Metabolite 6-mercaptopurine Research Articles

Azathioprine and 6-Mercaptopurine Alter the Nucleotide Balance in Endothelial Cells

Graft vascular disease after solid organ transplantation is a main complication that limits long-term survival of the graft. An injury of the endothelium and subsequent vascular response is considered to be responsible for smooth muscle cell hyperplasia with resulting luminal narrowing. What is less certain are the precise steps leading to endothelial injury and subsequent vessel disease. Since the immunosuppressive drug azathioprine is in clinical use due to its antiproliferative effect on lymphocytes, we were interested in how far it exerts effects on the vascular endothelium. Azathioprine and its metabolite 6-mercaptopurine, a potent inhibitor of purine salvage pathway enzymes, dose dependently led to decreased endothelial cell proliferation as well as to decreases in intracellular purine nucleotides adenosine-triphosphate and guanosine-triphosphate. By increasing the formation of the pyrimidine nucleotide uridine-triphosphate within 24 hours, azathioprine and its metabolite altered the endothelial nucleotide balance. Since not only the formation of toxic thio- and methylthiopurines (thio-guanosine-monophosphate, methyl-thio-inosine-monophosphate) was measured, the activity of the enzyme thiopurinemethyltransferase was induced (3.21±2.04 U per 10 9 cells, mean±SD). These findings indicate that the vascular endothelium plays an active role in the metabolization of the established immunosuppressant azathioprine that then exerts specific toxic effects on endothelial cells.

Azathioprine: an Update on Clinical Efficacy and Safety in Inflammatory Bowel Disease

Background: The approval of azathioprine (AZA) for the long-term therapy of patients with Crohn's disease in The Netherlands. Methods: Review and update of the literature on clinical efficacy and safety of AZA in inflammatory bowel disease. Results: AZA and its metabolite 6-mercaptopurine (6-MP) are effective in the treatment of active Crohn's disease. However, the onset of the clinical response is delayed, requiring combination with other active medication in the early phase of treatment. Maintenance therapy with AZA/6-MP is also effective in the prevention of relapses in patients with Crohn's disease in remission. Indications for AZA/6-MP therapy further include refractory, fistulizing and steroid-dependent Crohn's disease. It is not known whether or when AZA/6-MP can be withdrawn in patients in long-term remission, but most clinicians discontinue therapy after 3-5 years. Although fewer data are available, AZA/6-PM appears to be effective also in the therapy of patients with ulcerative colitis. Side effects of AZA/6-MP occur in about 15% of patients and include skin rash, pancreatitis and hepatitis, dose-related neutropenia and thrombocytopenia, and risk of increased occurrence and severity of infections. Recent data suggest that the risk of malignancy, other than colorectal cancer, is not increased. Safety in pregnancy has not been studied extensively, but no increased prevalence of birth defects has been reported. Conclusions: AZA/6-MP therapy is efficacious in patients with active Crohn's disease, but the drug is especially valuable in the long-term treatment of patients with Crohn's disease and ulcerative colitis. Drug-related side effects are frequent and require discontinuation or dose-reduction of the AZA/6-MP therapy. Due to an increased risk of infections secondary to myelosuppression, careful follow-up is mandatory. Insufficient data are available on safety in pregnancy and on the risk of malignancies, but the limited data available in patients with inflammatory bowel disease appear to be reassuring.

6-Mercaptopurine metabolism in Crohn’s disease

Editor,—I have read with interest the article by Cuffari et al ( Gut 1996; 39 :401–6) and do not feel that the conclusions of their study are supported by the results. The major aim of their study was to determine whether erythrocyte 6 mercaptopurine metabolite (6-TG) concentrations correlated with disease activity in patients with Crohn’s disease. The authors state that their results show an inverse correlation ( r = −0.457, p < 0.05) between 6-mercaptopurine nucleotide concentrations and the Harvey-Bradshaw index of disease activity. They conclude that these results support the immunosuppressive role of 6-TG metabolites and …

Increased Concentrations of Methylated 6-Mercaptopurine Metabolites and 6-Thioguanine Nucleotides in Human Leukemic Cells In Vitro by Methotrexate

The effect of methotrexate (MTX) on 6-mercaptopurine (6-MP) metabolism was studied in four human leukemic cell lines in vitro. CCRF-CEM, WI-L2, TBJ, and HL-60 all expressed thiopurine methyltransferase (TPMT) activity. The cells were grown in horse serum-supplemented RPMI 1640 medium to which was added 4 μM of 6-MP or 4 μM of 6-MP and 20 nM of MTX. The presence of MTX resulted in a 2.1-, 1.7-, 2.4- and 8-fold increase in the concentrations of methylmercaptopurine ribonucleotides (MMPRP) in CEM, WI-L2, TBJ, and HL-60 cells, respectively ( P < 0.0008). The concentrations of 6-thioguanine nucleotides (6 TGN) increased 1.9-, 1.4-, 2.4- and 1.9-fold in the same cell lines ( P < 0.02). The four cell lines differed with respect to the effect of MTX on the consumption of 6-MP from the medium; CEM consumed more 6-MP and WI-L2 less 6-MP from media containing MTX than from media containing 6-MP only ( P = 0.005 and 0.02, respectively). MTX did not affect the consumption of 6-MP by TBJ cells ( P = 0.17). Media in which HL-60 cells had been grown did not contain detectable amounts of 6-MP at the end of the experiment. The simultaneous increase in methylated 6-MP metabolites and 6-TGN represents a possible explanation for the synergism of MTX and 6-MP; however, the clinical importance of increased MMPRP remains to be elucidated.

Azathioprine in inflammatory bowel disease, a safe alternative?

Azathioprine and its metabolite 6-mercaptopurine are effective in the treatment of inflammatory bowel disease. They are mostly used for reduction of the use of steroids, maintenance therapy after remission induction by cyclosporin and treatment of fistulae in Crohn's disease. Adverse effects occur in about 15% of patients. The main side effects are pancreatitis, allergic reactions, fever and bone marrow suppression. Symptoms, management and prevention are discussed. A blood monitoring schedule is suggested. Azathioprine and 6-mercaptopurine seem to be safe in pregnancy. There may be a slight increased risk for developing a non-Hodgkin's lymphoma.

Immunosuppressive treatment for refractory ulcerative colitis--where do we stand and where are we going?

Effective medical treatment of ulcerative colitis is available. However, 20-40% of patients remain refractory and become steroid dependent, or have chronic activity. Azathioprine and its metabolite 6-mercaptopurine have been found to be effective in this setting, although duration of treatment and doses are not entirely clear. Neither is widely used in Europe for this indication. Methotrexate has no definitive place in the treatment of refractory colitis. Intravenous cyclosporin A induces remission in a considerable number of patients; follow-up treatment is, however, not defined. This approach may be useful to allow elective surgery. A number of other treatments have been proposed including chloroquine, interferons and anti-cytokines. None of these can currently be recommended for clinical practice. Anti-inflammatory cytokines such as interleukin-10 may be good candidates.

8 Safety of corticosteroids and immunosuppressive agents in ulcerative colitis

For many years, corticosteroids have been the mainstay for treating acute ulcerative colitis. In patients with refractory disease, immunosuppressive therapy may be indicated, including azathioprine or its metabolite 6-mercaptopurine, cyclosporin and possibly methotrexate. Their benefits in ulcerative colitis must be weighed up against their possible adverse effects, the availability of surgical cure for this condition, and the long-term risk of carcinoma complicating colitis that applies in patients with chronic extensive disease. Information about the safety of corticosteroids and immunosuppressive agents has accumulated as a result of their extensive use in inflammatory bowel disease, organ transplantation and various other disorders.

Intracellular metabolites of mercaptopurine in children with lymphoblastic leukaemia: a possible indicator of non-compliance?

As part of a programme assessing the pharmacokinetics of oral thiopurines given for lymphoblastic leukaemia, we assayed intracellular metabolites of mercaptopurine in children from all over the United Kingdom who were given a standard dose of the drug. The metabolites we measured, thioguanine nucleotides and methylmercaptopurines, are products of two competing metabolic pathways and would be expected to show an inverse correlation. A total of 327 children from 17 centres in the UK were studied. All were on the same therapeutic schedule of mercaptopurine. All had been on an unattenuated full protocol-directed dose (at least 75 mg m-2) for a minimum of 7 days before assay. There was a very wide variation in the concentration of the two metabolites measured; the thioguanine nucleotides ranged from 0 to 1255 pmol per 8 x 10(8) red cells (median 289, lower quartile 210, upper quartile 377) and the methylmercaptopurine metabolites ranged from 0 to 46.3 nmol per 8 x 10(8) red cells (median 5.18, lower quartile 2.31, upper quartile 11.59). The anticipated negative correlation was not apparent, but the ratio between the two was not randomly distributed. No child had both metabolite concentrations in the upper quartiles, but in 32 (10%) children the concentration of both metabolites was in the lower quartile. Of the 32, only one metabolite was detected in four and none at all in six. The most likely explanation for these findings is that a minority of children with lymphoblastic leukaemia fail to take oral mercaptopurine either totally or intermittently. The extent of the problem is unknown, but we suspect it may be clinically important in at least 10% of patients.

The accumulation of mercaptopurine metabolites in age fractionated red blood cells.

1. Red blood cells from four children with lymphoblastic leukemia were age fractionated on Percoll density gradients into 'young', 'middle-aged' and 'old' cells. 2. The rates of accumulation of the mercaptopurine (MP) metabolites thioguanine nucleotides (TGNs) and methylmercaptopurine nucleotides (MeMPs) were measured in the cell fractions from the start of MP continuing chemotherapy. 3. TGNs and MeMP metabolites were present in all the red cell fractions after 3 days oral MP. There was no significant difference between the metabolite concentrations measured in either young, middle-aged or old cells (Mann-Whitney P = 1.0 to 0.12). 4. These observations suggest that MP metabolites do not enter red cells at the stem cell level at the start of therapy. 5. With respect to the monitoring of therapy, these results suggest that the concentration of TGNs after 7 to 10 days MP could be used to predict eventual steady-state concentrations using a simple model.

6-mercaptopurine (6-MP) metabolite measurement in IBD patients' neutrophils correlates with drug efficacy: [formula omitted]∗, E. Seidman∗, Y. Théorêt †. Division of Gastroenterology, Dept. of Pediatrics∗, and Dept. of Pharmacology †, Centre de Recherche, Hôpital Ste-Justine, Université de Montréal, Montreal, Canada

6-mercaptopurine (6-MP) metabolite measurement in IBD patients' neutrophils correlates with drug efficacy: [formula omitted]∗, E. Seidman∗, Y. Théorêt †. Division of Gastroenterology, Dept. of Pediatrics∗, and Dept. of Pharmacology †, Centre de Recherche, Hôpital Ste-Justine, Université de Montréal, Montreal, Canada

The Role of Genetic Variation in Thiopurine Methyltransferase Activity and the Efficacy and/or Side Effects of Azathioprine Therapy in Dermatologic Patients

Thiopurine methyltransferase (TPMT) is one of three major enzymes involved in the metabolism of azathioprine and its active metabolite 6-mercaptopurine. Thiopurine methyltransferase activity is determined by an allelic polymorphism for either high (TPMTH) or low (TPMTL) enzyme activity. Homozygotes for the low activity allele are known to be at risk for profound myelosuppression with azathioprine. Heterozygotes may be at risk for myelosuppression. Homozygotes for the high activity allele may be inadequately immunosuppressed with conventional, empiric doses of azathioprine. We analyzed TPMT activity in red blood cell (RBC) lysates and determined the TPMT genotypes (based on normal population screening) of 28 dermatologic patients. This information was correlated with the observed efficacy and side effects of azathioprine therapy. Two patients with TPMT levels of less than 12.5 U/mL RBCs (both TPMTH heterozygotes) experienced leukopenia (white blood count < 4.0 x 10(9)/L) with azathioprine doses around 1.5 mg/kg. A third patient who experienced leukopenia had a TPMT level at the lower end of the homozygous range (15.5 U/mL RBCs) but received the highest dose of azathioprine (2.6 mg/kg) of all patients in this series. Ten patients with TPMT levels of more than 18.5 U/mL RBCs (all TPMTH homozygotes) receiving less than 1.5 mg/kg of azathioprine were judged to have a poor clinical response. In comparison, seven patients with TPMT levels between 12.5 and 18.5 U/mL RBCs (six TPMTH homozygotes and one TPMTH heterozygote) receiving 0.9 to 1.8 mg/kg of azathioprine had a favorable clinical response. Adverse effects of gastrointestinal upset and liver function test abnormalities did not appear to correlate with TPMT activity. The TPMTH heterozygotes may be at increased risk for myelosuppression with standard, empiric doses of azathioprine. On the other hand, homozygotes for TPMTH, particularly those with TPMT levels at the upper end of the homozygous range, may have a poor clinical response to azathioprine due to inadequate empiric dosing.

Determination of Intracellular Levels of 6-Mercaptopurine Metabolites in Erythrocytes Utilizing Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Capillary electrophoresis proved to be a useful technique for the analysis of intracellular levels of 6-thioguanosine mono-, di-, and triphosphate with analysis times of 20 min. Conditions required for baseline separation of the thioguanine nucleotides consisted of a 25 mM KH 2PO 4 (pH 8.0) buffer and a separation voltage of +28 kV. Laser-induced fluorescence detection (λ ex = 325 nm, λ em = 410 nm) of the thioguanine nucleotide metabolites of 6-mercaptopurine (6-MP) was possible following oxidation of the thiol functionality. Tedious extraction procedures involving mercury cellulose resins or phenyl mercury adduct formation, which had been required previously for the selective extraction of thiopurines from erythrocytes, were unnecessary due to the overall specificity of the approach. However, the inclusion of 50 mM EDTA in the sample preparation was required to inhibit the anabolic/catabolic enzymatic activity, which was responsible for the degradation of the analytes. The method demonstrated linearity from 5 to 1700 pmol/100 μl red blood cells for the three analytes (RSDs ≤ 8%). The feasibility of the method was demonstrated for the quantitation of 6-thioguanine nucleotides in patients receiving either oral or intravenous 6-MP therapy.

Mercaptopurine metabolism and risk of relapse in childhood lymphoblastic leukaemia

Many months' treatment with daily oral mercaptopurine is an important part of therapy for nearly all children with lymphoblastic leukaemia (ALL). Even when prescribed the same dose based on body surface area, patients have widely different intracellular concentrations of drug metabolites. Whether this variation matters in terms of disease control is not yet clear. To find out, we followed up a large group of children with ALL in whom mercaptopurine-derived thioguanine nucleotides in the red cells were measured during treatment with an identical dose of mercaptopurine early in first remission. 172 unselected children (100 boys, 72 girls) were recruited between 1980 and 1992. At median follow-up of 5 years from diagnosis 42 (24%) had relapsed; 30 had erythrocyte thioguanine nucleotide concentrations below the group median (284 pmol per 8 × 108 erythrocytes) and 12 had values above the median. The actuarial relapse-free survival at 5 years was 63% in the below-median group and 84% in the above-median group (difference 21% [95% Cl 3-39%], p=0·0018). Multivariate analysis showed that erythrocyte thioguanine nucleotide concentration was independent of other prognostic variables including age, leukaemia immunophenotype, white-blood-cell count at diagnosis, trial protocol, and sex. Whatever the cause, in childhood ALL variable formation of intracellular mercaptopurine metabolites seems to be clinically important. Therapeutic schedules that include long-term daily oral mercaptopurine might be more effective if such metabolites are monitored.

Thiopurine methyltransferase activity in American white subjects and black subjects.

Thiopurine methyltransferase (TPMT) is a cytoplasmic enzyme that preferentially catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including 6-mercaptopurine. TPMT exhibits genetic polymorphism in white populations, with 89% of individuals having high TPMT activity, 11% having intermediate activity, and one in 300 having extremely low or absent activity. TPMT activity is inversely correlated with formation of active 6-mercaptopurine metabolites (thioguanine nucleotides), thereby influencing 6-mercaptopurine toxicity and efficacy. To investigate ethnic and gender differences in TPMT, we measured erythrocyte TPMT activity in 209 white healthy subjects and 196 black healthy subjects (202 women and 303 men). The black population had lower TPMT activity than the white population (median, 14.4 versus 16.8 units/ml packed erythrocytes; p < 0.001). Maximum likelihood estimation of TPMT activity distribution identified 91.9% and 93.9% with high activity and 7.7% and 6.1% with intermediate activity in the white and black groups, respectively. These data indicate that TPMT activity is similarly polymorphic in American black subjects and white subjects, although median TPMT activity is approximately 17% lower in black subjects.

Effect of dexamethasone, 6-mercaptopurine and cyclosporine a on intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression

The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) is increased in immune-mediated diseases. These adhesion molecules promote the adherence of inflammatory cells to parenchymal target cells. We have examined the effect of immunosuppressants on the expression of ICAM-1 and VCAM-1 in a renal tubular epithelial cell line. We found that dexamethasone (4–40 μg/mL), the azathioprine metabolite 6-mercaptopurine (10– 100 μg/mL) and cyclosporine A (0.1–1 μg/mL) have no effect on the basal and the tumor necrosis factor-α- or interleukin-1-stimulated expression of these adhesion molecules. We conclude that these immunosuppressants do not directly influence the expression of ICAM-1 and VCAM-1 by renal tubular epithelium.

High-performance liquid chromatographic assay of the methyl and nucleotide metabolites of 6-mercaptopurine: quantitation of red blood cell 6-thioguanine nucleotide, 6-thioinosinic acid and 6-methylmercaptopurine metabolites in a single sample

A reversed-phase high-performance liquid chromatographic assay was developed to quantify intracellular metabolites of the cytotoxic drug 6-mercaptopurine in the human red blood cell. The 6-thioguanine nucleotides, 6-thioinosinic acid and 6-methylmercaptopurine metabolites are measured in a single sample. A similar assay measures the parent thiopurine compounds. The limit of quantitation of the assay is 0.03, 0.03 and 0.12 nmol per 8 · 10 8 red blood cells for the 6-thioguanine nucleotides, 6-thioinosinic acid and the 6-methylmercaptopurine metabolites, respectively.

Altered mercaptopurine metabolism, toxic effects, and dosage requirement in a thiopurine methyltransferase-deficient child with acute lymphocytic leukemia

Thiopurine methyltransferase deficiency, inherited as an autosomal codominant trait, is associated with aberrant mercaptopurine metabolism leading to excessive cellular accumulation of 6-thioguanine nucleotides, the active metabolites of mercaptopurine. We describe a case of severe thiopurine methyltransferase deficiency (activity less than 1 U/8 x 10(8) erythrocytes) in a young girl with acute lymphocytic leukemia. The level of 6-thioguanine nucleotide in the patient's erythrocytes was seven times the population median value, and she had intolerable hematologic toxic effects during postremission therapy with a standard dosage of mercaptopurine (75 mg/m2 per day). Subsequent therapy with 6% of this dosage (10 mg/m2 three times weekly) yielded erythrocytic 6-thioguanine nucleotide concentrations consistently above the population median but not associated with prohibitively toxic effects. This case demonstrates that thiopurine methyltransferase deficiency does not absolutely contraindicate mercaptopurine therapy, and it also provides insight into the mechanism of excessive toxic effects of mercaptopurine sometimes observed in children with acute lymphocytic leukemia.

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.

Clinical pharmacodynamics of anticancer drugs: a basis for extending the concept of dose-intensity.

The studies reviewed herein support the precept that "systemic dose-intensity" (i.e., systemic exposure) may be more informative than "administered dose-intensity" for certain anticancer drugs. This does not mean that the administered dose-intensity should be ignored; in fact these data indicate the importance of documenting and assessing administered dose-intensity as an initial step toward identifying those situations where systemic dose-intensity may be most important. The studies described in this review were selected as representative examples of successful clinical pharmacodynamic studies; other published examples include vincristine AUC versus severity of neurotoxicity, etoposide systemic exposure versus leukopenia, red cell concentration of mercaptopurine metabolites versus neutropenia in children with ALL, and ARA-CTP retention in leukemic blasts versus clinical response in acute non-lymphocytic leukemia. As is the case with other types of clinical trials in cancer patients, there are also examples of negative pharmacodynamic studies (i.e., no relationship found between concentration and effects). There are several possible reasons for such negative findings, including the lack of such a relationship for some drugs, measuring the inappropriate drug moiety (e.g., failure to measure all active metabolites), measuring drug concentrations in the wrong biological fluid, evaluating systemic exposure over too narrow a range (i.e., all patients have either sub- or supra-therapeutic systemic exposure), selecting inappropriate sampling times or pharmacokinetic parameters, inadequately assessing drug toxicity or response, or simply studying an inadequate number of patients or patients with drug-resistant cancers. Therefore, negative findings in some pharmacodynamic studies should not deter the investigation of other drugs and/or other malignant diseases, just as negative therapeutic trials do not preclude subsequent clinical trials in oncology. Also, finding a relation between systemic exposure and drug toxicity, in the absence of a clear relation to antitumor effects, is potentially of great clinical utility. Such data should allow more objective escalation of drug dosages in individual patients, to ensure maximum dose-intensity while avoiding host toxicity. Obviously, if such dose escalation could be guided by more easily measured patient characteristics (e.g., age, weight, CrCl, shoe size, etc.), then using drug concentrations in individual patients might be obviated.(ABSTRACT TRUNCATED AT 400 WORDS)

A clinically useful ion-pairing high-performance liquid chromatographic assay for the monophosphate metabolites of thioguanine and mercaptopurine in human neoplastic cells

A sensitive and rapid assay for the quantitation of thioinosine monophosphate and thioguanosine monophosphate, the major intracellular active metabolites of mercaptopurine and thioguanine, respectively, has been developed. Neoplastic cells are extracted with trichloracetic acid, and the neutralized acid extracts are analyzed by ion-pairing high-performance liquid chromatography with dual-channel uv-wavelength detection. This technique provides a lower limit of sensitivity of 30 pmol of thioinosine monophosphate and 10 pmol of thioguanosine monophosphate. The number of cells assayed per sample was 2 × 10 7. This assay makes it possible to detect and quantitate low levels of thioinosine monophosphate and thioguanosine monophosphate present in neoplastic cells obtained directly from patients receiving mercaptopurine or thioguanine chemotherapy.