine metabolite levels may be useful as a clinical tool to optimize thiopurine treatment of paediatric inflammatory bowel disease (IBD). AIM: The authors evaluated correlations between 6-thioguanine nucleotide (6-TGN) and therapeutic response, metabolite levels and drug toxicity. METHODS: Fifty-six paediatric IBD patients treated with thiopurines had 326 metabolite level measurements and were retrospectively reviewed. Clinical status and laboratory parameters were compared with metabolite levels. RESULTS: There was significant correlation between 6-TGN levels and therapeutic response, with higher median 6-TGN levels among patients with therapeutic response than those with non-therapeutic response (194 vs. 146 pmol/8 x 10(8) RBC; P = 0.0004). Patients with 6-TGN levels >235 pmol/8 x 10(8) RBC were more likely to achieve therapeutic response than those below the cut-off (odds ratio, 2.5; 95% CI, 1.5-4.1). Patients who developed leukopenia tended to have higher median 6-TGN levels than those without leukopenia (261 vs. 160 pmol/8 x 10(8) RBC) but the difference was not statistically significant. There was no correlation between 6-methylmercaptopurine levels and hepatotoxicity. Two patients developed acute pancreatitis. Metabolite level measurements were helpful in identifying non-compliance in nine patients. CONCLUSIONS: Monitoring of thiopurine metabolite levels is useful to guide and optimize dosing, as an adjunct to clinical judgement, blood count and liver biochemistry measurements to minimize the risk of drug toxicity and to confirm non-compliance.     

Allopurinol safely and effectively optimizes tioguanine metabolites in inflammatory bowel disease patients not responding to azathioprine and mercaptopurine

BACKGROUND: Many non-responders to azathioprine or mercaptopurine (6-mercaptopurine) have high normal thiopurine methyltransferase activity and preferentially metabolize mercaptopurine to produce 6-methylmercaptopurine instead of the active 6-tioguanine (6-tioguanine) metabolites. AIM: To describe the use of allopurinol in mercaptopurine/azathioprine non-responders to deliberately shunt metabolism of mercaptopurine towards 6-tioguanine. METHODS: Fifteen thiopurine non-responders whose metabolites demonstrated preferential metabolism towards 6-methylmercaptopurine are described. Subjects were commenced on allopurinol 100 mg po daily and mercaptopurine/azathioprine was reduced to 25-50% of the original dose. Patients were followed clinically and with serial 6-tioguanine and 6-methylmercaptopurine metabolite measurements. RESULTS: After initiating allopurinol, 6-tioguanine levels increased from a mean of 185.73 +/- 17.7 to 385.4 +/- 41.5 pmol/8 x 10(8) red blood cells (P < 0.001), while 6-methylmercaptopurine decreased from a mean of 10 380 +/- 1245 to 1732 +/- 502 pmol/8 x 10(8) RBCs (P < 0.001). Allopurinol led to a decrease in white blood cell from a mean of 8.28 +/- 0.95 to 6.1 +/- 0.82 x 10(8)/L (P = 0.01). CONCLUSIONS: The addition of allopurinol to thiopurine non-responders with preferential shunting to 6-methylmercaptopurine metabolites appears to be an effective means to shift metabolism towards 6-tioguanine.     

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.     

Determinants of mercaptopurine toxicity in paediatric acute lymphoblastic leukemia maintenance therapy

AIMS

6-mercaptopurine (6-MP) is used in the treatment of childhood acute lymphoblastic leukaemia (ALL). Its red blood cell (RBC) metabolite concentrations (6-thioguanine [6-TGN] and 6-methylmercaptopurine nucleotides [6-MMPN]) are related to drug response. We investigated the impact of non-genetic covariates and pharmacogenetic polymorphisms affecting thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) on 6-MP metabolism and response.

METHODS

Sixty-six children with ALL treated according to EORTC 58951 protocol were included in this study. Six patients had a heterozygous genotype for the most common TPMT polymorphisms, nine for ITPA 94 C > A and 17 for ITPA IVS2+21 A > C. 6-MP metabolites concentrations were analyzed by mixed model analysis.

RESULTS

During maintenance, steady-state RBC 6-TGN concentrations were lower in patients aged 6 years or younger (493 pmol/8 × 10⁸RBC) than in older children (600 pmol/8 × 10⁸RBC). 6-MMPN concentrations were low in patients with TPMT variant/wild-type ITPA (1862 pmol/8 × 10⁸RBC), intermediate in wild-type patients and high (16468 pmol/8 × 10⁸RBC) in patients wild-type TPMT/variant ITPA. A 6-MMPN threshold of 5000 pmol/8 × 10⁸RBC was associated with an increased risk of hepatotoxicity.

CONCLUSION

In this study, age and both TPMT and ITPA genotypes influenced 6-MP metabolism. High 6-MMPN was associated with hepatotoxicity. These pharmacological tools should be used to monitor ALL treatment in children.     

Variable correlation between 6-mercaptopurine metabolites in erythrocytes and hematologic toxicity: implications for drug monitoring in children with acute lymphoblastic leukemia

Nineteen pediatric patients affected by acute lymphoblastic leukemia (ALL) were examined weekly with respect to 6-mercaptopurine nucleotide (6-MPN) and 6-thioguanine nucleotide (6-TGN) levels in erythrocytes during the course of maintenance treatment with 6-MP 50 mg/m2 per d and results were related to various parameters of bone marrow function to assess, in the same individual, the level of reliability of 6-MP metabolites in predicting a later change in peripheral blood cell counts. Median values for 6-MPN and 6-TGN were 57 and 200 pmol/8 x 10(8) erythrocytes, respectively, as measured by reversed-phase high-performance liquid chromatography (HPLC). 6-TGN levels in erythrocytes were inversely related with white blood cell count (r = -0.463, p < 0.0001, n = 361), absolute neutrophil count (r = -0.386, p < 0.0001, n = 347), erythrocyte (r = -0.354, p < 0.0001, n = 287), and platelet counts (r = -0.24, p < 0.0001, n = 319) in the majority of patients (n = 10-12), while no correlation was found for 6-MPN. In the remaining children, no evidence of correlation was demonstrated between 6-TGN levels and myelotoxicity. The results confirm the role of 6-TGN as the reference cytotoxic metabolite for evaluating the exposure to 6-MP and identifying treatment compliance in ALL children but indicate the limits of a follow-up based solely on metabolite levels and suggest that a more correct approach remains the double monitoring of 6-TGN and blood cell count with differential.     

Influence of 5-aminosalicylic acid on 6-thioguanosine phosphate metabolite levels: a prospective study in patients under steady thiopurine therapy

Background and purpose:

5-aminosalicylate (5-ASA) raises levels of 6-thioguanine nucleotides (6-TGN), the active metabolites of thiopurines such as azathioprine (AZA). Changes in levels of each individual TGN – 6-thioguanosine mono-, di- and triphosphate (6-TGMP, 6-TGDP, 6-TGTP) – and of 6-methylmercaptopurine ribonucleotides (6-MMPR) after 5-ASA are not known.

Experimental approach:

Effects of increasing 5-ASA doses on AZA metabolites were investigated prospectively in 22 patients with inflammatory bowel disease in 4-week study periods. Patients started with 2 g 5-ASA daily, and then were increased to 4 g daily and followed by a washout period. Thiopurine doses remained unchanged throughout the entire study. Levels of 6-TGMP, 6-TGDP, 6-TGTP and 6-MMPR as well as of 5-ASA and N-acetyl-5-aminosalicylic acid (N-Ac-5-ASA) were determined each study period.

Key results:

Median baseline levels in 17 patients of 6-TGDP, 6-TGTP and 6-MMPR were 52, 319 and 1676 pmol per 8 × 10⁸ red blood cells respectively. After co-administration of 2 g 5-ASA daily, median 6-TGDP and 6-TGTP levels increased but median 6-MMPR levels were unchanged. Increasing 5-ASA to 4 g daily did not affect median 6-TGDP and 6-TGTP levels, but median 6-MMPR levels decreased. After discontinuation of 5-ASA, both 6-TGDP and 6-TGTP levels decreased and median 6-MMPR levels increased. The 6-TGTP/(6-TGDP+6-TGTP)-ratio did not change during the study, but 6-MMPR/6-TGN ratios decreased.

Conclusions and implications:

Individual 6-TGN metabolites increased after addition of 5-ASA, but 6-MMPR-levels and the 6-MMPR/6-TGN ratios decreased. Further studies are needed to decide whether this pharmacokinetic interaction would result in improvement of efficacy and/or increased risk of toxicity of AZA.     

Intestinal microbiota-mediated biotransformations alter the pharmacokinetics of the major metabolites of azathioprine in rats after oral administration

Adverse reactions to azathioprine (AZA) vary greatly among individuals, which is associated with the variable levels of its major metabolites 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP). The intestinal microbiota has been proven to contain AZA-metabolizing enzymes, although the explicit role of the intestinal microbiota in AZA metabolism in vivo remains poorly comprehended. In this study, the pharmacokinetic behaviours of 6-TGN and 6-MMP were assessed in the pseudo germ-free (PGF) group and control group following oral administration of AZA. The AUC_0-t and C_max of 6-TGN in the PGF group were significantly decreased by 34.0% and 35.0% (P < 0.05) compared with those in the control group. Additionally, the AUC_0-t and C_max of 6-MMP were reduced by 27.9% and 34.2% in the PGF group, although the differences were not significant. The TPMT and NUDT15 genotypes of rats in the two groups were genetically identical. The expression levels of key AZA-metabolizing enzymes in liver were not different between two groups. Furthermore, the major metabolites of AZA in the incubation system with intestinal microbial enzymes were identified. In summary, shifts in the composition of the intestinal microbiota may regulate the exposure of 6-TGN in vivo by altering the gut microbial metabolism of AZA.     

6-tioguanine monitoring in steroid-dependent patients with inflammatory bowel diseases receiving azathioprine

BACKGROUND: 6-Thioguanine (6-tioguanine) nucleotides are the active metabolites of azathioprine. AIM: The aim of the study was to evaluate the rate of clinical remission without steroids in steroid-dependent Crohn's disease and ulcerative colitis patients receiving azathioprine, the medium- and long-term efficacy and the predictive factors of clinical response when monitoring 6-tioguanine. METHODS: Steroid-dependent Crohn's disease and ulcerative colitis patients receiving either azathioprine or not (treated later with a daily dose of 2.5 mg/kg) were prospectively included. 6-tioguanine was monitored at 1 and 2 months and every 3 months thereafter for 1 year. The azathioprine dose was adapted to reach a 6-tioguanine level of >250 pmol/8 x 10(8) red blood cells. Thiopurine methyltransferase genotype/phenotype was evaluated in some patients. RESULTS: A total of 106 patients were prospectively included (70 Crohn's disease, 36 ulcerative colitis). The clinical remission rate without steroids in patients receiving azathioprine, in intention-to-treat analysis, was 72% and 59% at 6 and 12 months, respectively. The remission rate was significantly higher in patients with 6-tioguanine >250 pmol/8 x 10(8) RBC (86% and 69% at 6 and 12 months, respectively; P < 0.01). No significant difference was observed between Crohn's disease and ulcerative colitis patients whether treated by azathioprine or not on inclusion. In the univariate analysis, the absence of Crohn's disease stenosis, a 6-tioguanine level >250 pmol/8 x 10(8) RBC, and an increase of erythrocyte mean corpuscular volume were the factors predictive of a favourable clinical response. In the multivariate analysis, only a 6-tioguanine level of >250 pmol/8 x 10(8) red blood cells was a predictive factor of favourable clinical remission. CONCLUSIONS: Clinical remission without steroids is significantly more likely when monitoring 6-tioguanine so as to reach a level of >250 pmol/8 x 10(8) red blood cells in steroid-dependent Crohn's disease and ulcerative colitis patients receiving azathioprine (86% and 69% at 6 and 12 months, respectively).     

Pharmacokinetics of 6-thiouric acid and 6-mercaptopurine in renal allograft recipients after oral administration of azathioprine

Summary The immunosuppressive activity of azathioprine (AZA) is unpredictable and depends on the formation of intracellular thiopurine ribonucleotides. However, the quantification of these active thiopurines presents difficult analytical problems. It has recently been postulated that plasma concentrations of 6-thiouric acid (6-TU) and 6-mercaptopurine (6-MP), metabolites of AZA, may provide more readily measurable indices of the pharmacologic activity of AZA. In order to evaluate the utility of 6-TU and 6-MP plasma concentrations in monitoring AZA therapy, we studied their pharmacokinetics in 6 renal transplant patients, and their in vitro immunosuppressive potency in a mixed lymphocyte proliferation assay.A peak plasma 6-TU concentration of 710.7 ng/ml was observed at 3.8 h after oral dosing. Good correlation was observed between the elimination t_1/2 of 6-TU and serum creatinine, and between AUC over 24 h and serum creatinine. However, we did not observe a second peak in plasma 6-TU concentration that could be attributed to the degradation of active AZA metabolites. 6-MP plasma concentrations in the patients were low (mean peak concentration 36.0 ng/ml) and rapidly disappeared within 8 h. In vitro immunosuppressive activity could not be demonstrated for 6-TU over a concentration range of 1.25 ng/ml to 0.25 mg/ml.We conclude that 6-TU is pharmacologically inert and is primarily eliminated by the kidneys. Our findings currently do not support the use of plasma concentrations of 6-TU or 6-MP to monitor AZA therapy. In order to optimize AZA therapy, analytical techniques that are technically feasible and that can directly quantify the active intracellular thiopurines are being explored.