mercaptopurine and 6-methylthiopurine

The thiopurine drugs azathioprine and mercaptopurine are effective in the treatment of disorders of immune regulation and acute lymphoblastic leukaemia. Although developed in the 1950s, thiopurines remained relevant in the anti-tumour necrosis factor biologic era, finding widespread use as a co-immunomodulator. Step changes in the management of patients treated with thiopurines have reduced the incidence of severe, sometimes life-threatening toxicity. Testing for thiopurine methyltransferase (TPMT) deficiency directs a safe initial dose for therapy. The introduction of red cell thioguanine nucleotide (TGN) monitoring provides a basis for dose adjustment and the identification of patients with high levels of red cell methylmercaptopurine (MMP) and an increase in the MMP:TGN ratio. These patients are at risk for hepatotoxicity and where TGN levels are sub-therapeutic, non-response to therapy. Switching thiopurine hypermethylators to low-dose thiopurine and allopurinol combination therapy resolves hepatoxicity and increases sub-therapeutic TGN levels to regain clinical response.

Topics: Azathioprine; Drug Hypersensitivity; Erythrocytes; Female; Genotype; Humans; Male; Mercaptopurine; Methyltransferases; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Purine-Pyrimidine Metabolism, Inborn Errors; Purines

Thiopurine drugs, including azathioprine and 6-mercaptopurine, are used commonly in patients with inflammatory bowel disease for maintenance of remission. Although generally well tolerated, adverse effects lead to discontinuation in a significant minority of patients. Pharmacogenomic studies have suggested that metabolic breakdown of azathioprine in an individual is genetically determined. Coupled with the fact that certain thiopurine metabolites, notably 6-thioguanine nucleotide and 6-methylmercaptopurine, are associated with antiinflammatory effects and adverse effects, respectively, some investigators have examined intentionally shunting the metabolism of azathioprine toward increasing 6-thioguanine nucleotide levels by using low doses of the xanthine oxidoreductase inhibitor allopurinol to improve efficacy and decrease toxicity of azathioprine in patients with inflammatory bowel disease. We performed a search of the MEDLINE and Embase databases for basic and clinical research reports of this modality. Pertinent articles were retrieved, reviewed, and assessed by the authors. Case series, cohort studies, and one randomized trial have investigated adding allopurinol to azathioprine therapy in patients with inflammatory bowel disease. Most reports primarily examined metabolite levels in these patients. In general, the literature suggests that this modality was successful at significantly increasing 6-thioguanine nucleotide levels while decreasing 6-methylmercaptopurine levels. Several small reports have suggested that patients with increased 6-thioguanine nucleotide levels had improved symptoms or symptom remission. Adverse effects and discontinuation rates remained similar or were improved in patients who were taking a thiopurine and started allopurinol. In conclusion, the addition of allopurinol may be an option for optimizing thiopurine metabolite production in select patients with low 6-thioguanine nucleotide levels. Appropriate care and monitoring of these patients are mandatory to prevent neutropenia or other adverse effects.

Topics: Allopurinol; Drug Therapy, Combination; Enzyme Inhibitors; Guanine Nucleotides; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Mercaptopurine; Methyltransferases; Prospective Studies; Retrospective Studies; Thionucleotides

Azathioprine and mercaptopurine have a pivotal role in the treatment of inflammatory bowel disease (IBD). However, because of their complex metabolism and potential toxicities, optimal use of biomarkers to predict adverse effects and therapeutic response is paramount.. To provide a comprehensive review focused on pharmacogenetics and pharmacokinetics for safe and effective thiopurine therapy in IBD.. A literature search up to July 2015 was performed in PubMed using a combination of relevant MeSH terms.. Pre-treatment thiopurine S-methyltransferase typing plus measurement of 6-tioguanine nucleotides and 6-methylmercaptopurine ribonucleotides levels during treatment have emerged with key roles in facilitating safe and effective thiopurine therapy. Optimal use of these tools has been shown to reduce the risk of adverse effects by 3-7%, and to improve efficacy by 15-30%. For the introduction of aldehyde oxidase (AOX) into clinical practice, the association between AOX activity and AZA dose requirements should be positively confirmed. Inosine triphosphatase assessment associated with adverse effects also shows promise. Nucleoside diphosphate-linked moiety X-type motif 15 variants have been shown to predict myelotoxicity on thiopurines in East Asian patients. However, the impact of assessments of xanthine oxidase, glutathione S-transferase, hypoxanthine guanine phosphoribosyltransferase and inosine monophosphate dehydrogenase appears too low to favour incorporation into clinical practice.. Measurement of thiopurine-related enzymes and metabolites reduces the risk of adverse effects and improves efficacy, and should be considered part of standard management. However, this approach will not predict or avoid all adverse effects, and careful clinical and laboratory monitoring of patients receiving thiopurines remains essential.

Topics: Azathioprine; Humans; Inflammatory Bowel Diseases; Mercaptopurine; Methyltransferases; Pharmacogenetics; Thioinosine; Thionucleotides

This article reviews current data to optimize the use of both older and newer drugs in inflammatory bowel disease. For patients with severe ulcerative colitis (UC), steroid dosing has been clarified, and a mega-analysis of steroid outcomes and toxicities has been reported. In regard to mesalamine, recent information has suggested benefit of a higher dose of pH-dependent release mesalamine for patients with moderate UC. Also, a once-daily formulation with Multi-Matrix System (MMX) technology (Shire Pharmaceuticals, Wayne, PA), has been approved. In regard to cyclosporine, two centers have reported an increased rate of colectomy over a long-duration follow-up of a cyclosporin A course given for UC. Additional information regarding thiopurines has been published, including the use of metabolite testing and duration of therapy for these drugs. Lastly, additional information regarding the optimal method for using anti-tumor necrosis factor therapy continues to accumulate.

Topics: Absorptiometry, Photon; Adalimumab; Adrenal Cortex Hormones; Anti-Inflammatory Agents, Non-Steroidal; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Azathioprine; Colectomy; Colitis, Ulcerative; Combined Modality Therapy; Crohn Disease; Cyclosporine; Humans; Inflammatory Bowel Diseases; Infliximab; Mercaptopurine; Mesalamine; Patient Compliance; Prednisone; Treatment Failure

The thiopurine drugs azathioprine and 6-mercaptopurine (6-MP) are well-established in the treatment of inflammatory bowel disease (IBD). However, there is a wide inter- and intra-patient variation in the concentrations of active and toxic metabolites due to their complex metabolism and genetic polymorphisms in metabolizing enzymes. Serious drug toxicity leads to cessation of therapy in 9-25% of patients, and there is failure to achieve efficacy in approximately 15% of cases. Advances in the understanding of thiopurine drug metabolism have led to new genetic and metabolite tests to help clinicians optimize thiopurine use. Thiopurine methyltransferase (TPMT) enzyme activity can predict life-threatening myelotoxicity in the one in 300 patients who are TPMT-deficient. However, myelotoxicity can also occur in the presence of normal TPMT activity so blood count monitoring should remain standard practice. TPMT testing may also aid in dose individualization. 6-Thioguanine nucleotides (6-TGN) are thought to be the predominant active metabolites of the thiopurines. 6-thioguanine nucleotide concentration is correlated with bone marrow toxicity and may also correlate with efficacy in IBD. Measurement of 6-TGN and 6-methylmercaptopurine (6-MMP) concentration is most useful in determining why a patient is not responding to a standard dose of a thiopurine drug and may help in avoiding myelosuppression. The ratio of these metabolites can help distinguish non-compliance, under-dosing, thiopurine-resistant and thiopurine-refractory disease. Some of these investigations are entering routine clinical practice but more research is required to determine their optimal use in patients with IBD.

Topics: Antimetabolites, Antineoplastic; Azathioprine; Bone Marrow; Erythrocytes; Guanine Nucleotides; Humans; Inflammatory Bowel Diseases; Inosine Triphosphatase; Mercaptopurine; Methyltransferases; Polymorphism, Genetic; Pyrophosphatases; Thionucleotides

Chronotherapy is the timing of medication according to biological rhythms of the host to optimize drug efficacy and minimize toxicity. Efficacy and myelosuppression of azathioprine/6-mercaptopurine (AZA/6-MP) are correlated with the metabolite 6-thioguanine, while the metabolite 6-methylmercaptopurine correlates with hepatotoxicity.. This was a single-center, 10-week prospective crossover trial involving 26 participants with inactive inflammatory bowel disease (IBD) on a stable dose and time of AZA or 6-MP therapy. Participants were switched to the opposite delivery time (morning or evening) for 10 weeks, and metabolite measurements were at both time points.. In the morning vs evening dosing, 6-thioguanine levels were 225.7 ± 155.1 vs 175.0 ± 106.9 ( P < 0.01), and 6-methylmercaptopurine levels were 825.1 ± 1,023.3 vs 2,395.3 ± 2,880.3 ( P < 0.01), with 69% (18 out of 26) of participants had better metabolite profiles in the morning. Participants with optimal dosing in the morning had an earlier chronotype by corrected midpoint of sleep.. In the first study on a potential role of chronotherapy in IBD, we found (i) morning dosing of AZA or 6-MP resulted in more optimal metabolite profiles and (ii) host chronotype could help identify one-third of patients who would benefit from evening dosing. Circadian regulation of metabolic enzymes of AZA/6-MP activity in the liver is the likely cause of these differences. This pilot study confirms the need to incorporate chronotherapy in future multicenter clinical trials on IBD disease.

Topics: Azathioprine; Chronotherapy; Cross-Over Studies; Humans; Inflammatory Bowel Diseases; Mercaptopurine; Pilot Projects; Prospective Studies; Thioguanine

Hepatotoxicity, gastrointestinal complaints and general malaise are common limiting adverse reactions of azathioprine and mercaptopurine in IBD patients, often related to high steady-state 6-methylmercaptopurine ribonucleotide (6-MMPR) metabolite concentrations.. To determine the predictive value of 6-MMPR concentrations 1 week after treatment initiation (T1) for the development of these adverse reactions, especially hepatotoxicity, during the first 20 weeks of treatment.. The cohort study consisted of the first 270 IBD patients starting thiopurine treatment as part of the Dutch randomised-controlled trial evaluating pre-treatment thiopurine S-methyltransferase genotype testing (ClinicalTrials.gov NCT00521950). Blood samples for metabolite assessment were collected at T1. Hepatotoxicity was defined by alanine aminotransaminase elevations >2 times the upper normal limit or a ratio of alanine aminotransaminase/alkaline phosphatase ≥5.. Forty-seven patients (17%) presented hepatotoxicity during the first 20 weeks of thiopurine treatment. A T1 6-MMPR threshold of 3615 pmol/8 × 10. In more than 80% of patients, thiopurine-induced hepatotoxicity could be explained by elevated T1 6-MMPR concentrations and the independent risk factors age, gender and BMI, allowing personalised thiopurine treatment in IBD to prevent early failure.

Topics: Adult; Aged; Aged, 80 and over; Azathioprine; Chemical and Drug Induced Liver Injury; Cohort Studies; Early Diagnosis; Female; Genotype; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Middle Aged; Prognosis; Risk Factors; Thioinosine; Thionucleotides; Treatment Outcome; Young Adult

Several studies have demonstrated a favourable safety profile for thiopurine use for inflammatory bowel disease (IBD) during pregnancy. We performed a study in pregnant patients with IBD who were using thiopurines, in order to determine the influence of pregnancy on thiopurine metabolism and to assess intrauterine exposure of the fetus to thiopurines.. Female patients with IBD receiving steady-state thiopurines and planning a pregnancy were prospectively enrolled. 6-Thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine (6-MMP) concentrations were determined, combined with routine laboratory tests, before, during and after pregnancy. Thiopurine metabolites were measured in umbilical cord blood immediately after delivery.. Thirty patients who were using azathioprine (28 patients, median dose 1.93 mg/kg) or mercaptopurine (two patients, doses 1.32 and 0.94 mg/kg) were included. During pregnancy, median 6-TGN decreased over time (p=0.001). while 6-MMP increased, without causing myelotoxicity or hepatotoxicity. After delivery, both 6-TGN and 6-MMP levels returned to preconception baseline levels. Fetal 6-TGN concentrations correlated positively with maternal 6-TGN levels (p<0.0001). No 6-MMP was detected in the newborns, except one born with pancytopenia and high alkaline phosphatase activity; the mother of this infant had severe pre-eclampsia. All infants had normal Apgar scores, but 60% had anaemia at birth. No major congenital abnormalities were observed.. Pregnancy has a major effect on maternal thiopurine metabolism. In utero the unborn child is exposed to 6-TGN, but not to 6-MMP. Sixty per cent of the infants were born with anaemia, which raises the question whether infants should be tested for possible anaemia immediately after birth.

Topics: Abnormalities, Drug-Induced; Adolescent; Adult; Anemia, Neonatal; Azathioprine; Biomarkers; Female; Fetal Blood; Follow-Up Studies; Guanine Nucleotides; Humans; Immunosuppressive Agents; Infant, Newborn; Inflammatory Bowel Diseases; Mercaptopurine; Middle Aged; Pregnancy; Pregnancy Complications; Pregnancy Outcome; Prospective Studies; Thionucleotides; Treatment Outcome; Young Adult

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.. A stepwise dose-escalation of allopurinol was performed in 11 azathioprine-pretreated IBD patients with inadequately low 6-TGN concentrations (<235 pmol/8 × 10(8) erythrocytes) and/or elevated MMPN concentrations (>5,000 pmol/8 × 10(8) 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.. 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 × 10(8) 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.. 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.

Topics: Adult; Aged; Allopurinol; Azathioprine; Female; Guanine Nucleotides; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Middle Aged; Thionucleotides

Thiopurines are crucial in the treatment of inflammatory bowel disease. The phenotype of pivotal metabolic enzymes determines whether thioguanine nucleotides (6-TGN) are generated in clinically sufficiently high levels. The first step in activation of thiopurine prodrugs to 6-TGN is catalysis by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Often, patients exhibit a clinically unfavorable metabolism, leading to discontinuation of conventional thiopurine therapy. The combination of allopurinol and low-dose thiopurine therapy may optimize this variant metabolism, presumably by affecting enzyme activities. We performed a prospective pharmacodynamic study to determine the effect of combination therapy on the activity of HGPRT. The activity of HGPRT and 6-TGN concentrations was measured in red blood cells during thiopurine monotherapy and after 4 weeks of combination therapy. The activity of HGPRT was also measured after 12 weeks of combination therapy. From the results, we conclude that combination therapy increases the activity of HGPRT and subsequently 6-TGN concentrations.

Topics: Adolescent; Adult; Allopurinol; Azathioprine; Child; Dose-Response Relationship, Drug; Drug Therapy, Combination; Female; Gout Suppressants; Humans; Hypoxanthine Phosphoribosyltransferase; Inflammatory Bowel Diseases; Male; Mercaptopurine; Middle Aged; Young Adult

A prospective randomized trial in patients with Crohn disease studied whether 6-thioguanine nucleotide (6-TGN) concentration-adapted azathioprine (AZA) therapy is clinically superior to a standard dose of 2.5 mg/kg/day AZA.. After 2 weeks of standard therapy, patients (n = 71) were randomized into standard (n = 32) or adapted-dose (n = 25) groups; 14 patients dropped out before randomization. In the adapted group, the AZA dose was adjusted to maintain 6-TGN concentrations between 250 and 400 pmol/8 x 10(8) erythrocytes (Ery). Response criteria were the number of patients in remission after 16 weeks without steroids (primary) and remission after 24 weeks, frequency of side effects, and quality of life (secondary).. After 16 weeks, 14 of 32 (43.8%) patients in the standard group vs 11 of 25 (44%) in the adapted group were in remission without steroids (intent-to-treat analysis). After 24 weeks, 43.8% vs 40% were in remission. No significant differences were found concerning quality of life, disease activity, 6-TGN concentrations, AZA dose, or dropouts due to side effects. Sixty-six patients had a wild-type thiopurine S-methyltransferase (TPMT) genotype, with TPMT activities of 8 to 20 nmol/(mL Ery x h). Five patients (dropouts after randomization) were heterozygous, with TPMT activities <8 nmol/(mL Ery x h). 6-Methyl mercaptopurine (6-MMP) concentrations >5700 pmol/8 x 10(8) Ery were not associated with hepatotoxicity.. Standard and adapted dosing with the provided dosing scheme led to identical 6-TGN concentrations and remission rates. Adapted dosing had no apparent clinical benefit for patients with TPMT activity between 8 and 20 nmol/(mL Ery x h). Additionally, 6-MMP monitoring had no predictive value for hepatotoxicity.

Topics: Adolescent; Adult; Aged; Chemical and Drug Induced Liver Injury; Crohn Disease; Dose-Response Relationship, Drug; Erythrocyte Indices; Female; Genotype; Guanine Nucleotides; Humans; Leukopenia; Male; Mercaptopurine; Methyltransferases; Middle Aged; Remission Induction; Thionucleotides; Thrombocytopenia

6-Mercaptopurine (6-MP) and azathioprine (AZA) have proven efficacy in the treatment of inflammatory bowel disease (IBD). However, adverse events leading to discontinuation may occur in 10-20% of patients. The efficacy of AZA and 6-MP is based on formation of their active metabolites, the 6-thioguaninenucleotides (6-TGNs). Therefore, 6-thioguanine (6-TG), an agent leading more directly to the formation of 6-TGNs and until recently used only in patients suffering from leukaemia, may be an alternative in AZA or 6-MP intolerance. The purpose of our study was to assess the short-term safety of 6-TG.. Thirty-two IBD patients with previously established AZA or 6-MP intolerance were treated with 6-TG in doses of 20 mg (n = 19) or 40 mg (n = 13) once daily. Safety parameters were obtained at 0, 1, 2, 4 and 8 weeks after start of medication. Primary outcome measures were the ability to tolerate 6-TG and the occurrence of adverse events. Secondary outcome definitions included laboratory parameters.. Twenty-six (81%) patients were able to tolerate 6-TG during the first 8 weeks. In three of six patients, side effects leading to discontinuation were probably (n = 2) or obviously (n = 1) related to 6-TG. No clinically relevant haematological events or hepatotoxicity occurred in the observed period. Steady-state 6-TG levels were significantly higher with 40 mg once daily (1621 +/- 828 picomol/8 x 10(8) red blood cells (RBC)) than with 20 mg once daily (937 +/- 325 picomol/8 x 10(8) RBC; n = 0.001).. 6-TG treatment seems promising in AZA- or 6-MP-intolerant IBD patients. However, long-term safety and efficacy have yet to be determined.

Topics: Adult; Azathioprine; Chemical and Drug Induced Liver Injury; Dose-Response Relationship, Drug; Drug Monitoring; Erythrocytes; Female; Gastrointestinal Agents; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Middle Aged; Prospective Studies; Thioguanine

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.

Topics: Adolescent; Antimetabolites, Antineoplastic; Child; Child, Preschool; Dose-Response Relationship, Drug; Erythrocytes; Female; Genotype; Humans; Infant; Infections; Leukocyte Count; Male; Mercaptopurine; Methyltransferases; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Thioguanine

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/8x10(8) RBC (median value, 144) and those of 6-mMP metabolites from <150 to 19000 pmol/8x10(8) 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.

Topics: Adolescent; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Child; Child, Preschool; Erythrocytes; Female; Guanine Nucleotides; Humans; Leukocyte Count; Male; Mercaptopurine; Methotrexate; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Thioguanine

Topics: Azathioprine; Erythrocytes; Graft Rejection; Guanine Nucleotides; Humans; Immunosuppressive Agents; Kidney Transplantation; Mercaptopurine; Methyltransferases; Prospective Studies; Thionucleotides

6-mercaptopurine is a mainstay of acute lymphoblastic leukemia treatment. It has a narrow therapeutic window, dictated by its metabolite, thioguanine and 6-methylmercaptopurine. Skin manifestations usually consist of mild facial rash or hypersensitivity exanthems. We report a child who developed a painful acral rash and mucositis while undergoing maintenance therapy for B-cell acute lymphoblastic leukemia without infectious or known drug etiology. Thiopurine metabolites were skewed toward 6-methylmercaptopurine. Two weeks after allopurinol was added and 6-mercaptopurine (6-MP) dose adjusted, the cutaneous manifestations and other constitutional symptoms resolved. We posit that the rash was because of 6-MP toxicity related to skewed metabolism, adding to the growing list of toxicity related to altered 6-MP metabolism.

Topics: Allopurinol; Burkitt Lymphoma; Child; Exanthema; Humans; Mercaptopurine; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Thioguanine

In the recent era of growing availability of biological agents, the role of thiopurines needs to be reassessed with the focus on toxicity.. We assessed the incidence and predictive factors of thiopurine-induced adverse events (AE) resulting in therapy cessation in pediatric inflammatory bowel disease (IBD), related to thiopurine metabolites and biochemical abnormalities, and determined overall drug survival.. We performed a retrospective, single-center study of children diagnosed with IBD between 2000 and 2019 and treated with thiopurine therapy. The incidence of AE and overall drug survival of thiopurines were evaluated using the Kaplan-Meier method. Correlations between thiopurine metabolites and biochemical tests were computed using Spearman's correlation coefficient.. Of 391 patients with IBD, 233 patients (162 Crohn's disease, 62 ulcerative colitis, and 9 IBD-unclassified) were prescribed thiopurines (230 azathioprine and 3 mercaptopurine), of whom 50 patients (22%) discontinued treatment, at least temporary, due to thiopurine-induced AE (median follow-up 20.7 months). Twenty-six patients (52%) were rechallenged and 18 of them (70%) tolerated this. Sixteen patients (6%) switched to a second thiopurine agent after azathioprine intolerance and 10 of them (63%) tolerated this. No predictive factors for development of AE could be identified. Concentrations of 6-thioguanine nucleotides (6-TGN) were significantly correlated with white blood cell and neutrophil count, 6-methylmercaptopurine (6-MMP) concentrations with alanine aminotransferase and gamma-glutamyltranspeptidase.. Approximately 20% of pediatric patients with IBD discontinued thiopurine treatment due to AE. A rechallenge or switch to mercaptopurine is an effective strategy after development of AE. Concentrations of 6-TGN and 6-MMP are associated with biochemical abnormalities.

Topics: Adolescent; Antimetabolites; Azathioprine; Biomarkers, Pharmacological; Child; Cohort Studies; Colitis, Ulcerative; Crohn Disease; Drug Substitution; Drug-Related Side Effects and Adverse Reactions; Female; Guanine Nucleotides; Humans; Male; Mercaptopurine; Netherlands; Retrospective Studies; Thionucleotides; Withholding Treatment

The aim of this study was to describe the long-term health outcomes of children born to mothers with inflammatory bowel disease (IBD) and to assess the impact of maternal IBD medication use on these outcomes.. We performed a multicentre retrospective study in The Netherlands. Women with IBD who gave birth between 1999 and 2018 were enrolled from 20 participating hospitals. Information regarding disease characteristics, medication use, lifestyle, pregnancy outcomes and long-term health outcomes of children was retrieved from mothers and medical charts. After consent of both parents, outcomes until 5 years were also collected from general practitioners. Our primary aim was to assess infection rate and our secondary aims were to assess adverse reactions to vaccinations, growth, autoimmune diseases and malignancies.. We included 1000 children born to 626 mothers (381 (61%) Crohn's disease, 225 (36%) ulcerative colitis and 20 (3%) IBD unclassified). In total, 196 (20%) had intrauterine exposure to anti-tumour necrosis factor-α (anti-TNF-α) (60 with concomitant thiopurine) and 240 (24%) were exposed to thiopurine monotherapy. The 564 children (56%) not exposed to anti-TNF-α and/or thiopurine served as control group. There was no association between adverse long-term health outcomes and in utero exposure to IBD treatment. We did find an increased rate of intrahepatic cholestasis of pregnancy (ICP) in case thiopurine was used during the pregnancy without affecting birth outcomes and long-term health outcomes of children. All outcomes correspond with the general age-adjusted population.. In our study, we found no association between in utero exposure to anti-TNF-α and/or thiopurine and the long-term outcomes antibiotic-treated infections, severe infections needing hospital admission, adverse reactions to vaccinations, growth failure, autoimmune diseases and malignancies.

Topics: Adalimumab; Adult; Anti-Bacterial Agents; Autoimmune Diseases; Cesarean Section; Child Development; Child, Preschool; Congenital Abnormalities; Drug Prescriptions; Drug Therapy, Combination; Female; Gastrointestinal Agents; Humans; Infant; Infant, Newborn; Infant, Small for Gestational Age; Infections; Inflammatory Bowel Diseases; Infliximab; Mercaptopurine; Neoplasms; Netherlands; Patient Admission; Pregnancy; Pregnancy Complications; Premature Birth; Prenatal Exposure Delayed Effects; Retrospective Studies; Tumor Necrosis Factor Inhibitors; Vaccines

The additional value of azathioprine concomitant treatment on infliximab pharmacokinetics in children is not well described yet.. In the present study, we aimed to describe the relationship between thiopurine metabolite levels, infliximab trough levels, anti-IFX antibody formation, and clinical and laboratory markers of disease activity in pediatric patients with Crohn's disease, and to assess non-adherence.. Data were collected prospectively during repeated visits from pediatric patients followed for Crohn's disease in two Czech pediatric inflammatory bowel disease centers between January 2016 and June 2017. Thiopurine metabolites (6-thioguanine and 6-methylmercaptopurine) were measured by high-performance liquid chromatography. Infliximab trough levels and anti-IFX antibody serum levels were measured routinely by ELISA. The risk of loss of response to infliximab therapy was also assessed.. A significant association between infliximab serum levels and 6-thioguanine erythrocyte levels was observed when tested as categorical variables (63 patients, 321 observations). To predict infliximab levels > 5 µg/mL, we propose a 6-thioguanine cutoff of 278 pmol/8 × 10. Thiopurine metabolite monitoring in pediatric patients with Crohn's disease is useful when optimizing combination therapy. Pediatric patients with undetectable 6-thioguanine levels are more likely to lose response to infliximab therapy. When targeting optimal infliximab levels, the 6-thioguanine cutoff levels in children appear to be higher than in adults.

Topics: Adolescent; Azathioprine; Biomarkers; Child; Crohn Disease; Drug Therapy, Combination; Female; Humans; Immunologic Factors; Infliximab; Longitudinal Studies; Male; Mercaptopurine; Prospective Studies

Thiopurines are drugs widely used in patients for the maintenance of remission in inflammatory bowel disease. The optimal plasma levels are known, but there is controversy about whether the need for other drugs is reduced or is cost-effective. The aim of this study is to describe the use of the optimised treatment with thiopurines in paediatric patients with inflammatory bowel disease followed up in this Unit since the introduction of determining the drug levels.. A descriptive retrospective study was conducted in which the plasma values of 6-thioguanine (6-TGN), 6-methyl-mercapto-purine (6-MMP), and their ratios were analysed using liquid chromatography. Other variables were collected, such as clinical status, analytical and demographic variables of patients with inflammatory bowel disease followed up in this Unit.. A total of 72 patients were included, and 149 determinations of metabolites were performed. The 6-TGN levels were found to below the therapeutic range in 61.5% of patients (in 7 cases due to lack of adherence to therapy), and 6-MMP was in the toxicity range in 7.4%. After the determination of 77 specimens, some action was taken, such as modifying the dose, change of formula, or withdrawing the drug. Only 9 patients were scaled to a biological drug (13.4% of the total on single therapy). No association was found between the activity of the disease and the thiopurine levels.. In our experience, the monitoring of thiopurine levels helped to modify the drug dose that the patient received, adjusting their therapeutic levels, and potentially avoiding the addition of new drugs.

Topics: Adolescent; Child; Child, Preschool; Chromatography, Liquid; Colitis, Ulcerative; Crohn Disease; Dose-Response Relationship, Drug; Drug Monitoring; Female; Humans; Immunosuppressive Agents; Infant; Infant, Newborn; Male; Mercaptopurine; Retrospective Studies; Thioguanine; Treatment Outcome

Thiopurine and allopurinol in combination are associated with clinical remission in inflammatory bowel diseases but their influence on subsequent outcomes is unclear. We compared outcomes during exposure to both thiopurines and allopurinol versus thiopurines alone.. We established a nationwide cohort of patients with inflammatory bowel diseases exposed to thiopurines ± allopurinol during 1999-2014, using registry data. Patients were followed until hospitalization, surgery, anti-TNFα, or death (as a primary composite outcome). We used Poisson regression analyses to calculate incidence rate ratios overall and stratified by calendar period (assuming the combined exposure was unintended before 2009).. A total of 10,367 patients with inflammatory bowel diseases (Crohn's disease,. Our nationwide inflammatory bowel disease cohort study shows that concomitant thiopurine-allopurinol is as safe to use as thiopurines alone, with a tendency towards a positive effect on clinical outcomes in recent calendar periods when combined use was intended.

Topics: Adult; Allopurinol; Azathioprine; Colitis, Ulcerative; Crohn Disease; Denmark; Drug Therapy, Combination; Female; Follow-Up Studies; Hospitalization; Humans; Immunosuppressive Agents; Male; Mercaptopurine; Middle Aged; Remission Induction; Severity of Illness Index; Signal Transduction; Thioguanine; Treatment Outcome

6-Mercaptopurine (6-MP) is widely used to treat pediatric acute lymphoblastic leukemia (ALL). Mini-tablets of 5 mg per tablet were developed for precision individual therapy for children and individuals with poor thiopurine S-methyltransferase (TPMT) or nucleoside diphophate-linked moiety X-type motif 15 (NUDT15) metabolism. This study investigated the pharmacokinetic profiles of mini-tablets and conventional tablets with an improved ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method.. After giving 8 healthy beagle dogs 50 mg 6-MP in different dosage forms, plasma samples collected at different time points were analyzed for pharmacokinetic evaluation. The samples were precipitated by methanol with 0.05% formic acid and separated on a Waters Atlantis T3 column (2.1 × 150 mm, 3 μm particles) using 0.1% formic acid in water and methanol at a flow rate of 0.4 mL/min in 4 min.. This method showed good linearity, accuracy, precision and stability with a detection range of 5.0-500.0 ng/mL for 6-MP, 6-methylmercaptopurine (6-MMP) and 6-thioguanine (6-TG). The main parameters, half-life of apparent terminal disposition, maximum observed plasma concentration, total AUC extrapolated to infinity, AUC since initiation of the experiment, mean residence time, distribution volume and clearance were 1.62 ± 0.87 hours, 90.58 ± 60.43 ng/mL, 151.20 ± 94.18 ng·h/mL, 292.06 ± 184.02 ng·h2/mL, 1.90 ± 0.92 hours, 864.08 ± 538.52 L, and 432.75 ± 360.64 L/h for conventional tablets and 1.70 ± 1.10 hours, 84.15 ± 39.50 ng/mL, 147.70 ± 51.80 ng·h/mL, 300.92 ± 124.48 ng·h2/mL, 2.07 ± 0.50 hours, 756.90 ± 324.00 L, and 340.75 ± 125.81 L/h for minitablets, respectively. Paired t-tests showed no significant difference in any of the evaluated pharmacokinetic parameters between the two types tablets (P > 0.05).. Two dosage forms showed the same pharmacokinetic characteristics. This developing, novel formulation will help to provide a more accurate and optimal dosing regimen of 6-MP for humans in the future.

Topics: Animals; Chromatography, High Pressure Liquid; Chromatography, Liquid; Dogs; Mercaptopurine; Plasma; Tandem Mass Spectrometry; Thioguanine

Concentrations of 6-thioguanine (6TG) nucleotides and 6-methylmercaptopurine (6MMP) nucleotides in RBCs were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This assay was validated for clinical use and was applied to blood samples from patients taking mercaptopurine (6MP).. RBCs were hemolyzed and deproteinized using perchloric acid, followed by heating for the hydrolysis of nucleotides, and the resultant base was measured using LC-MS/MS. Precision, recovery, linearity, matrix effect, and limit of quantification was validated for clinical application. Our results were compared with another institution's established LC-MS/MS assay. We measured the concentrations of 6TG and 6MMP in RBCs of pediatric patients with acute lymphoblastic leukemia (ALL), and the clinical impact of those metabolites was investigated.. The imprecision coefficient of variations of 6TG and 6MMP were 5.7%-8.1%, and the bias was within 5%. Lower limits of quantification were set at 54 ng/mL for 6TG and 1036 ng/mL for 6MMP. Correlation coefficients for 6TG and 6MMP were 0.997 and 1.0 in a comparison study. For clinical proof-of-concept, 74 blood samples were collected from 37 pediatric ALL patients receiving maintenance therapy. Concentration of 6TG ranged from 16.1 to 880 pmol/8 × 10 RBCs and that of 6MMP from 55 to 20,937 pmol/8 × 10 RBCs. The 6MP metabolites were not correlated with WBC or absolute neutrophil count. On the other hand, the higher 6MMP level was associated with elevated alanine aminotransferase and aspartate aminotransferase.. In this study, an assay for the quantification of 6TG and 6MMP in RBCs was established and applied to pediatric ALL patients. Interindividual variability in 6MP metabolite concentrations was considerable and associated with elevation of liver enzymes, which may be useful in the clinical monitoring of 6MP maintenance therapy in pediatric ALL patients.

Topics: Adolescent; Antimetabolites, Antineoplastic; Child; Child, Preschool; Chromatography, Liquid; Erythrocytes; Female; Humans; Male; Mercaptopurine; Nucleotides; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Tandem Mass Spectrometry; Thioguanine

Thiopurines are key drugs in maintenance therapy for treating inflammatory bowel disease (IBD). Time-dependent 5-aminosalicylates (5-ASA) releasing preparations (time-dependent 5-ASA) increase 6-thioguanine nucleotide (6-TGN), an active metabolite of thiopurines. However, the effects of pH-dependent 5-ASA releasing preparations (pH-dependent 5-ASA) on thiopurine metabolism were not reported.. We conducted a retrospective study of 134 IBD patients who received thiopurine treatment. The 6-methylmercaptopurine (6-MMP)/6-TGN values after taking the same dose of thiopurine preparations for at least 28 days were included.. There was a significant decrease in the 6-MMP/6-TGN ratio in time-dependent 5-ASA compared with group without 5-ASA preparations and the pH-dependent 5-ASA group (p = 0.008 and < 0.001 respectively). Spearman's rank correlation coefficient indicated a negative relationship between the daily oral dose of time-dependent 5-ASA and the 6-MMP/6-TGN ratio (r = -0.362, p = 0.003). Multivariate logistic regression analysis was performed in the groups with 6-MMP/6-TGN ratios of 1 or more and less than 1. The use of time-dependent 5-ASA and concomitant allopurinol negatively affected the independent 6-MMP/6-TGN ratio (p = 0.006 and 0.007 respectively).. Our study revealed that time-dependent but not pH-dependent 5-ASA decreases the 6-MMP/6-TGN ratio. We also confirmed that concomitant allopurinol results in a low 6-MMP/6TGN ratio.

Topics: Administration, Oral; Adolescent; Adult; Aged; Allopurinol; Anti-Inflammatory Agents, Non-Steroidal; Drug Interactions; Drug Liberation; Female; Guanine Nucleotides; Humans; Hydrogen-Ion Concentration; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Mesalamine; Middle Aged; Retrospective Studies; Thionucleotides; Time Factors; Young Adult

Thiopurines are drugs widely used for the treatment of autoimmune conditions, inflammatory bowel disease or acute lymphoblastic leukemia. Determination of thiopurine methyltransferase activity (TPMT), a major determinant of thiopurines toxicity, has been suggested before implementing thiopurine treatment. An ultraperformance liquid chromatography (UPLC) method was developed and validated for the quantification of TPMT enzyme activity based on the conversion of 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-L-methionine (SAM) as methyl donor in red blood cell lysates (RBC). This method was improved from a previous laborious high performance liquid chromatography (HPLC) method, using a lower volume of injection and with a shorter runtime. After incubation and protein precipitation 6-MMP was separated on a HSS-T3 (2.1 × 50 mm, 1.8 μm) column and monitored by UV detection (290 nm). A change on the organic solvent used to dissolve 6-MP resulted in a reduction of interference by endogenous or non-enzymatic methylated 6-MMP. A full validation of the 6-MMP assay was performed according to the FDA and EMA guidelines. The method was linear from 0.125 to 2 nmol/mL, with acceptable values of accuracy and precision. The method was applied in 106 patients treated with thiopurines whose TPMT activity was previously quantified by HPLC. Evaluation through Bland-Altman plot showed that TPMT activities were in agreement between both methods.

Topics: Chromatography, High Pressure Liquid; Drug Monitoring; Enzyme Assays; Erythrocytes; Humans; Limit of Detection; Linear Models; Mercaptopurine; Methyltransferases; Reproducibility of Results; S-Adenosylmethionine

Azathioprine is frequently used in severe eczema. It is converted in the liver into active metabolites, including 6-thioguanine nucleotide (6-TGN) and methylated 6-methylmercaptopurine (6-MMP). In the past, the therapeutic potential of azathioprine may have not been fully utilized. Recent investigations on inflammatory bowel disease have led to a better understanding of azathioprine metabolism and optimizing treatment.. To investigate whether measuring thiopurine metabolites in circulation can improve the effectiveness and safety of azathioprine treatment in patients with atopic dermatitis and/or chronic hand/foot eczema.. Azathioprine metabolite levels were measured in eczema patients during maintenance treatment (Part I) and dose escalation (Part II). Clinical effectiveness, hepatotoxicity, and bone marrow suppression were analyzed and TPMT genotype was assessed.. A wide variation in metabolite levels in all dose groups was observed. In Part I (32 patients), there were no significant differences in 6-TGN levels between clinical responders and non-responders (p = .806). No hepatoxicity or myelotoxicity was observed. In Part II, all 6-TGN and 6-MMP levels increased during dose escalation. Hypermethylation was observed in 2/8 patients.. For individual eczema patients treated with azathioprine, routinely measuring 6-TGN and 6-MMP can be helpful in optimizing azathioprine dose, improving clinical effectiveness, and preventing side effects.

Topics: Adult; Azathioprine; Chromatography, High Pressure Liquid; Dermatitis, Atopic; Eczema; Female; Guanine Nucleotides; Humans; Immunosuppressive Agents; Male; Mercaptopurine; Middle Aged; Thionucleotides; Treatment Outcome

Topics: Adult; Allopurinol; Azathioprine; Chronic Disease; Dermatitis, Atopic; Drug Therapy, Combination; Eczema; Female; Foot Dermatoses; Guanine Nucleotides; Hand Dermatoses; Humans; Immunosuppressive Agents; Male; Mercaptopurine; Middle Aged; Prospective Studies; Thionucleotides; Time Factors; Treatment Outcome

Interactions between principal cytotoxic thiopurine metabolites, that is 6-thioguanine nucleotides [6-TGN], and infliximab [IFX] and anti-IFX antibodies [Abs] may contribute to higher effectiveness of IFX-thiopurine combination therapy than monotherapies in inflammatory bowel disease.. To examine if thiopurine metabolites influenced trough IFX and anti-IFX Abs, 89 patients previously assessed for anti-IFX Abs were included. To assess if IFX influenced thiopurine metabolites, eight patients who had responded to 12 weeks of intensified IFX at a constant thiopurine dosing were included.. In the first cohort, IFX-thiopurine combination therapy reduced anti-IFX Ab detection [8/40; 20%] as compared with IFX monotherapy [22/49; 45%], odds ratio [OR] 0.31 [0.12-0.80], p < 0.05. 6-TGN was significantly lower in anti-IFX Ab-positive patients (50 pmol/8 × 108 red blood cells [RBC] vs 105, p < 0.01). All anti-IFX Ab-positive patients had 6-TGN < 117 pmol/8 × 108 RBC (sensitivity 100% [63-100], specificity 47% [29-65], area under the curveROC = 0.82, p < 0.01). Trough IFX was similar between anti-IFX Ab-negative patients in IFX monotherapy and IFX-thiopurine combination therapy [5.1 μg/mL vs 4.9, p = 0.76]. 6-TGN and IFX did not correlate [rP = 0.04, p = 0.83; rS = 0.02, p = 0.89, respectively]. In the second cohort, trough IFX increased during IFX intensification [ΔIFX median 6.5 μg/mL, p = 0.02], but 6-TGN was stable [6-TGN at Weeks 0, 4, 8, 12: 90 pmol/8 × 108 RBC, 93, 101, 90; p > 0.05]. Methylated mercaptopurine metabolite associations were consistently negative.. Superior effect of IFX-thiopurine combination therapy over monotherapies partly relates to decrease in anti-IFX Abs, which associates with 6-TGN levels and has a lower therapeutic threshold than during thiopurine monotherapy. Additional benefit likely ascribes to synergy between different anti-inflammatory modes of action rather than direct drug interactions.

Topics: Adult; Antibodies; Drug Synergism; Drug Therapy, Combination; Erythrocytes; Female; Gastrointestinal Agents; Guanine Nucleotides; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Infliximab; Male; Mercaptopurine; Methyltransferases; Middle Aged; Retrospective Studies; Thionucleotides

Variation in metabolism, toxicity and therapeutic efficacy of thiopurine drugs is largely influenced by genetic polymorphisms in the thiopurine S-methyltransferase (TPMT) gene. Determination of TPMT activity is routinely performed in patients to adjust drug therapy.. We further optimized a previously established high-performance liquid chromatography (HPLC) method by measuring TPMT activity in whole blood instead of isolated erythrocytes, which is based on conversion of 6-mercaptopurine to 6-methylmercaptopurine using S-adenosyl-methionine as methyl donor.. The simplified TPMT whole-blood method showed similar or better analytical and diagnostic performance compared with the former erythrocyte assay. The whole-blood method was linear for TPMT activities between 0 and 40 nmol/(mL·h) with a quantification limit of 0.1 nmol/(mL·h). Within-day imprecision and between-day imprecision were ≤5.1% and ≤8.5%, respectively. The optimized method determining TPMT activity in whole blood (y) showed agreement with the former method determining TPMT activity in erythrocytes (x) (n=45, y=1.218+0.882x; p>0.05). Phenotype-genotype concordance (n=300) of the whole-blood method was better when TPMT activity was expressed per volume of whole blood (specificity 92.2%), whereas correction for hematocrit resulted in lower genotype concordance (specificity 86.9%). A new cutoff for the whole-blood method to distinguish normal from reduced TPMT activity was determined at ≤6.7 nmol/(mL·h).. This optimized TPMT phenotyping assay from whole blood using 6-MP as substrate is suitable for research and routine clinical analysis.

Topics: Chromatography, High Pressure Liquid; Genotype; Healthy Volunteers; Humans; Mercaptopurine; Methyltransferases; Phenotype; Substrate Specificity

Measurement of thiopurine metabolites is helpful to monitor adverse effects and assess compliance in patients on thiopurine treatment. The purpose of this study was to develop and validate an analytical method for measurement of thiopurine metabolites, thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine nucleotide (6-MMPN), in RBCs. We developed and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of 6-TGN and 6-MMPN and evaluated the stability of the thiopurine metabolites in RBC and whole blood states without any preprocessing at various storage conditions. The linear range was 0.1-10 μmol/L and 0.5-100 μmol/L for 6-TGN and 6-MMPN, respectively. The mean extraction recovery at the two concentrations was 71.0% and 75.0% for 6-TGN, and 102.2% and 96.4% for 6-MMPN. Thiopurine metabolites in preprocessed RBC samples were stable at 25°C and 4°C after storage for 4 hours and at -70°C for up to 6 months. However, 6-TGN decreased by 30% compared with the initial concentration when stored at -20°C for 180 days. In whole blood states, 6-TGN decreased by about 20% at four days after storage at 4°C. We validated a reliable LC-MS/MS method and recommend that the patient's whole blood sample be preprocessed as soon as possible.

Topics: Chromatography, High Pressure Liquid; Erythrocytes; Humans; Mercaptopurine; Tandem Mass Spectrometry; Temperature; Thioguanine

Identification of patients with thiopurine S-methyltransferase (TPMT) deficiency prior to thiopurine drug therapy has become routine clinical practice worldwide. To measure TPMT activity, traditional radiochemical assays have been replaced by chromatographic methods.. Inspired by the increasing number of isotope labelled sources that may be of benefit for the TPMT assay, a new LC-MS/MS method for TPMT activity was developed and validated. Isotope labelled d3-S-adenosyl-l-methionine (d3-SAM) was selected for the enzymatic methylation of mercaptopurine during sample incubation; d3-6-methylmercaptopurine (d3-6-MMP) with d2-2, 8-hypoxanthine as the internal standard was quantified to ascertain individual TPMT activity.. The validation of the analytical part of this method showed good linearity (coefficient of determination 0.9999 in the range of 1-500 ng/mL) with the intra-and inter-day impression CV% between 7.6% and 9.1% and 3.7% and 9.2%, respectively. Recovery ranged from 94.9% to 112.3%. The specificity of the enzymatic reaction was validated by using 108 clinical check samples. After compared with traditional radiochemical assay and genotype results, all homozygous and heterozygous deficiency clinical checks fitted into the nominal groups, inter-batch and intra-batch impression CV% were between 2.3% and 9.7%.. With the inclusion of isotope labelled substrate, interfering non-enzymatic methylation no longer results in potential false assignment of abnormal patients. Furthermore, the method can be applied to patients who have already been prescribed thiopurine drugs. This new LC-MS/MS is therefore a favourable clinical routine application to test TPMT activity, as it shows excellent performance in identifying patients with TPMT deficiency.

Topics: Chromatography, Liquid; Humans; Mass Spectrometry; Mercaptopurine; Methyltransferases

The thiopurines azathioprine and mercaptopurine remain pivotal maintenance treatments in inflammatory bowel disease (IBD); however, up to 15%-20% of patients preferentially produce the hepatotoxic metabolite 6-methylmercaptopurine (6MMP) at the expense of the therapeutic 6-thioguanine nucleotides (6TGN). This metabolic shunting usually begins within 3 months of therapy. We noted patients developing shunting many months or years after starting treatment and aimed to determine how often this late shunting occurs and whether this could be explained by patient factors or concomitant medications.. The New Zealand database of thiopurine metabolite results from 2002 to 2016 (19085 6TGN/6MMP pairs from 7130 patients) was interrogated to identify patients developing a 6MMP/6TGN ratio >20 after at least 4 months treatment. Dosing history, concomitant therapy, and comorbidity data were assessed.. Fifteen percent of database patients developed preferential 6-MMP production, and of these, 29 patients had late-onset shunting with sufficient data available for validation. This extrapolates to 90 patients in total, representing 1.7% of IBD patients on thiopurines, or 10% of all those with preferential 6-MMP production. Time from starting therapy to shunting was 5 months to 10.4 years (median, 21 months). Eleven patients had abnormal liver function when shunting was recognized, all with 6MMP >5900 pmol/8 × 108 red blood cells. No common factors were found to explain the late onset.. Some IBD patients develop preferential 6MMP production many months or years after commencing therapy. This is important when considering frequency of metabolite monitoring, failure of therapy, or abnormal liver function. 10.1093/ibd/izx081_video1izx081.video15746667546001.

Topics: Adult; Aged; Aged, 80 and over; Azathioprine; Female; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Middle Aged; New Zealand; Thioguanine; Young Adult

Thiopurines are commonly used in the maintenance of remission for children with inflammatory bowel diseases (IBDs). Variation in drug metabolism may affect hepatotoxicity or therapeutic effect. We aimed to describe our center's experience with thiopurine optimization through the use of reduced thiopurine dosing in combination with allopurinol upon hepatotoxicity, drug metabolite levels, and clinical outcomes in children with IBD.. Patients aged 2 to 21 years with IBD treated with the combination of thiopurines/allopurinol between 2008 and 2015 were retrospectively reviewed. Patients previously treated with antitumor necrosis factor therapy were excluded. Demographic data, transaminase levels (aspartate transaminase, alanine transaminase), drug metabolites levels (6-thioguanine [6-TG], 6-methylmercaptopurine), physician global assessment, and corticosteroid use were recorded at baseline, 6, and 12 months.. Fifty-two patients (29 girls, 56%) met inclusion criteria. Thirty-two of 52 (62%) remained on the combination for 12 months. In those remaining on the thiopurine/allopurinol combination, median alanine transaminase and aspartate transaminase levels were reduced (P < 0.001) and median 6-TG levels were increased (P < 0.001) at both 6 and 12 months. Corticosteroid use was decreased at both 6 (P < 0.001) and 12 months (P < 0.001) compared to use at baseline. Remission rates also improved at both 6 (P = 0.013) and 12 months (P = 0.003). Twenty of the 52 patients (38%) had discontinued the thiopurine/allopurinol combination within 12 months of initiation with 17 of 52 (33%) initiating antitumor necrosis factor therapy.. Low-dose thiopurines in combination with allopurinol improved hepatotoxicity and increased 6-TG levels in children with IBD. Corticosteroid use was reduced and remission rates improved in those patients remaining on this combination for 1 year. However, approximately 40% of patients required a change in therapy within 12 months.

Topics: Adolescent; Adult; Allopurinol; Azathioprine; Child; Child, Preschool; Dose-Response Relationship, Drug; Drug Therapy, Combination; Female; Follow-Up Studies; Free Radical Scavengers; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Retrospective Studies; Treatment Outcome; Young Adult

Up to 20% of patients with inflammatory bowel disease (IBD) who are refractory to thiopurine therapy preferentially produce 6-methylmercaptopurine (6-MMP) at the expense of 6-thioguanine nucleotides (6-TGN), resulting in a high 6-MMP:6-TGN ratio (>20). The objective of this study was to evaluate whether genetic variability in guanine monophosphate synthetase (GMPS) contributes to preferential 6-MMP metabolizer phenotype.. Exome sequencing was performed in a cohort of IBD patients with 6-MMP:6-TGN ratios of >100 to identify nonsynonymous single nucleotide polymorphisms (nsSNPs). In vitro assays were performed to measure GMPS activity associated with these nsSNPs. Frequency of the nsSNPs was measured in a cohort of 530 Caucasian IBD patients.. Two nsSNPs in GMPS (rs747629729, rs61750370) were detected in 11 patients with very high 6-MMP:6-TGN ratios. The 2 nsSNPs were predicted to be damaging by in silico analysis. In vitro assays demonstrated that both nsSNPs resulted in a significant reduction in GMPS activity (P < 0.05). The SNP rs61750370 was significantly associated with 6-MMP:6-TGN ratios ≥100 (odds ratio, 5.64; 95% confidence interval, 1.01-25.12; P < 0.031) in a subset of 264 Caucasian IBD patients.. The GMPS SNP rs61750370 may be a reliable risk factor for extreme 6MMP preferential metabolism.

Topics: Adult; Azathioprine; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor; Cohort Studies; Female; Guanine Nucleotides; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Middle Aged; Polymorphism, Single Nucleotide; Risk Factors; Thionucleotides; Young Adult

Thiopurine therapy can be optimised by determining the concentration of the drug's metabolites.. Retrospective analysis on a prospective database of 31 patients with inflammatory bowel disease who failed therapy with thiopurines. Thiopurine metabolites (6-thioguanine, 6-TGN and 6-methylmercaptopurine, 6-MMP) were measured by high-performance liquid chromatography (Laboratorios Cerba, Barcelona) and treatment was duly adjusted in accordance with the results. Clinical response was reassessed after six months.. Despite the appropriate theoretical dose of thiopurines being administered, the dose was insufficient in 45.6% of patients (nonadherence to treatment suspected in 6.45%) and 16.2% received an excessive dose or the drug was metabolised by other metabolic pathways. After treatment was optimised based on metabolite levels, only 25.8% (8/31) were prescribed a biological agent, while 74.2% of cases (23/31) were managed through dose optimisation alone.. Monitoring thiopurine metabolite levels may help clinicians to assess non-responsive patients before adding or switching to another drug (generally a biological agent), thereby avoiding any additional costs or potential toxicity. This strategy may also help to identify patients receiving an insufficient dose and those with an alternative metabolic pathway, who could be candidates for low-dose AZA with allopurinol, as well as patients who are suspected of being non-adherent. In three out of four patients, switching to a biological agent can be avoided.

Topics: Adult; Aged; Azathioprine; Biotransformation; Dose-Response Relationship, Drug; Drug Monitoring; Female; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Middle Aged; Retrospective Studies; Thioguanine; Young Adult

Azathioprine (AZA) is the mainstay of maintenance therapy in pediatric autoimmune hepatitis (AIH). The use of thiopurines metabolites to individualize therapy and avoid toxicity has not, however, been clearly defined.. Retrospective analysis of children ≤18 years diagnosed with AIH between January 2001 and 2016. Standard definitions were used for treatment response and disease flare. Thiopurine metabolite levels were correlated with the corresponding liver function test.. A total of 56 children (32 girls) were diagnosed with AIH at a median age of 11 years (interquartile range [IQR] 9). No difference in 6-thioguanine-nucleotide (6-TG) levels (271[IQR 251] pmol/8 × 10 red blood cell vs 224 [IQR 147] pmol/8 × 10 red blood cell, P = 0.06) was observed in children in remission when compared with those who were not in remission. No correlation was observed between the 6-TG and alanine aminotransferase levels (r = -0.179, P = 0.109) or between 6-methyl-mercaptopurine (6-MMP) and alanine aminotransferase levels (r = 0.139, P = 0.213). The 6-MMP/6-TG ratio was significantly lower in patients who were in remission (2[7] vs 5 (10), P = 0.04). Using a quartile analysis, we found that having a ratio of <4 was significantly associated with being in remission with OR 2.50 (95% confidence interval 1.02-6.10), P = 0.047. Use of allopurinol with low-dose AZA in 6 children with preferential 6-MMP production brought about remission in 5/6 (83.3%).. Thiopurine metabolite levels should be measured in patients with AIH who have experienced a loss of remission. A 6-MMP/6-TG ratio of <4 with the addition of allopurinol could be considered in these patients.

Topics: Allopurinol; Antimetabolites; Azathioprine; Child; Female; Guanine Nucleotides; Hepatitis, Autoimmune; Humans; Liver Function Tests; Maintenance Chemotherapy; Male; Mercaptopurine; Retrospective Studies; Thionucleotides; Treatment Outcome

Big data analytics leverage patterns in data to harvest valuable information, but are rarely implemented in clinical care. Optimising thiopurine therapy for inflammatory bowel disease [IBD] has proved difficult. Current methods using 6-thioguanine nucleotide [6-TGN] metabolites have failed in randomized controlled trials [RCTs], and have not been used to predict objective remission [OR]. Our aims were to: 1) develop machine learning algorithms [MLA] using laboratory values and age to identify patients in objective remission on thiopurines; and 2) determine whether achieving algorithm-predicted objective remission resulted in fewer clinical events per year.. Objective remission was defined as the absence of objective evidence of intestinal inflammation. MLAs were developed to predict three outcomes: objective remission, non-adherence, and preferential shunting to 6-methylmercaptopurine [6-MMP]. The performance of the algorithms was evaluated using the area under the receiver operating characteristic curve [AuROC]. Clinical event rates of new steroid prescriptions, hospitalisations, and abdominal surgeries were measured.. Retrospective review was performed on medical records of 1080 IBD patients on thiopurines. The AuROC for algorithm-predicted remission in the validation set was 0.79 vs 0.49 for 6-TGN. The mean number of clinical events per year in patients with sustained algorithm-predicted remission [APR] was 1.08 vs 3.95 in those that did not have sustained APR [p < 1 x 10-5]. Reductions in the individual endpoints of steroid prescriptions/year [-1.63, p < 1 x 10-5], hospitalisations/year [-1.05, p < 1 x 10-5], and surgeries/year [-0.19, p = 0.065] were seen with algorithm-predicted remission.. A machine learning algorithm was able to identify IBD patients on thiopurines with algorithm-predicted objective remission, a state associated with significant clinical benefits, including decreased steroid prescriptions, hospitalisations, and surgeries.

Topics: Adolescent; Adult; Algorithms; Area Under Curve; Azathioprine; Drug Prescriptions; Female; Hospitalization; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Machine Learning; Male; Medication Adherence; Mercaptopurine; Middle Aged; Remission Induction; Retrospective Studies; ROC Curve; Treatment Outcome; Young Adult

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of thiopurine methyltransferase (TPMT) activity in human whole blood lysate, based on conversion of 6-mercaptopurine (6-MP) by TPMT to 6-methylmercaptopurine (6-MMP) using S-adenosyl-l-methionine (SAM) as the methyl donor. This method was improved from the previous laborious method for washing of red cell lysate preparation to develop whole blood EDTA lysate. In addition, the TPMT incubation was optimized and the chromatography was performed in a short runtime of 7min on a C18-column by detection via triple quadrupole mass spectrometry. The MS/MS was optimally tuned to monitor mass to charge a ratio (m/z) for 6-MMP 167.2→151.9 and the isotope 6-MMP-d3 with m/z of 170.5→152.2 were applied as an internal standard. The calibration curve covered the range of 2.5-360ng/ml and the correlation coefficient was greater than 0.999. The accuracy of this method was determined in four concentrations of control of quality that ranged between 99.33 and 106.33%. The intra-assay coefficient of variation (CV) was less than 4.41% and the inter-assay was less than 5.43%. This method developed for measuring TPMT by LC-MS/MS is a reliable, safe, and simple with a small volume requirement (100μl of whole blood EDTA). The assay was used to study TPMT activity in 132 Thai children with a range from 29.0 to 89.1nmol 6-MMP/g Hb/h with means and median values of TPMT activity 55.9±12.47nmol 6-MMP/g Hb/h and 54.2nmol 6-MMP/g Hb/h. The genotype-phenotype association of TPMT was evaluated for common ethnic Thai single nucleotide polymorphisms (SNP) in 30 samples and demonstrated good concordance.

Topics: Chromatography, Liquid; Genotype; Humans; Mercaptopurine; Methyltransferases; Phenotype; Tandem Mass Spectrometry

Early-onset inflammatory bowel disease (IBD) is generally aggressive, with a high probability of complications and need of surgery. Despite the introduction of highly effective biological drugs, treatment with azathioprine continues to be important even for early-onset IBD; however, in these patients azathioprine response seems to be reduced. This study evaluated azathioprine doses, metabolite concentrations, and their associations with patients' age in children with IBD treated at 6 tertiary pediatric referral centers.. Azathioprine doses, metabolites, and clinical effects were assessed after at least 3 months of therapy in 17 early-onset (age < 6 yr, cases) and 51 nonearly-onset (aged > 12 and <18 yrs, controls) patients with IBD. Azathioprine dose was titrated on therapeutic efficacy (response and adverse effects). Azathioprine metabolites and thiopurine methyltransferase activity were determined by high-performance liquid chromatography with ultra violet-vis detection (HPLC-UV) methods.. Frequency of patients in remission was similar among early-onset and control groups, respectively (82% and 84%, P value = 0.72). Early-onset patients required higher doses of azathioprine (median 2.7 versus 2.0 mg·kg·d, P value = 1.1 × 10). Different doses resulted in comparable azathioprine active thioguanine nucleotide metabolite concentrations (median 263 versus 366 pmol/8 × 10 erythrocytes, P value = 0.41) and methylmercaptopurine nucleotide concentrations (median 1455 versus 1532 pmol/8 × 10 erythrocytes, P value = 0.60). Lower ratios between thioguanine nucleotide metabolites and azathioprine doses were found in early-onset patients (median 98 versus 184 pmol/8 × 10 erythrocytes·mg·kg·d, P value = 0.017). Interestingly, early-onset patients presented also higher thiopurine methyltransferase activity (median 476 versus 350 nmol methylmercaptopurine/mg hemoglobin/h, P-value = 0.046).. This study demonstrated that patients with early-onset IBD present increased inactivating azathioprine metabolism, likely because of elevated activity of the enzyme thiopurine methyltransferase.

Topics: Adolescent; Age of Onset; Antimetabolites; Azathioprine; Case-Control Studies; Child; Child, Preschool; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Erythrocytes; Female; Guanine Nucleotides; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Thioguanine

6-Mercaptopurine (6-MP) and its prodrug azathioprine (AZA) are used in many autoimmune diseases and after solid-organ transplantation. Their properties are mediated by active metabolites, 6-thioguanine nucleotides (6-TGN), and 6-methylmercaptopurine (6-MMP). The most common adverse effects are myelo- and hepato-toxicity. The aim of the study was quantification of 6-TG and 6-MMP, with the use of liquid chromatography combined with tandem mass spectrometry (LC/MS/MS) in solid-organ transplant recipients.. In 33 patients, kidney transplant recipient (n = 25) and liver transplant recipient (n = 8) intra-erythrocyte concentrations of 6-TG and 6-MMP were measured with the use of LC/MS/MS.. The mean concentration of 6-TG was 205.35 ± 157.62 pmol/8 × 10(8) red blood cells (RBC); median concentration of 6-MMP was 1064.1 (35.78-11,552.9) pmol/8 × 10(8) RBC. There were no correlations between 6-TG levels and peripheral blood parameters (white blood cell count, WBC; hemoglobin, Hb concentration; PLT, blood platelet count) or alanine aminotransferase activity (AlAT) activity. Relationships between 6-MMP concentrations and peripheral blood parameters (WBC, Hb, PLT) or AlAT activity have not been found. Subgroups with leukopenia, anemia, thrombocytopenia, and liver dysfunction did not differ in concentrations of 6-TG or 6-MMP. We have observed a negative correlation between daily azathioprine dose and WBC count (r = -0.37, P = .04).. Relationships between concentrations of azathioprine metabolites and myelotoxicity or hepatotoxicity have not been confirmed. Further studies on larger groups of patients would be helpful in a more accurate understanding of the impact of azathioprine metabolites on parameters of bone marrow and liver function.

Topics: Adult; Azathioprine; Chromatography, Liquid; Erythrocytes; Female; Guanine Nucleotides; Humans; Immunosuppressive Agents; Leukocyte Count; Male; Mercaptopurine; Organ Transplantation; Tandem Mass Spectrometry; Thioguanine; Thionucleotides

Clinical efficacy and risk of complications are associated with intracellular levels of thiopurine metabolites 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurines (6-MMP) in patients with Crohn's disease. Therapeutic monitoring of thiopurine metabolites is not widely available. Surrogate markers such as hematologic indices (MCV, leukopenia) have been proposed.. To evaluate accuracy of hematologic indices for prediction of therapeutic levels of thiopurine metabolites.. A retrospective cross-sectional study. We included patients treated with thiopurines for IBD between February 2008 and November 2013. Hematologic indices were correlated with thiopurine metabolites and compared to pre-treatment levels.. A total of 168 patients with 608 measurements were included. Macrocytosis was observed in 4.5 % of the patients. On multivariate analysis, macrocytosis was associated with 6-TGN levels >235 pmol/8 × 10(8) erythrocytes and 6-mmp levels >5,700 pmol/8 × 10(8) erythrocytes. Therapeutic 6-TGN levels were associated with MCV, ΔMCV, macrocytosis and lymphocyte count. Sensitivity and Spearman's r correlation for prediction of therapeutic metabolite levels were poor for all hematologic indices.. Although macrocytosis and an elevated MCV are associated with therapeutic 6-TGN levels, the correlation is weak. None of the evaluated hematologic indices is a reliable surrogate marker for thiopurine metabolite levels.

Topics: Anti-Inflammatory Agents; Azathioprine; Biomarkers; Colitis, Ulcerative; Crohn Disease; Cross-Sectional Studies; Drug Monitoring; Erythrocyte Indices; Female; Guanine Nucleotides; Hematologic Tests; Hemoglobins; Humans; Lymphocyte Count; Male; Mercaptopurine; Platelet Count; Predictive Value of Tests; Retrospective Studies; Thionucleotides; Treatment Outcome

Preferential conversion of azathioprine or 6-mercaptopurine into methylated metabolites is a major cause of thiopurine resistance. To seek potentially Mendelian causes of thiopurine hypermethylation, we recruited 12 individuals who exhibited extreme therapeutic resistance while taking azathioprine or 6-mercaptopurine and performed whole-exome sequencing (WES) and copy-number variant analysis by array-based comparative genomic hybridisation (aCGH). Exome-wide variant filtering highlighted four genes potentially associated with thiopurine metabolism (ENOSF1 and NFS1), transport (SLC17A4) or therapeutic action (RCC2). However, variants of each gene were found only in two or three patients, and it is unclear whether these genes could influence thiopurine hypermethylation. Analysis by aCGH did not identify any unusual or pathogenic copy-number variants. This suggests that if causative mutations for the hypermethylation phenotype exist they may be heterogeneous, occurring in several different genes, or they may lie within regulatory regions not captured by WES. Alternatively, hypermethylation may arise from the involvement of multiple genes with small effects. To test this hypothesis would require recruitment of large patient samples and application of genome-wide association studies.

Topics: Adult; Azathioprine; Colitis, Ulcerative; Comparative Genomic Hybridization; Crohn Disease; DNA Copy Number Variations; Drug Resistance; Exome; Female; Genome-Wide Association Study; Hepatitis, Autoimmune; High-Throughput Nucleotide Sequencing; Humans; Male; Mercaptopurine; Metabolic Networks and Pathways; Methyltransferases; Middle Aged; Mutation

Mercaptopurine (6-MP), a critical component of acute lymphoblastic leukemia (ALL) therapy, is metabolized to 6-thioguanine (6-TGN) which is responsible for its anti-leukemic effect, and to 6-methylmercaptopurine nucleotides (6-MMPN/6-MMP) which can be hepatotoxic. Some patients preferentially metabolize 6-MP to 6-MMPN which may increase the risk of liver injury, reduce serum levels of 6-TGN and potentially increase the risk of relapse. The addition of allopurinol to oral 6-MP has been shown to optimize metabolism towards 6-TGN in patients with inflammatory bowel disease (IBD); however, this use has not been reported in patients undergoing treatment for ALL.

Topics: Adolescent; Allopurinol; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Biotransformation; Chemical and Drug Induced Liver Injury; Child, Preschool; Drug Evaluation; Female; Guanine Nucleotides; Humans; Hyperbilirubinemia; Hypoxanthine Phosphoribosyltransferase; Maintenance Chemotherapy; Male; Mercaptopurine; Methotrexate; Methyltransferases; Neutropenia; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Thionucleotides; Xanthine Oxidase

Symptomatic fasting hypoglycemia has been reported as an unusual side effect in patients with acute lymphoblastic leukemia (ALL) on maintenance therapy. We evaluated the relation of the red cell 6-mercaptopurine (6-MP) metabolite 6-methyl-mercaptopurine (6MMP) with hypoglycemia.. We retrospectively reviewed charts of three patients with ALL and symptomatic hypoglycemia while fasting who were noted to have high levels of 6MMP. All patients had an empiric trial of switching from evening to morning 6-MP administration, and two patients were subsequently switched to twice daily dosing. Patients also received complex carbohydrates at bedtime.. Switching 6-MP from evening to morning administration reduced 6MMP levels yet preserved adequate levels of the active metabolite red cell 6-thioguanine nucleotide (6TGN). All patients had decreased hypoglycemic events when changed from evening to morning dosing. Two patients showed a rebound in 6MMP levels with return of hypoglycemic symptoms. Both were then switched to twice daily 6-MP dosing with one having a decrease in 6MMP and hypoglycemic symptoms.. High levels of 6MMP are associated with symptomatic hypoglycemia which may be mitigated by switching to morning or twice daily 6-MP dose administration.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Biotransformation; Blood Glucose; Child; Child, Preschool; Dexamethasone; Dietary Carbohydrates; Drug Administration Schedule; Fasting; Female; Guanine Nucleotides; Humans; Hyperglycemia; Hypoglycemia; Hypoxanthine Phosphoribosyltransferase; Infant; Liver Glycogen; Maintenance Chemotherapy; Male; Mercaptopurine; Methotrexate; Methylation; Methyltransferases; Polymorphism, Genetic; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma; Prodrugs; Retrospective Studies; Thionucleotides

To report the use of febuxostat in order to potentiate thiopurines' metabolism in a patient on azathioprine (AZA) therapy with low metabolite 6-thioguanine nucleotides (6-TGN) levels and elevated metabolite 6-methylmercaptopurine (6-MMP) levels.. A 44-year-old woman with a history of anti-signal recognition particle necrotizing myopathy was treated with AZA-allopurinol combination therapy. When she developed an atypical drug-induced hypersensitivity syndrome, allopurinol was replaced by the new xanthine oxidase (XO) inhibitor febuxostat, at a daily dose of 40 mg. Febuxostat-AZA combination was successful with 6-TGN reaching therapeutic levels while 6-MMP levels remained low. After 5 months, she developed similar manifestations that she had presented on AZA-allopurinol combination. Febuxostat and AZA were then stopped.. AZA and 6-MP are both inactive pro-drugs that undergo a complex metabolic transformation leading to active 6-TGN and potentially hepatotoxic 6-MMP. Some patients with unfavorable thiopurine metabolism might benefit from addition of XO inhibitor allopurinol in order to potentiate 6-TGN and reduce 6-MMP levels. It is likely that febuxostat, via its XO inhibition, would exhibit the same effect on thiopurines' metabolism.. It has been shown that low dose of febuxostat was able to prevent hypermethylation and to potentiate 6-TGN levels in an AZA-treated patient. Thus, febuxostat could be useful in optimizing thiopurines' metabolism, but more data are needed before this practice can be recommended. The mechanisms by which febuxostat optimizes thiopurines' metabolism remain to be confirmed. Also, the optimal dose of febuxostat for this use remains to be determined.

Topics: Adult; Azathioprine; Drug Interactions; Enzyme Inhibitors; Febuxostat; Female; Guanine Nucleotides; Humans; Immunosuppressive Agents; Mercaptopurine; Muscular Diseases; Thiazoles; Thionucleotides; Xanthine Oxidase

Thiopurines are used as a maintenance therapy in patients with ulcerative colitis (UC). For some patients the metabolism of thiopurines is unfavorable, leading to increased adverse effects, including hepatotoxicity. There are many reports in the adult literature concerning the manipulation of thiopurine metabolism with allopurinol; however, there is only 1 publication in this respect for pediatric UC. We present 3 pediatric cases of UC wherein the combination of allopurinol and low-dose 6-mercaptopurine allowed for shunting of thiopurine metabolites to a more favorable pattern. This intervention supported clinical remission in all, including one case poorly responsive to infliximab.

Topics: Adolescent; Allopurinol; Antimetabolites; Child; Colitis, Ulcerative; Drug Therapy, Combination; Female; Guanine Nucleotides; Humans; Immunosuppressive Agents; Male; Mercaptopurine; Thionucleotides

Thiopurines (azathioprine, 6-mercaptopurine) are a mainstay of treatment in Crohn disease (CD). Monitoring intracellular metabolite (6-thioguanine nucleotides [6-TGN] and 6-methylmercaptopurine [6-MMP]) levels can help optimize therapeutic efficacy and minimize potential toxicity. Determination of 6-MMP/6-TGN ratios may provide additional useful information, such as the identification of individuals with excessive thiopurine methyltransferase activity and disadvantageous 6-MMP overproduction. These patients are at increased risk of therapeutic failure and hepatotoxicity. The aim of the study was to evaluate the correlation of 6-MMP/6-TGN ratios with therapeutic efficacy and risk of hepatotoxicity in CD.. The present study was a single-center cross-sectional study including pediatric patients with CD studied prospectively with clinical and laboratory assessments along with serial measurements of 6-MMP and 6-TGN. Clinical response was determined using established clinical indices.. The study included 238 pediatric patients with CD with a total of 1648 evaluation points. The patients were in steroid-free remission at 59.1% of the evaluation points. 6-MMP/6-TGN ratios of 4 to 24 were protective against relapse (odds ratio [OR] 0.52, 95% confidence interval [CI] -0.39 to 0.69, P = 0.001). Hepatotoxicity was associated with high 6-MMP levels (>3919  pmol/8 × 10 red blood cell count: OR 7.65, 95% CI 3.7-15.9, P = 0.001) and high 6-MMP/6-TGN ratios (>24: OR 5.35, 95% CI -3.43 to 8.43, P = 0.001).. We observed significant associations between 6-MMP/6-TGN ratios and clinical response, and risk of hepatotoxicity. Our results suggest that determination of thiopurine metabolite ratios is a valuable tool for identification of patients at increased risk of therapeutic failure and hepatotoxicity.

Topics: Adolescent; Azathioprine; Chemical and Drug Induced Liver Injury; Child; Crohn Disease; Female; Guanine Nucleotides; Humans; Immunosuppressive Agents; Male; Mercaptopurine; Methyltransferases; Odds Ratio; Thionucleotides; Treatment Failure

Topics: Adult; Anti-Inflammatory Agents, Non-Steroidal; Colitis, Ulcerative; Drug Interactions; Erythrocytes; Feces; Female; Guanine Nucleotides; Humans; Immunosuppressive Agents; Leukocyte L1 Antigen Complex; Mercaptopurine; Mesalamine

Previous studies have reported no or only a very poor correlation between 6-methylmercaptopurine/6-thioguanine nucleotide (6-MeMPN/6-TGN) and azathioprine (AZA) dose in the treatment of inflammatory bowel disease (IBD). However, metabolite levels are often repeatedly measured yielding a hierarchical data structure that requires more appropriate data analysis.. This study explored the relationship between the weight-based dosage of AZA and metabolites levels in 86 pediatric IBD patients using multilevel analysis. Other covariates related to patient characteristics and treatment were evaluated.. This is the first study to demonstrate positive correlations between AZA dose and 6-TGN and 6-MeMPN levels and 6-MeMPN/6-TGN ratio (P < 0.001) in IBD children. Other novel predictors of metabolites were reported. Younger children exhibited lower 6-TGN and 6-MeMPN levels, probably suggesting age-related differences in metabolism and/or absorption of thiopurines. Coadministration of infliximab resulted in a significant increase in 6-TGN levels (P = 0.023). Moreover, alanine aminotransferase values positively correlated with 6-MeMPN levels (P = 0.032). The duration of AZA therapy, gender, and thiopurine methyltransferase activity were associated with metabolite levels. The wide interindividual variability in metabolite levels that accounted for 67.7%, 48.6%, and 49.4% of variance in the 6-TGN and 6-MeMPN levels and the ratio, respectively, were confirmed.. The reliable AZA dose-metabolites relationship is useful for clinicians to guide the dosing regimen to maximize clinical response and minimize side effects or to consider alternative therapies when patients have preferential production of the toxic 6-MeMPN. These results may be of potential interest for optimizing thiopurine therapy to achieve safe and efficacious AZA use in pediatric IBD patients.

Topics: Adolescent; Age Factors; Azathioprine; Child; Dose-Response Relationship, Drug; Female; Guanine Nucleotides; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Multilevel Analysis; Retrospective Studies; Thionucleotides

Thiopurine drugs (azathioprine, 6-mercaptopurine) show wide interindividual variability and a narrow therapeutic range thus making therapeutic monitoring of their active metabolite 6-thioguanine nucleotides (6-TGN) desirable. We improved the currently available laborious and complex methodology of therapeutic drug monitoring of 6-TGN and the metabolite 6-methylmercaptopurine (6-MMP) in washed erythrocytes (ery) based on a whole-blood method.. The analytes were hydrolyzed and extracted from 25-µL ethylenediaminetetraacetic acid-anticoagulated whole-blood spiked with isotope labeled 6-TG-C2N and 6-MMP-d3 internal standards. Chromatography was performed in 5.1 minutes on a C18 reverse phase column followed by detection via electrospray interface-coupled API 4000 mass spectrometer set up in the positive multiple reaction monitoring mode. The hemoglobin concentration was measured in 20 µL of the original sample (AHD575 method), and the results were standardized to 120 g/L of hemoglobin.. Calibration curves were linear with r > 0.999 (6-TGN and 6-MMP up to 10,000 pmol/0.2 mL). The limit of quantification was 30 pmol/0.2 mL for 6-TGN and 6-MMP. Intraassay and interassay imprecision was <7.5% at 3 tested levels for 6-TGN and 6-MMP, respectively. Method comparisons were as follows: Ery 6-TGN: y = 1.3x - 11 and ery 6-MMP y = 1.1x - 124.. The new method compares favorably with established ones, allowing for rapid single run determination of 6-TGN and 6-MMP from <50 µL of fresh or frozen whole blood. Linearity and limits of quantification cover the clinically relevant range. Variability during sample preparation and matrix effects are compensated by the use of isotope-labeled internal standards. The whole-blood method is hemoglobin standardized to avoid falsely low results in the case of anemia. The method correlates well with 6-TGN measured in washed erythrocytes, but it requires significantly less hands-on time. Preliminary therapeutic ranges for the most common indications of azathioprine and 6-MP are provided.

Topics: Calibration; Chromatography, High Pressure Liquid; Drug Monitoring; Erythrocytes; Guanine Nucleotides; Humans; Limit of Detection; Mercaptopurine; Tandem Mass Spectrometry; Thionucleotides; Time Factors

Up to 1/5 of patients with wildtype thiopurine-S-methyltransferase (TPMT) activity prescribed azathioprine (AZA) or mercaptopurine (MP) demonstrate a skewed drug metabolism in which MP is preferentially methylated to yield methylmercaptopurine (MeMP). This is known as thiopurine hypermethylation and is associated with drug toxicity and treatment non-response. Co-prescription of allopurinol with low dose AZA/MP (25-33%) circumvents this phenotype and leads to a dramatic reduction in methylated metabolites; however, the biochemical mechanism remains unclear. Using intact and lysate red cell models we propose a novel pathway of allopurinol mediated TPMT inhibition, through the production of thioxanthine (TX, 2-hydroxymercaptopurine). In red blood cells pre-incubated with 250 μM MP for 2h prior to the addition of 250 μM TX or an equivalent volume of Earle's balanced salt solution, there was a significant reduction in the concentration of MeMP detected at 4h and 6h in cells exposed to TX (4 h, 1.68, p=0.0005, t-test). TX acts as a direct TPMT inhibitor with an apparent Ki of 0.329 mM. In addition we have confirmed that the mechanism is relevant to in vivo metabolism by demonstrating raised urinary TX levels in patients receiving combination therapy. We conclude that the formation of TX in patients receiving combination therapy with AZA/MP and allopurinol, likely explains the significant reduction of methylated metabolites due to direct TPMT inhibition.

Topics: Adult; Allopurinol; Azathioprine; Case-Control Studies; Drug Therapy, Combination; Erythrocytes; Female; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Oxypurinol; Prospective Studies; Xanthines

Few data on azathioprine (AZA) therapy for inflammatory bowel disease (IBD) exist for children. We evaluated whether the 6-thioguanine nucleotides (6-TGN) level predicts AZA refractoriness in children with IBD and whether children benefit an AZA dose escalation. Seventy-eight children with IBD initially treated with an AZA dose of 1.5-2.5 mg/kg/day were retrospectively included. The dose was adjusted based on the clinical status. The receiver operating characteristic curve and logistic regression were used to determine predictors for AZA resistance. Initially, 18 of 40 (45%) patients receiving a dose of <2 mg/kg/day and 11 of 38 (28.9%) patients receiving a dose of 2-2.5 mg/kg/day achieved remission. The 6-TGN level above 250 pmol/8.10(8) RBCs was associated with a higher remission rate, though non-significant. Among 35 patients with a dose escalation due to treatment failure, 12 (34.3%) achieved remission (the median 6-TGN level increased from 260 to 394 pmol/8.10(8) RBCs [P = .002]), 23 (67.6%) were AZA refractory. A 6-TGN level above 405 pmol/8.10(8) RBCs was the only predictor for AZA resistance (sensitivity 78.3%, specificity 75%, OR 10.8 [95% CI: 2.1-55.7, P = .004]). Serial metabolite monitoring is useful to identify children with IBD resistant to AZA. Children who cannot achieve remission despite a 6-TGN level above 405 pmol/8.10(8) RBCs should receive alternative therapies than dose increase.

Topics: Adolescent; Antimetabolites; Azathioprine; Child; Child, Preschool; Drug Resistance; Female; Humans; Inflammatory Bowel Diseases; Leukopenia; Male; Mercaptopurine; Thioguanine

Azathioprine is prescribed as a corticosteroid-sparing agent for many inflammatory conditions, including refractory atopic dermatitis (AD). There are limited prospective data on its appropriate use and monitoring for children with AD.. This study was designed to assess clinical response to azathioprine, determine the necessity for repeated measurement of thiopurine methyltransferase (TPMT) activity during treatment, and test the utility of measuring levels of the metabolites 6-thioguanine nucleotide and 6-methylmercaptopurine.. Twelve children with severe, recalcitrant AD were treated with oral azathioprine and followed prospectively. Disease severity was determined by the SCORing AD index. Baseline TPMT activity was measured and this was repeated along with 6-thioguanine nucleotide and 6-methylmercaptopurine measurement at times of stable improvement, inadequate response, or change in response.. Azathioprine therapy was associated with clinical improvement in all but 1 patient. There were few adverse effects. Three patients showed a significant change in TPMT activity during treatment: 2 had a mild decrease and 1 demonstrated enzyme inducibility with an increase from the intermediate to the normal activity range. These changes, but not 6-thioguanine nucleotide or 6-methylmercaptopurine levels, inversely correlated with the clinical response to therapy.. Small sample size is a limitation.. Azathioprine can be of benefit in the treatment of recalcitrant pediatric AD. Repeat assessment of TPMT activity may be helpful for evaluation of nonresponse or change in response and warrants further study. In contrast, measurement of thiopurine metabolites during treatment was not clinically useful.

Topics: Administration, Oral; Adolescent; Azathioprine; Child; Child, Preschool; Dermatitis, Atopic; Female; Guanine Nucleotides; Humans; Immunosuppressive Agents; Male; Mercaptopurine; Methyltransferases; Prospective Studies; Severity of Illness Index; Thionucleotides

The thiopurines azathioprine and 6-mercaptopurine are recommended for maintenance of remission in inflammatory bowel disease (IBD). Measurement of concentrations of the metabolites 6-thioguanine nucleotide and 6-methylmercaptopurine helps delineate interindividual variation in metabolism that may underlie variability in efficacy and toxicity.. We aimed to perform a retrospective observational study to determine the utility of thiopurine metabolite testing following its introduction into South Australia.. All patients having thiopurine metabolite tests done at Flinders Medical Centre between November 2008 and January 2010 were identified. Case notes of patients with testing done in the context of treatment for IBD were interrogated to determine the reason for testing, clinical context and outcome.. One hundred and fifty-one patients were identified with thiopurine metabolite testing for IBD with 157 testing episodes. Eighty (51.0%) had testing done for flare or inefficacy, 18 (11.5%) for adverse effects, 5 (3.2%) for a combination of inefficacy and adverse effects, and 54 (34.4%) for routine or other reasons. Testing was followed by improved outcomes of increased efficacy, reduced toxicity or change to alternative therapy in 55.0% of the inefficacy/flare group, 27.8% of the suspected adverse reaction group, 60.0% of the combination group, and 13.0% of the routine/other group. Allopurinol was used as cotherapy in 16 patients and led to marked improvements in metabolite concentrations.. Thiopurine metabolite testing has quickly become established in South Australia. When used for inefficacy or adverse effects, it often leads to improved outcomes. Prospective studies are needed to determine whether routine testing to guide dosing is of benefit.

Topics: Adult; Biomarkers; Disease Management; Female; Guanine Nucleotides; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Middle Aged; Retrospective Studies; Thionucleotides

Ten percent of patients with autoimmune hepatitis (AIH) are nonresponsive or intolerant to thiopurine therapy. A skewed metabolism, leading to the preferential generation of (hepato)toxic thiopurine metabolites (6-MMPs) instead of the metabolic active 6-tioguanine (thioguanine) nucleotides (6-TGNs), may explain this unfavourable outcome. Co-administration of allopurinol to low-dose thiopurine therapy may effectively revert this deviant metabolism, as has been shown in inflammatory bowel disease.. To describe the effect of adding allopurinol to low-dose thiopurine therapy in patients with AIH with intolerance or nonresponse to normal thiopurine dosages due to a skewed metabolism.. We describe the clinical efficacy and tolerability of allopurinol-thiopurine combination therapy with allopurinol 100 mg and low-dose thiopurine (25-33% of the original dosage) in eight AIH patients with a skewed thiopurine metabolism. Patients were switched because of dose-limiting intolerance (n = 3), nonresponse (n = 3) or loss of response (n = 2) to conventional thiopurine treatment.. All eight patients showed biochemical improvement with a reduction in median alanine aminotransferase (ALT) levels of 62 U/L at start to 35 U/L at 1 month (P = 0.03). This clinical benefit was sustained in seven patients. Allopurinol-thiopurine combination therapy effectively bypassed thiopurine side effects in four of five patients. Median 6-tioguanine nucleotides levels increased from 100 to 200 pmol/8 × 10(8) red blood cells (RBC) at 3 months (P = 0.04). Median 6-MMP levels decreased in all patients from 6090 to 175 pmol/8 × 10(8) RBC (P = 0.01).. Allopurinol safely and effectively optimises thiopurine therapy in patients with autoimmune hepatitis with intolerance and/or nonresponse due to an unfavourable thiopurine metabolism.

Topics: Adult; Aged; Alanine Transaminase; Allopurinol; Antimetabolites; Drug Interactions; Drug Therapy, Combination; Female; Hepatitis, Autoimmune; Humans; Male; Mercaptopurine; Methyltransferases; Middle Aged; Salvage Therapy; Thioguanine

Thiopurine therapy effectively maintains remission in inflammatory bowel disease. However, many patients are unable to achieve optimum benefits from azathioprine or 6-mercaptopurine because of undesirable metabolism related to high thiopurine methyltransferase (TPMT) activity characterized by hepatic transaminitis secondary to increased 6-methylmercaptopurine (6-MMP) production and reduced levels of therapeutic 6-thioguanine nucleotide (6-TGN). Allopurinol can optimize this skewed metabolism. We discuss two brothers who were both diagnosed with ulcerative colitis (UC). Their disease remained active despite oral and topical mesalamines. Steroids followed by 6-mercaptopurine (MP) were unsuccessfully introduced for both patients and both were found to have high 6-MMP and low 6-TGN levels, despite normal TMPT enzyme activity, accompanied by transaminitis. Allopurinol was introduced in combination with MP dose reduction. For both brothers addition of allopurinol was associated with successful remission and optimized MP metabolites. These siblings with active UC illustrate that skewed thiopurine metabolism may occur despite normal TPMT enzyme activity and can lead to adverse events in the absence of disease control. We confirm previous data showing that addition of allopurinol can reverse this skewed metabolism, and reduce both hepatotoxicity and disease activity, but we now also introduce the concept of a family history of preferential MP metabolism as a clue to effective management for other family members.

Topics: Allopurinol; Antimetabolites; Colitis, Ulcerative; Dose-Response Relationship, Drug; Drug Therapy, Combination; Guanine Nucleotides; Humans; Liver; Male; Mercaptopurine; Methyltransferases; Steroids; Thionucleotides; Treatment Outcome; Young Adult

High concentrations of methylated thiopurine metabolites, such as 6-methyl mercaptopurine, are associated with hepatotoxicity during administration of the conventional thiopurines azathioprine or 6-mercaptopurine in IBD patients. Metabolization of the non-conventional thiopurine 6-thioguanine does not generate 6-methyl mercaptopurine. Hence, the aim of our study was to evaluate hepatotoxicity during 6-thioguanine in IBD patients who previously failed conventional thiopurines due to 6-methyl mercaptopurine associated hepatotoxicity.. A retrospective single center intercept cohort study was performed of IBD patients using 6-thioguanine between January 2006 and July 2010 after failing conventional thiopurine therapy due to 6-methyl mercaptopurine associated hepatotoxicity. The primary outcome was the occurrence of 6-thioguanine induced hepatotoxicity, scaled according to the Common Terminology Criteria for Adverse Events.. Nineteen patients were included. Median duration of 6-thioguanine therapy (median daily dosage 21 mg (9-24)) was 23 weeks (6-96). Hepatotoxicity did not reoccur in 15 out of 19, whereas grade 1 toxicity persisted in 4 patients (p<0.001). Median aspartate aminotransferase and alanine aminotransferase concentrations decreased from 34 U/l (20-59) and 64 U/l (15-175) to 23 U/l (18-40; p=0.003) and 20 U/l (14-48; p=0.019), respectively.. Hepatotoxicity does not reoccur during 6-thioguanine treatment in most IBD patients who failed conventional thiopurines due to 6-methyl mercaptopurine associated hepatotoxicity. Hence, at least at short-term, 6-thioguanine appears a justifiable alternative thiopurine for these IBD patients.

Topics: Antimetabolites, Antineoplastic; Azathioprine; Chemical and Drug Induced Liver Injury; Female; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Retrospective Studies; Thioguanine; Treatment Outcome

Up to 20% of patients on thiopurine therapy fail to achieve adequate drug response. Many of these patients preferentially produce the toxic 6-methylmercaptopurine metabolites (6-MMP) rather than the active 6-thioguanine nucleotides (6-TGN) resulting in a high 6-MMP/6-TGN ratio (>20) and increased risk of hepatotoxicity.. To determine the prevalence of preferential 6-MMP producers and define the relationships between 6-TGN, 6-MMP and thiopurine methyltransferase (TPMT).. The database of 6-TGN, 6-MMP and TPMT measurements from patients throughout New Zealand was used to calculate patients' 6-MMP/6-TGN ratios and identify those with high (>20) or normal ratio (≤20).The TPMT enzyme activity was compared amongst the groups.. Of 1879 patients with TPMT, 6-TGN and 6-MMP results, 349 (19%) had a 6-MMP/6-TGN ratio >20. The mean TPMT enzyme activity was slightly lower for those with a 6-MMP/6-TGN ratio ≤20 vs. >20, which achieved statistical significance (12.2 vs. 13.2; P < 0.001). However, the distributions of TPMT enzyme activity were similar, with 97% of TPMT results falling between 5.0 and 17.6 IU/mL for both groups. In all, 17% of those with 6-MMP/6-TGN ratio ≤20 were intermediate TPMT metabolisers (TPMT 5.0-9.2 IU/mL) vs. 7% in those with a ratio >20.. In this patient population with measured 6-MMP/6-TGN ratios, 19% of patients were preferential 6-MMP producers. The results show that high TPMT enzyme activity is not the major reason for preferential 6-MMP production in most patients with a high metabolite ratio. This suggests that there are one or more important alternative mechanisms for preferentially producing 6-MMP.

Topics: Antimetabolites, Antineoplastic; Azathioprine; Chromatography, High Pressure Liquid; Drug Resistance; Erythrocyte Membrane; Female; Genotype; Guanine Nucleotides; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Statistics as Topic; Thioguanine; Thionucleotides

Mercaptopurine and azathioprine (AZA) are efficacious in treating IBD. 6-tioguanine (6-TGN) levels correlate with therapeutic efficacy, whereas high 6-methylmercaptopurine (6-MMP) levels are associated with hepatotoxicity and myelotoxicity. Some IBD patients exhibit dose-limiting preferential 6-MMP production, which may lead to undesired side effects and impact efficacy.. To review the outcomes of thiopurine split-dosing in patients with preferential 6-MMP metabolism.. A retrospective chart review of 179 IBD patients treated at the Cedars-Sinai IBD Center with AZA or mercaptopurine was performed. Preferential 6-MMP metabolisers with 6-MMP levels greater than 7000 pmol/8 × 10(8) erythrocytes who underwent split-dosing were identified and assessed for biochemical and clinical responses to these dose modifications.. A total of 20 of 179 patients met the criteria for preferential 6-MMP metabolism and underwent thiopurine split-dosing. Dividing the total daily thiopurine dose led to a reduction in 6-MMP levels (11785 vs. 5324 pmol/8 × 10(8) erythrocytes; P < 0.0001) without negatively affecting clinical disease activity or 6-TGN levels (239 vs. 216 pmol/8 × 10(8) erythrocytes; P = N.S.) and led to resolution of 6-MMP associated side effects (elevated transaminases, leucopenia and flu-like symptoms) in all but two patients. After mean follow-up of 36 months, 12 patients remained in clinical remission on split-dose mercaptopurine. Five of the remaining eight patients escalated to anti-TNF therapy, two progressed to surgery, and one switched to tioguanine therapy.. Split-dose administration of mercaptopurine/AZA represents an alternative option in IBD patients with preferential 6-MMP metabolism who might otherwise require steroid exposure or escalation of therapy.

Topics: Adolescent; Adult; Azathioprine; Dose-Response Relationship, Drug; Erythrocytes; Female; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Middle Aged; Retrospective Studies; Treatment Outcome; Young Adult

Circulating concentrations of 6-thioguanine nucleotide (6-TGN) and 6-methyl mercaptopurine (6-MMP) are associated with thiopurine efficacy and may predict toxicity. This study aimed to examine retrospectively the utility of measuring metabolite concentrations in patients with inflammatory bowel disease (IBD) who had continuing symptoms despite stable thiopurine treatment.. Concentrations of 6-TGN and 6-MMP were measured in lysates of washed red cells by high-performance liquid chromatography in peripheral blood drawn from 63 symptomatic patients with IBD (63% men, mean age 37, range 14-74 years, 67% Crohn's disease, 33% ulcerative colitis) treated with azathioprine or 6-mercaptopurine. Short-term clinical outcomes were examined.. 6-TGN concentrations weakly correlated with the thiopurine dose (r = 0.28, P = 0.08). On weight-based criteria, 50% of patients were underdosed. However, metabolite patterns suggested 7 (11%) patients were noncompliant, 18 (29%) were being underdosed, 33 (52%) were refractory to treatment with either appropriate (41%) or elevated (11%) metabolite concentrations, and 6 (10%) had a raised 6-MMP:6-TGN ratio consistent with aberrant thiopurine metabolism. The clinical outcome improved in 40 of 46 (87%) of patients in whom the course of action taken was as recommended by a metabolite-directed algorithm, while 3 of 17 patients (18%) improved where discordant actions were taken (P = 0.0001; Fisher's exact test). Fifteen patients (24%) avoided inappropriate escalation of therapy.. Dose-optimization or toxicity-avoidance strategies frequently result from metabolite testing in patients with inadequate efficacy from thiopurines, with evidence of better outcomes. Thiopurine metabolite testing is a potentially powerful tool for optimizing thiopurine usage in IBD.

Topics: Adolescent; Adult; Aged; Azathioprine; Chromatography, High Pressure Liquid; Colitis, Ulcerative; Crohn Disease; Female; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Middle Aged; Thioguanine; Treatment Failure; Young Adult

We performed a worldwide survey to evaluate the extent to which gastroenterologists who are experts in the field of inflammatory bowel diseases (IBDs) are utilizing thiopurine metabolism in practice.. This was a Web-based cross-sectional survey consisting of 12 multiple-choice and open-ended questions.. Between December 2009 and April 2010, 175 questionnaires were received. The proportion of practitioners with access and reimbursement for thiopurine S-methyltransferase (TPMT) genotype, TPMT phenotype, 6-thioguanine nucleotides (6-TGN) levels, and 6-methylmercaptopurine ribonucleotides (6-MMP) levels was 48%, 54%, 44%, and 35%, respectively. Before azathioprine initiation, TPMT genotype and phenotype were performed by only 30% and 43% of responders, respectively. In patients on thiopurine therapy, 6-TGN and 6-MMP levels were determined by 54% and 44% of responders, respectively. Only 27% of physicians always wait for TMPT activity/genotype results before initiating azathioprine and 81% do not routinely recheck metabolite levels after dose escalation or reduction. In cases of very high or low TPMT activity, 75% and 74% of practitioners take into account TMPT activity result, respectively. If access to all azathioprine metabolite measurements was available and if all these tests were reimbursed by public health insurance, 47% of responders would use these tests more often in their practice. The availability and reimbursement of TPMT status and azathioprine metabolites strongly influenced experts' attitudes.. Thiopurine testing is relatively underutilized by IBD gastroenterologists. The availability and reimbursement of TPMT status and azathioprine metabolites strongly influence the management of IBD patients treated with thiopurines.

Topics: Azathioprine; Biomarkers; Cross-Sectional Studies; Gastroenterology; Genetic Testing; Genotype; Global Health; Humans; Inflammatory Bowel Diseases; Mercaptopurine; Methyltransferases; Phenotype; Practice Patterns, Physicians'; Surveys and Questionnaires

In this contribution, the great potential of surface enhanced Raman spectroscopy (SERS) in a lab-on-a-chip (LOC) device for the detection of analyte molecules in a complex environment is demonstrated. Using LOC-SERS, the enzyme activity of thiopurine S-methyltransferase (TPMT) is analysed and identified in lysed red blood cells. The conversion of 6-mercaptopurine to 6-methylmercaptopurine catalysed by TPMT is observed as it gives evidence for the enzyme activity. Being able to determine the TPMT activity before starting a treatment using 6-mercaptopurine, an optimized dosage can be applied to each patient and serious toxicity appearing within thiopurine treatment will be prevented.

Topics: Erythrocytes; Humans; Lab-On-A-Chip Devices; Mercaptopurine; Methyltransferases; Spectrum Analysis, Raman

Clinical response to thiopurine medication is related to the concentration of its metabolites. Proxy measures are traditionally used to assess dose adequacy. We present our experience of using tioguanine (previously known/formerly referred to as thioguanine) metabolite measurements in paediatric patients and evaluate their effect on clinical practice.. To report our experience of using tioguanine metabolite measurements in paediatric patients and to evaluate their effects on clinical practice.. The 6-tioguanine nucleotide (6-TGN) and 6-methylmercaptopurine (6-MMP) were measured in children prescribed thiopurine medication for at least 3 months. Data were collected on thiopurine methyl transferase (TPMT) genotype, drug dose, laboratory indices and management changes. Therapeutic 6-TGN levels were defined as 235-400 pmol/8 × 10(8) RBCs. Seventy individuals (30 males) with a median age of 15 years. Underlying diagnoses were 'IBD' (68/70) and two cases of eosinophilic colitis. Sixty-three were treated with azathioprine and seven with mercaptopurine. A total of 103 separate measurements were made.. On initial measurement, 68% of patients had 6-TGN levels outside therapeutic levels despite standard thiopurine dosing. Initial 6-TGN levels were significantly higher in patients with TPMT mutations. Toxicity occurred in seven cases. The 6-TGN levels were significantly higher in those with signs of marrow toxicity. The 6-TGN level correlated with WBC, leukocyte count, mean corpuscular volume (MCV) and ΔMCV; however, the ability of each of these to predict therapeutic 6-TGN levels was poor. After initial measurement, management was changed in 25/70 cases (36%).. 6-TGN levels were therapeutic in a minority of those patients who were tested. Proxy measures perform poorly in predicting therapeutic 6-TGN levels. Measuring thiopurine metabolites is useful for dosage adjustment in children, and for the detection of potential toxicity.

Topics: Adolescent; Azathioprine; Biomarkers; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Female; Guanine Nucleotides; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Retrospective Studies; Thionucleotides; Time Factors; Treatment Outcome

Levels of the thiopurine metabolites 6-thioguanine nucleotide (6-TGN) and 6-methylmercaptopurine commonly are monitored during thiopurine therapy for inflammatory bowel disease despite this test's high cost and poor prediction of clinical response (sensitivity, 62%; specificity, 72%). We investigated whether patterns in common laboratory parameters might be used to identify appropriate immunologic responses to thiopurine and whether they are more accurate than measurements of thiopurine metabolites in identifying patients who respond to therapy.. We identified 774 patients with inflammatory bowel disease on thiopurine therapy using metabolite and standard laboratory tests over a 24-hour time period. Machine learning algorithms were developed using laboratory values and age in a random training set of 70% of the cases; these algorithms were tested in the remaining 30% of the cases.. A random forest algorithm was developed based on laboratory and age data; it differentiated clinical responders from nonresponders in the test set with an area under the receiver operating characteristic (AUROC) curve of 0.856. In contrast, 6-TGN levels differentiated clinical responders from nonresponders with an AUROC of 0.594 (P < .001). Algorithms developed to identify thiopurine nonadherence (AUROC, 0.813) and thiopurine shunters (AUROC, 0.797) were accurate.. Algorithms that use age and laboratory values can differentiate clinical response, nonadherence, and shunting of thiopurine metabolism among patients who take thiopurines. This approach was less costly and more accurate than 6-TGN metabolite measurements in predicting clinical response. If validated, this approach would provide a low-cost, rapid alternative to metabolite measurements for monitoring thiopurine use.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Algorithms; Artificial Intelligence; Child; Child, Preschool; Drug Monitoring; Female; Guanine Nucleotides; Humans; Immunologic Factors; Inflammatory Bowel Diseases; Male; Mercaptopurine; Middle Aged; Thionucleotides; Young Adult

Monitoring of thiopurine metabolites 6-thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine (6-MMP) is used to assess compliance and explain adverse reactions in IBD-patients. Correlations between dosage, metabolite concentrations and therapeutic efficacy or toxicity are contradictive. Research is complicated by analytical problems as matrices analyzed and analytical procedures vary widely. Moreover, stability of thiopurine metabolites is not well documented, yet pivotal for interpretation of analytical outcomes. Therefore, we prospectively investigated metabolite stability in blood samples under standard storage conditions.. Stability at room temperature and refrigeration (22 degrees C, 4 degrees C) was investigated during 1 week and frozen samples (-20 degrees C, -80 degrees C) were analyzed during 6 months storage. Ten patient samples were analyzed for each study period.. Median 6-TGN concentrations on day 7 decreased significantly to 53% and 90% during storage at ambient temperature or refrigeration. Median 6-MMP concentrations on day 7 decreased significantly to 55% and 86%, respectively. Samples stored at -20 degrees C also showed significant decreases in both 6-TGN and 6-MMP in comparison with baseline values. At -80 degrees C, only 6-MMP showed a significant decrease in values compared to baseline.. The stability of thiopurine metabolites is clearly a limiting factor in studies investigating utilisation of TDM and correlations with therapeutic outcome in IBD-patients. This has to be accounted for in clinical practice and (multi-center) trials investigating thiopurine drugs.

Topics: Chromatography, High Pressure Liquid; Drug Stability; Humans; Inflammatory Bowel Diseases; Mercaptopurine; Reproducibility of Results; Specimen Handling; Statistics, Nonparametric; Thioguanine

Azathioprine is commonly used in the treatment of autoimmune hepatitis (AIH). Few data are available on drug monitoring of azathioprine metabolites in patients with AIH, especially in pediatric patients. The purpose of this study was to investigate intracellular thiopurine metabolites in children with AIH and to assess the relevance of drug monitoring compared with the efficacy and toxicity. Data from 28 patients with AIH treated by azathioprine for at least 3 months were recorded. 6-Thioguanine nucleotides (6-TGN) and 6-methyl mercaptopurine nucleotides (6-MeMPN) concentrations and TPMT activity were determined by high-performance liquid chromatography. Blood cell counts and liver function tests were also collected and the clinical outcome was documented. A wide interindividual variability in 6-TGN and 6-MeMPN concentrations was observed with values ranging from 51 to 1966 pmol/8 x 10(8) red blood cells (RBCs) for 6-TGN and from 42 to 8189 pmol/8 x 10(8) RBCs for 6-MeMPN. A total of 61.4% of the patients achieved remission and only 32.6% of these children had 6-TGN values within the target range proposed for inflammatory bowel disease (250-450 pmol/8 x 10(8) red blood cells). No difference in metabolite concentrations was observed between children in remission and those with active disease. Azathioprine dosage was significantly related to 6-TGN and 6-MeMPN levels (r = 0.308, P < 0.001 and r = 0.405, P < 0.001, respectively). A significant negative correlation was observed between 6-TGN concentrations and erythrocyte and lymphocyte counts, whereas 6-MeMPN was not related to blood cell counts. Although leukocyte counts were not related to 6-TGN concentrations, patients with leucopenia exhibited higher 6-TGN values than those without leucopenia (median values 438 pmol/8 x 10(8) RBCs versus 405 pmol/8 x 10(8) RBCs, respectively). Thiopurine metabolite monitoring appears useful in identifying the myelotoxicity and the hepatotoxicity as a result of azathioprine with disease recurrence and to assess adherence to therapy. A further larger study will be required to confirm the optimal recommended target for thiopurine metabolites to achieve remission in patients with AIH.

Topics: Adolescent; Adrenal Cortex Hormones; Anti-Inflammatory Agents; Azathioprine; Biotransformation; Blood Cell Count; Child; Child, Preschool; Chromatography, High Pressure Liquid; Drug Monitoring; Drug Therapy, Combination; Female; Hepatitis, Autoimmune; Humans; Immunosuppressive Agents; Liver Function Tests; Male; Mercaptopurine; Thioguanine

Monitoring of thiopurine metabolites is important due to a complex metabolism with large interindividual variation, but the suitability of currently used methods has been questioned. The drawbacks include poor reproducibility, the inability to differentiate between the different analytes, as well as the use of a nontarget matrix. Further research should be directed toward measuring thiopurine metabolites in mononuclear cells, measuring the different nucleotides specifically, as well as measuring the incorporation of thioguanine into DNA. The studies should not be limited to thioguanosine nucleotides but include methylthioinosine nucleotides as well.

Topics: Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Mercaptopurine; Purines; Thioguanine

6-mercaptopurine (6-MP) is used for the induction and maintenance of remission of inflammatory bowel disease (IBD). 6-MP is converted into 6-methylmercaptopurine (6-MMP) or 6-thioguanine nucleotides (6-TGN) intracellularly. Treatment response in IBD patients correlates with 6-TGN levels. This study prospectively evaluated the effect of allopurinol on 6-MP metabolites in adult and pediatric IBD patients. Additionally, we quantified the prevalence of preferential metabolism towards 6-MMP through a retrospective analysis of IBD patients.. Twenty patients (10 adult; 10 pediatric) with evidence of preferential metabolism towards 6-MMP, (6-TGN<250 pmol/8×10⁸ RBCs and 6-MMP>5000 pmol/8×10⁸ RBCs) were prospectively treated with allopurinol 100 mg daily and up to 100 mg of 6-MP. 6-MP dose was adjusted after a 3-week metabolite measurement.. The median dose of 6-MP for adults decreased from 100mg daily (range: 37.5-150 mg) to 25mg daily (range: 12.5-50 mg). The median dose of 6-MP for pediatric patients decreased from 50 mg (range: 25-50 mg) to 10.7 mg (range: 10.7 to 21.4 mg). Mean 6-TGN levels in all subjects increased from 197.4 (± 59) to 284.8 (± 107) pmol/8×10⁸ RBCs (p=0.0005). Mean 6-MMP levels in all subjects decreased from a mean of 7719.8 (± 4716) to 404.8 (± 332) pmol/8×10⁸ RBCs (p=0.0004). There were no complications associated with allopurinol therapy. Eighty-eight (30.9%) of 285 IBD patients had evidence of preferential metabolism towards 6-MMP. The proportion of preferential metabolism was equal in adults and pediatric patients.. Our results indicate that the addition of allopurinol safely shifts metabolite production in both adult and pediatric IBD patients and that there is a high prevalence of preferential metabolism towards 6-MMP among IBD patients.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Allopurinol; Child; Child, Preschool; Drug Administration Schedule; Enzyme Inhibitors; Female; Guanine Nucleotides; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Middle Aged; Phenotype; Prevalence; Prospective Studies; Thionucleotides; Young Adult

Azathioprine is a thiopurine immunosuppressive antimetabolite used to chronically treat inflammatory bowel disease and autoimmune hepatitis. Azathioprine treatment is a long-term therapy and therefore it is at risk for non-adherence, which is considered an important determinant of treatment inefficacy. Measurement of 6-thioguanine and 6-methylmercaptopurine nucleotides has been recently suggested as a screener for non-adherence detection. We describe four young patients in which non-adherence to azathioprine therapy was detected only through the measurement of drug metabolite concentrations, and the criterion for non-adherence was undetectable metabolite levels. After the identification of non-adherence, patients and their families were approached and the importance of a correct drug administration was thoroughly enlightened and discussed; this allowed obtaining a full remission in all subjects. Our observations support the use of undetectable metabolite levels as indicators of non-adherence to therapy in azathioprine treated patients. The additional level of medical supervision given by this assay allows getting a better adherence to medical treatment, which results in an improvement in the response to therapy; these benefits may justify the costs associated with the assay.

Topics: Adolescent; Azathioprine; Child; Child, Preschool; Female; Guanine Nucleotides; Hepatitis, Autoimmune; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Medication Adherence; Mercaptopurine; Thionucleotides; Young Adult

Azathioprine (AZA) has a slow onset of action in treatment of pediatric inflammatory bowel disease (IBD). It is anticipated, that this delay correlates to the kinetics of 6-thioguanine nucleotides (6-TGN) accumulation. The aim of this study was to evaluate the time to steady state of 6-TGN concentration in red blood cells.. The inclusion criteria were: a) age 0-19 years b) IBD diagnosis c) AZA treatment initiation. High performance liquid chromatography was used for the 6-TGN analysis. Concentrations of metabolites were studied in weeks 0, 1, 2, 5, and 8 after beginning of treatment.. The inclusion criteria were matched to 18 patients with IBD. The median time to steady state of 6-TGN was 55.3 days. The mean 6-TGN concentration at the steady state achieved 326 (SD 154) pmol/8.108 erythrocytes. High erythrocyte TPMT activity corresponds to the low steady state 6-TGN concentration and vice versa. This correlation reached statistical significance (p<0.01) for the dose expressed in mg per square meter of body surface area.. The time to steady state of 6-TGN erythrocyte concentration is significantly shorter than would expected according to clinical observation describe earlier.

Topics: Adolescent; Azathioprine; Child; Child, Preschool; Female; Genotype; Guanine Nucleotides; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Prospective Studies; Severity of Illness Index; Thionucleotides; Treatment Outcome

Individualised doses of azathioprine (AZA) may be prescribed by monitoring the levels of the enzyme thiopurine methyltransferase (TPMT). The measurements of thiopurine metabolites of AZA, 6-thioguanine (6-TGN) and 6-methylmercaptopurine (6-MMP), have also been reported as new markers of AZA activity.. To describe TPMT phenotype in our population and to establish a relationship between thiopurine metabolites,and therapeutic activity and adverse effects.. Data on TPMT were retrospectively collected from 107 patients, and 6-TGN and 6-MMP levels in 18 patients currently on treatment with AZA (Crohn's disease 5, ulcerative colitis 5, autoimmune hepatitis 5).. Mean value of TPMT was 20.19U/ml. None of the patients had a TPMT activity<5U/ml. Of the 18 patients on treatment, 13 showed sub-therapeutic levels of 6-TGN (<235pmol/8x10(8) red blood cells). Clinical remission was maintained in 45% of patients. Mean levels of 6-TGN in patients with clinical remission were 259pmol/8x10(8) red blood cells versus 209pmol/8x10(8) red blood cells in non-responders (p=0.37). There was an inverse relationship (r=-0.28) between TPMT and 6-TGN levels. Toxic effects occurred in 6 of 18 patients, with leukopenia in 5 and hyperamylasemia in 1.. Determination of TPMT and monitoring of thiopurine metabolites allows AZA treatment to be optimised, although further studies are necessary to establish therapeutic effectiveness and toxicity ranges.

Topics: Adolescent; Azathioprine; Female; Hepatitis, Autoimmune; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Retrospective Studies; Thioguanine

Topics: Humans; Mercaptopurine; Patents as Topic; Pharmaceutical Preparations; Purines; Thioguanine

Thiopurines are a mainstay of immunomodulator therapy in inflammatory bowel disease (IBD). Despite their efficacy, some patients may have a poor response due to inability to achieve adequate levels of the active metabolite, 6-thioguanine (6-TGN). Others experience hepatotoxicity, which correlates with excessive 6-methylmercaptopurine (6-MMP) levels. Two adult studies have demonstrated successful manipulation of thiopurine metabolism with allopurinol, a xanthine oxidase inhibitor, to achieve more optimal thiopurine levels. The aim was to retrospectively characterize the utility of allopurinol to optimize thiopurine metabolite levels in pediatric IBD patients.. Thirteen patients received allopurinol daily (100 mg in patients >or=30 kg and 50 mg <30 kg), and their thiopurine dose was simultaneously reduced to 25%-50% of the previous maintenance dose. Metabolite levels and other screening labs were checked 2-4 weeks later.. The mean azathioprine dose was decreased from 148.1 to 59.6 mg daily (60% of the mean original dose). The mean 6-TGN level increased from 173 to 303 pmol/8 x 10(8) red blood cell count (RBC) (P = 0.03), and the mean 6-MMP level decreased from 7888 to 2315 pmol/8 x 10(8) RBC (P < 0.001). Elevated transaminase levels improved or resolved in all patients. Two patients experienced reversible neutropenia. At the conclusion of the study 9 patients (69%) remained on combination therapy with a mean duration of follow-up of 162.8 +/- 119.2 days.. Combination therapy successfully shunted thiopurine metabolites to a more favorable pattern. Reversible neutropenia was the most common side effect (2 patients). Long-term prospective studies are needed in this patient population.

Topics: Adolescent; Allopurinol; Antimetabolites; Child; Child, Preschool; Cohort Studies; Colitis, Ulcerative; Crohn Disease; Drug Therapy, Combination; Female; Follow-Up Studies; Humans; Male; Mercaptopurine; Prognosis; Remission Induction; Retrospective Studies; Treatment Outcome

To investigate the population pharmacokinetics of 6-mercaptopurine (6-MP) active metabolites in paediatric patients with acute lymphoblastic leukaemia (ALL) and examine the effects of various genetic polymorphisms on the disposition of these metabolites.. Data were collected prospectively from 19 paediatric patients with ALL (n = 75 samples, 150 concentrations) who received 6-MP maintenance chemotherapy (titrated to a target dose of 75 mg m(-2) day(-1)). All patients were genotyped for polymorphisms in three enzymes involved in 6-MP metabolism. Population pharmacokinetic analysis was performed with the nonlinear mixed effects modelling program (nonmem) to determine the population mean parameter estimate of clearance for the active metabolites.. The developed model revealed considerable interindividual variability (IIV) in the clearance of 6-MP active metabolites [6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-mMPNs)]. Body surface area explained a significant part of 6-TGNs clearance IIV when incorporated in the model (IIV reduced from 69.9 to 29.3%). The most influential covariate examined, however, was thiopurine methyltransferase (TPMT) genotype, which resulted in the greatest reduction in the model's objective function (P < 0.005) when incorporated as a covariate affecting the fractional metabolic transformation of 6-MP into 6-TGNs. The other genetic covariates tested were not statistically significant and therefore were not included in the final model.. The developed pharmacokinetic model (if successful at external validation) would offer a more rational dosing approach for 6-MP than the traditional empirical method since it combines the current practice of using body surface area in 6-MP dosing with a pharmacogenetically guided dosing based on TPMT genotype.

Topics: Adolescent; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Child; Child, Preschool; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Genotype; Humans; Male; Mercaptopurine; Metabolic Clearance Rate; Methyltransferases; Models, Biological; Pharmacogenetics; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Prospective Studies

Azathioprine and its initial metabolite, 6-mercaptopurine (6-MP), are associated with high rates of treatment cessation due to toxicity or inadequate response. Individualization of thiopurine dose based on concentrations of the active 6-thioguanine nucleotide (6-TGN) metabolites can help improve outcomes with this class. Some individuals, however, preferentially metabolize thiopurine drugs to the potentially hepatotoxic 6-methylmercaptopurine nucleotide (6-MMPN) metabolites rather than the 6-TGNs. For these patients, escalation in thiopurine dose is not likely to increase 6-TGN concentrations sufficiently but may lead to a disproportionate increase in exposure to the 6-MMPNs. We present three cases in whom thiopurine dose escalation based on clinical status and low 6-TGN concentrations (100-262 pmol/8 x 10 RBC) resulted in severe hepatotoxicity (liver failure in two cases) associated with unrecognized extremely high 6-MMPN concentrations of 26,000-40,000 pmol/8 x 10 RBC. These cases illustrate a risk with thiopurine dose adjustment based on monitoring of 6-TGN metabolites without also monitoring 6-MMPN.

Topics: Adult; Azathioprine; Chemical and Drug Induced Liver Injury; Drug Administration Schedule; Drug Monitoring; Female; Guanine Nucleotides; Humans; Immunosuppressive Agents; Mercaptopurine; Middle Aged; Thionucleotides

Topics: Bias; Guanine Nucleotides; Humans; Inflammatory Bowel Diseases; Mercaptopurine; Prospective Studies; Thionucleotides

Topics: Adult; Antimetabolites; Breast Feeding; Dose-Response Relationship, Drug; Female; Humans; Infant; Infant, Newborn; Inflammatory Bowel Diseases; Mercaptopurine; Milk, Human

To develop an HPLC method for the determination of 6-methylmercaptopurine concentration and then numerate the thiopurine methyltransferase (TPMT) activity in erythrocyte via formula and analysis of the TPMT activity difference between human and pig.. The chromatographic apparatus SHIMADZU GC-2010C was used. The stationary phase was a Symmetry C18 reverse-phase column (150 mm x 3.9 mm, 5 microm). The mobile phase was 0.1% acetic acid-methanol and the flow rate 1.0 mL/min. The samples were extracted by ethyl acetate and injected automatically. They were measured at UV 280 nm. 2-amino-6-methyl-mercaptopurine was used as the internal standard.. The retention times for 6-MP, AMMP and 6-MMP were 4.4 min, 7.7 min and 9.3 min respectively. The calibration curves were linear over the range of 6.25-100 nmol/mL. the methodology recovery was 98.08%-100.05%. The extraction recovery of 6-MMP was 88.1%-92.4%. The within-group RSD 1.0%-1.5% and inter-group RSD 1.0%-4.4%. The levels of TPMT activity of human, pig and Banna Minipig Inbred Line (BMI) were (17.45 +/- 3.62) U, (7.65 +/- 1.35) U and (8.73 +/- 1.55) U respectively.. The method is suitable for determination of TPMT activity in erythrocyte of different patients and species in that of clinical medicine and scientific research. TPMT activity of pig and BMI is obviously lower than human's.

Topics: Animals; Chromatography, High Pressure Liquid; Erythrocytes; Humans; Mercaptopurine; Methyltransferases; Species Specificity; Swine; Swine, Miniature

Methylmercaptopurine riboside (meMPR), a cellular metabolite of 6-mercaptopurine (6-MP), is a potent inhibitor of de novo purine synthesis (DNPS). Human MOLT4 T-lymphoblastic leukaemia cells that have acquired resistance to 6-MP or 6-thioguanine (6-TG) as a consequence of defective transport exhibit enhanced sensitivity to meMPR. HPLC-based analysis of the transport of meMPR revealed normal uptake of this compound by our thiopurine-resistant cell sublines, suggesting a route of transport distinct from that for 6-MP and 6-TG. Studies on the wild-type parental leukemic cells showed that adenosine, dipyridamole and nitrobenzylthioinosine inhibit uptake of meMPR to a significant extent, whereas Na+ ions have no influence on this process. Transfection of these leukemic cells with small interference RNA molecules targeting the gene encoding the first member of the family of equiliberative nucleoside transporters (ENT1) strongly reduced the initial rate of meMPR transport. Our resistant cell lines exhibited 30-52% reductions (p < 0.005) in their levels of mRNA encoding several proteins involved in de novo purine synthesis, i.e., aminoimidazole carboxamide ribonucleotide formyltransferase, glycinamide ribonucleotide transformylase and guanine monophosphate synthetase. Consequently, the rate of de novo purine synthesis in these resistant sublines was decreased by 50%. Furthermore, the levels of ribonucleoside triphosphates in these cells were significantly lower than in the non-resistant parental cells. In combination, a reduced rate of de novo purine synthesis together with low levels of ribonucleoside triphosphates can explain the enhanced sensitivity of our thiopurine-resistant cell lines to meMPR. In this manner, meMPR bypasses the mechanisms of resistance to thiopurines and is even more cytotoxic towards resistant than towards wild-type cells.

Topics: Adenosine Kinase; Cell Line, Tumor; Cell Survival; Down-Regulation; Drug Resistance, Neoplasm; Equilibrative Nucleoside Transporter 1; Gene Silencing; Humans; Inhibitory Concentration 50; Mercaptopurine; Polymerase Chain Reaction; Purines; Ribonucleotides; RNA, Messenger; RNA, Small Interfering; Sodium; T-Lymphocytes; Thioguanine

Azathioprine is a key drug in the management of autoimmune hepatitis (AIH), with effects mediated via conversion to 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP), the latter controlled by thiopurine methyltransferase (TPMT). Our aims were to evaluate the role of TPMT genotyping and phenotyping and to examine 6-TG and 6-MMP metabolite levels in patients with AIH.. TPMT genotyping and phenotyping was performed on 86 patients with AIH, and metabolites evaluated in assessable patients.. Eighty-six patients with AIH received azathioprine; 22 developed toxicity and 4/22 were heterozygous for TPMT alleles. Cirrhosis was more common amongst patients who developed toxicity (12/22 (54.5%) versus 19/64 (29.6%), P=0.043). Patients who required persistent prednisone at equivalent azathioprine doses had a higher mean fibrosis stage (P=0.044). TPMT activity, but not metabolites, was lower in patients with stage III/IV fibrosis versus stage I/II fibrosis (30+/-1.92 versus 35.2+/-1.93, P=0.044). Azathioprine dose significantly correlated with measured 6-TG levels (r=0.409, P<0.0001) and 6-MMP levels (r=0.387, P<0.001).. Advanced fibrosis but not TPMT genotype or activity predicts azathioprine toxicity in AIH. Overlap in 6-TG and 6-MMP metabolite levels is noted whether or not steroid therapy is used to maintain remission.

Topics: Adolescent; Adult; Aged; Anti-Inflammatory Agents; Azathioprine; Drug Monitoring; Female; Genotype; Hepatitis, Autoimmune; Humans; Immunosuppressive Agents; Liver; Liver Cirrhosis; Male; Mercaptopurine; Methyltransferases; Middle Aged; Phenotype; Prednisone; Thioguanine

We evaluated how our use of thiopurines was altered by determination of thiopurine methyltransferase (TPMT) level and drug dose adjustment guided by a 6-mercaptopurine metabolite assay. We further examined whether these resulted in better selection of the drug dose, improved control of disease, and decreased corticosteroid use in pediatric inflammatory bowel disease (IBD).. This is a retrospective review of 101 pediatric patients with IBD receiving a stable dose of azathioprine (AZA) for 4 months or longer. The study group (n = 64) consisted of patients who received AZA and had metabolite levels measured. The comparison group (n = 37) consisted of patients who were receiving AZA before the availability of metabolite measurement. The TPMT level was measured in study group patients before starting AZA.. Patients with normal TPMT level received a higher starting dose of AZA than in patients who were heterozygous for TPMT deficiency (1.7 vs 0.9 mg/[kg x d], P < 0.0001). Study group patients received a higher starting dose (1.6 vs 1.2 mg/[kg x d], P = 0.001) and a higher final dose of AZA (2.4 vs 1.7 mg/[kg x d], P < 0.0001) compared with patients in the comparison group. These patients also had more dose adjustments (0.8 vs 0.4 mg/kg, P < 0.002). The number of disease exacerbations per patient per year was 55% less in the study group (95% CI, 17%-76%, P < 0.0001). The study group patients received less prednisone (P < 0.0001) and had lower disease activity scores (P < 0.05). There was no difference between groups in infliximab use or surgery rate.. Azathioprine dose adjustment using a 6-mercaptopurine metabolite assay was associated with use of higher doses, improved control of disease and decreased corticosteroid use in pediatric patients with IBD.

Topics: Adolescent; Adult; Azathioprine; Child; Child, Preschool; Colitis, Ulcerative; Crohn Disease; Erythrocytes; Female; Guanine Nucleotides; Heterozygote; Humans; Infant; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Prednisone; Remission Induction; Retrospective Studies; Thionucleotides

To determine infant exposure to 6-thioguanine and 6-methylmercaptopurine nucleotides (6-TGN and 6-MMPN, respectively) during maternal use of azathioprine in breastfeeding.. Mother-infant pairs provided blood for determination of 6-TGN and 6-MMPN concentrations, and TPMT genotype.. Four women taking azathioprine 1.2-2.1 mg kg(-1) day(-1) and their infants were studied. All had the wild-type TPMT genotype. Maternal 6-TGN and 6-MMPN concentrations ranged from 234 to 291 and 284 to 1178 pmol per 8 x 10(8) red blood cells, respectively, and were consistent with those associated with improved therapeutic outcomes. Neither 6-TGN nor 6-MMPN was detected in any of the infants, despite a sensitive assay.. The data suggest that azathioprine may be 'safe' during breastfeeding in patients with the wild-type TPMT genotype ( approximately 90% of caucasian patients) taking 'normal' doses.

Topics: Adult; Azathioprine; Breast Feeding; Female; Humans; Immunosuppressive Agents; Infant; Male; Mercaptopurine; Methyltransferases; Mothers; Thioguanine

This study describes approaches for stacking a large volume of sample solutions containing a mixture of mercaptopurine monohydrate, 6-methylmercaptopurine, thioguanine, thioguanosine, and thioxanthine in capillary electrophoresis (CE). After filling the run buffer (60 mM borate buffer, pH 8.5), a large sample volume was loaded by hydrodynamic injection (2.5 psi, 99.9 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-15 kV). Monitoring the current and reversing the polarity when 95% of current recovered, the separation of anionic analytes was performed in a run buffer < 20 kV. Around 44- to 90-fold improvement of sensitivity for five analytes was achieved by large-volume stacking with polarity switching when compared with CE without stacking. This method was feasible for determination of the analytes spiked in plasma. Removing most of electrolytes from plasma is a key step for performing large-volume sample stacking. Solid-phase extraction was used for pretreatment of biological samples. To our knowledge, this study is one of few applications showing the possibilities of this stacking procedure to analyze biological samples by large-volume sample stacking with polarity switching (LVSSPS) in CE.

Topics: Electrophoresis, Capillary; Guanosine; Humans; Mercaptopurine; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Sensitivity and Specificity; Thioguanine; Thionucleosides; Xanthines

Polymorphic thiopurine S-methyltransferase (TPMT) is a major determinant of thiopurine toxicity.. We extracted 6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-MMPNs) from erythrocytes with perchloric acid and converted them to 6-thioguanine (6-TG) and a 6-methylmercaptopurine (6-MMP) derivative during a 60-min acid hydrolysis step. The liquid chromatography system consisted of a C(18) column with an ammonium acetate-formic acid-acetonitrile buffer. 8-Bromoadenine was the internal standard. Analytes were measured with positive ionization and multiple reaction monitoring mode. With PCR-restriction fragment length polymorphism analysis and TaqMan allelic discrimination, common TPMT alleles (*1, *2, *3A, *3B, *3C) were determined in 31 792 individuals. We used perchloric acid extraction, acid hydrolysis, and HPLC with ultraviolet detection to measure erythrocyte 6-TG and 6-MMP nucleotide concentrations in 6189 patients with inflammatory bowel disease receiving azathioprine/6-mercaptopurine therapy.. Intra- and interday imprecision were <10% at low and high analyte concentrations. The conversion of 6-TG and 6-MMP nucleoside mono-, di-, and triphosphates was complete after hydrolysis. Allelic frequency for TPMT variant alleles ranged from 0.0063% (*3B) to 3.61% (*3A). Compared with wild types, TPMT heterozygotes had an 8.3-fold higher risk for 6-TGNs >450 pmol/8 x 10(8) erythrocytes (concentration associated with increased risk for leukopenia), but an 8.2-fold lower risk for 6-MMPNs >5700 pmol/8 x 10(8) erythrocytes (concentration associated with increased risk for hepatotoxicity).. The liquid chromatography-tandem mass spectrometry method can be applied to the routine monitoring of thiopurine therapy. The association between TPMT genotype and metabolite concentrations illustrates the utility of pharmacogenetics in the management of patients undergoing treatment with thiopurines.

Topics: Adult; Azathioprine; Chromatography, Liquid; Cohort Studies; Drug Therapy, Combination; Erythrocytes; Female; Genetic Variation; Guanine Nucleotides; Humans; Hydrolysis; Inflammatory Bowel Diseases; Male; Mass Spectrometry; Mercaptopurine; Methyltransferases; Purine Nucleotides; Thionucleotides

To study the distribution of the thiopurine methyltransferase (TPMT) genotype among azathioprine (Aza)-tolerant and -intolerant patients with various disorders, and to investigate a possible relationship with the Aza metabolite levels.. Forty-six Aza-tolerant and six Aza-intolerant patients had the TPMT genotype distribution determined using a polymerase chain reaction (PCR) assay and the forty-six Aza-tolerant patients had the Aza metabolite levels determined using a high-pressure liquid chromatography (HPLC) analysis.. One non-functional TPMT mutant allele was demonstrated in 2 of the 46 Aza-tolerant patients (4.4%) and one or two non-functional mutant alleles in 2 of the 6 Aza-intolerant patients (33.3%). Of the 4 patients, with one or two non-functional mutant alleles 2 (50%) were intolerant to Aza compared with 4 of the 48 patients (8.3%) with no mutations detected. The time to hepatotoxicity did not differ significantly between the 2 patients with one or two non-functional mutant alleles and the remaining 3 patients ( P=0.5). The TPMT genotype distribution differed slightly in the three different categories of disorders ( P=0.05). The median E-6-TGN level among the 2 TPMT heterozygous patients was 275 pmol/8x10(8) RBC (range 240-310), whereas the remaining 44 patients had a median E-6-TGN level of 110 pmol/8x10(8) RBC (range 0-440) ( P=0.07).. Although TPMT genotyping cannot be recommended on behalf of the present study, it is to be expected that half of the patients with one or two non-functional TPMT mutant alleles will develop Aza intolerance leading to withdrawal of therapy. Thus, clinicians may anticipate about 5% of the patients to develop intolerance to Aza therapy solely for that reason.

Topics: Adjuvants, Immunologic; Adult; Aged; Aged, 80 and over; Anti-Inflammatory Agents, Non-Steroidal; Azathioprine; Female; Genotype; Guanine Nucleotides; Humans; Male; Mercaptopurine; Methyltransferases; Middle Aged; Mutation; Polymerase Chain Reaction; Thionucleotides

There are conflicting reports on the role of azathioprine (AZA) thioguanine nucleotide (TGN) metabolites in optimising therapy for inflammatory bowel disease (IBD). The aim of this study was to investigate TGN intrapatient variation, and the relationship between TGN concentrations and disease activity in IBD patients taking long term constant dose AZA.. TGN and methylmercaptopurine nucleotide (MeMPN) concentrations were measured at intervals over a two year period. Disease activity was assessed at each clinic visit using the Crohn's disease activity index or Walmsley simple index for ulcerative colitis.. Serial TGNs were measured in 159 patients (3-14 TGN assays, median 6). Intrapatient variation in TGN concentrations was 1-5-fold (median 1.6); the incidence of non-compliance was 13%. At the end of two years, 131 patients were evaluable at TGN steady state. Of this group, patients who remained in remission had significantly higher mean TGN concentrations than those patients who developed active disease (median TGNs 236 v 175, respectively; median difference 44 pmol (95% confidence interval 1-92); p = 0.04). MeMPN concentrations were not related to AZA efficacy or toxicity.. This study has shown that lower TGN concentrations were linked to the development of active disease, and that TGNs may act as useful markers of compliance. However, it is clear that repeat TGN measurements are required for an unambiguous index of active metabolite exposure. In view of the high intrapatient variability in TGN production over time, TGN measurements may not be currently advocated for routine clinical use.

Topics: Adolescent; Adult; Aged; Alanine Transaminase; Antimetabolites, Antineoplastic; Azathioprine; Child; Child, Preschool; Colitis, Ulcerative; Crohn Disease; Erythrocytes; Female; Humans; Inflammatory Bowel Diseases; Male; Mercaptopurine; Middle Aged; Patient Compliance; Thioguanine; Treatment Outcome

Standardization of thiopurine metabolite testing is currently lacking. This paper presents in-depth methodological analysis and optimization of two currently available HPLC procedures (Lennard-Singleton [J. Chromatogr. 583 (1992) 83] and Dervieux-Boulieu [Clin. Chem. 44 (1998) 551]) to improve precision, turn-around time, ruggedness, and cost effectiveness.. Reversed-phase chromatography with UV detection was performed on a Waters HPLC system. The two protocols were improved with regards to internal standardization (IS), chromatographic conditions, as well as reagent preparation, storage, and use. 6-Thioguanine nucleotides (6-TGNs) were analyzed by our optimized techniques in samples from patients on thiopurine therapy (n = 24) and the results were compared.. 6-Mercaptopurine (6-MP) was an ideal internal standard in either procedure. Isocratic elution with 5% acetonitrile (ACN) in 20 mmol/l phosphate buffer pH 2.5 allowed for minimal background interference in both protocols. 6-Thioguanine, 6-mercaptopurine, and 6-methylmercaptopurine (6-MMP) eluted at around 4, 5, and 6 min, respectively. Dithiothreitol (DTT) was critical only during the acid hydrolysis step. Less mercury-containing waste was generated in the Lennard-Singleton procedure. With our optimized protocols recovery of 6-TGNs was on average 1.38-fold higher in the Dervieux-Boulieu method over a range of 10-678 pmol/8 x 10(8) RBC and no interfering peaks hindered analysis. Specific extraction of thiopurines before their analysis as per Lennard-Singleton procedure may be redundant.. We improved the quality and cost effectiveness of two known procedures for thiopurine metabolite assay. Through common chromatographic conditions and internal standardization, future comparison studies are now facilitated a great deal. The less tedious Dervieux-Boulieu procedure for routine thiopurine metabolite testing is warranted.

Topics: Chromatography, High Pressure Liquid; Dithiothreitol; Drug Monitoring; Humans; Linear Models; Mercaptopurine

We aimed at determining the utility of measuring 6-thioguanine (6-TG) and 6-methylmercaptopurine (6-MMP) in inflammatory bowel disease (IBD) patients on azathioprine (AZA) or 6-mercaptopurine (6-MP), whether the described therapeutic range for 6-TG (235-400 pmol/8 x 10(8) red blood cells, RBC) correlated with clinical remission or leukopenia, and if 6-MMP level was a marker for hepatotoxicity (>5,700 pmol/8 x 10(8) RBC).. Study eligibility included an IBD diagnosis of >6 months and either active disease or disease remission of <6 months and the use of AZA/6-MP for >10 wk consecutively. Metabolite levels were evaluated against clinical status, CBC, and hepatic parameters.. Seventy-four of 166 AZA/6-MP users were eligible. 6-TG levels >235 pmol/8 x 10(8) RBC were found in 22/59 (38%) with active disease and in 7/15 with remission (47%, p= 0.16). There was a trend of higher 6-TG levels among those in remission versus those with active disease (mean 325 +/- 284 vs 223 +/- 159 pmol/8 x 10(8) RBC, p= 0.2). No hepatotoxicity was observed, although 12.2% had 6-MMP levels > 5,700 pmol/8 x 10(8) RBC. The correlation between 6-MP dose and 6-TG levels was weak (r = 0.22, p= 0.08). The 6-TG level did not correlate with WBC. There were five instances, each of markedly low levels of both 6-TG and 6-MMP, suggesting noncompliance and of marked 6-MMP levels versus 6-TG.. There was a poor correlation between 6-TG levels and remission. Nonetheless, the measurements of these levels are helpful when patients are on high doses but not achieving remission since noncompliance or metabolism favoring 6-MMP can be established.

Topics: Adult; Azathioprine; Biomarkers; Cohort Studies; Colitis, Ulcerative; Crohn Disease; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Humans; Inflammatory Bowel Diseases; Male; Maximum Tolerated Dose; Mercaptopurine; Middle Aged; Predictive Value of Tests; Prognosis; Prospective Studies; Risk Assessment; Severity of Illness Index; Thioguanine; Treatment Outcome

The immunosuppressive effects of thiopurine drugs are mainly mediated through their intracellular metabolism into active 6-thioguanine nucleotide (6-TGN) metabolites, which are incorporated into DNA. Erythrocyte 6-TGN (E-6TGN) levels have been proposed as an instrument for monitoring treatment. The aim of the study was to use erythrocyte E-6TGN, methylated mercaptopurine (MeMP) metabolites, and thiopurine methyltransferase (TPMT) measurements in a clinical setting to determine the clinical outcome in relation to thiopurine metabolism.. Fifty-five adult patients with inflammatory bowel disease were included in a prospective study and followed for 6 months. Metabolite levels were measured and correlated to outcome and AZA/6-MP dose.. The E-6TGN level was significantly related to the TPMT genotype (P = 0.008). Patients in disease remission had higher E-6TGN levels than patients with disease activity both at baseline (P < 0.05) and after 6 months (P = 0.02). Active disease was more frequent among subjects with E-6TGN < or = 125 nmol/mmol Hb at baseline (P = 0.04), but not at 6 months. AZA/6-MP drug dose was positively correlated to E-MeMP levels (r = 0.48; P < 0.001) and E-MeMP/E-6TGN ratio (r = 0.41; P = 0.002). Dose changes were positively correlated with the changes in E-MeMP levels (P = 0.01) and E-MeMP/E-6TGN ratio (P = 0.03).. E-6TGN level was the only factor in this study related to disease activity, while there was no relationship between AZA/6-MP dose and E-6TGN levels. This finding illustrates the clinical usefulness of E-6TGN monitoring in the evaluation of treatment intensity.

Topics: Adolescent; Adult; Azathioprine; Drug Monitoring; Erythrocytes; Female; Genotype; Guanine Nucleotides; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Methyltransferases; Middle Aged; Sulfasalazine; Thionucleotides

Thiopurine drugs are commonly used immunosuppressants in the treatment of inflammatory bowel disease (IBD) as well as in autoimmune hepatitis (AIH), rheumatic diseases and in transplantation medicine. The relatively narrow therapeutic range requires useful therapy control. Therefore, the purpose of this study was to further investigate the rationale and usefulness of therapeutic drug monitoring in the surveillance of thiopurine drug therapy in Crohn's disease, ulcerative colitis and autoimmune hepatitis.. 6-Thioguanine nucleotide (TGN) and 6-methylmercaptopurine nucleotide (MMPN) levels were measured in 182 IBD patients and 18 AIH patients using HPLC-UV.. In our cohort of IBD patients, 18% had TGN levels < 235 pmol/8 x 10 red blood cells (RBC) (recommended range, 235-450 pmol/8 x 10 RBC), 41% of these patients were sent for drug failure. Twenty-four per cent of the IBD patients had TGN levels > 450 pmol/8 x 10 RBC, but only 27% of these experienced adverse effects. Fifty-nine per cent of the patients having drug failure had TGN levels in the recommended range and could therefore be classified as non-responders. In the AIH cohort 33% of the patients had TGN levels below the recommended range but showed clinical response to therapy. MMPN levels increased with the duration of treatment and could be useful for controlling compliance. There was 8.8% of IBD patients who were heterozygous for non-functional TPMT alleles.. TGN monitoring did not identify significant differences between patient groups but allowed the identification of non-responders from non-compliant patients and allowed the differentiation of mild side effects, such as malaise, from genuine toxicity caused by highly increased TGN levels.

Topics: Adolescent; Adult; Aged; Child; Child, Preschool; Chromatography, High Pressure Liquid; Cohort Studies; Colitis, Ulcerative; Crohn Disease; Drug Monitoring; Female; Guanine Nucleotides; Hepatitis, Autoimmune; Heterozygote; Humans; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Mercaptopurine; Middle Aged; Purines; Thioguanine; Thionucleotides

Azathioprine (AZA) and its active metabolite mercaptopurine (MP) are frequently used in the management of inflammatory bowel disease. Measurement of the AZA/MP metabolites, thioguanine (TG) and methylmercaptopurine (MMP), has been suggested as a means to optimize therapy with AZA/MP in inflammatory bowel disease.. To evaluate the results of initial AZA/MP metabolite panels sent by gastroenterologists during the first year of its widespread availability.. Initial AZA/MP metabolite panels sent by gastroenterologists to a single laboratory were reviewed and the metabolite panels were interpreted.. Initial metabolite levels were reviewed for 9187 patients. Noncompliance was detected in 263 patients (3%) and under-dosing in 4260 patients (46%). 534 patients (6%) had levels that were consistent with preferential metabolism via the TPMT pathway. The therapeutic goal was achieved in 2444 patients (27%) and an additional 552 patients (6%) had appropriate TG levels but potential hepatotoxicity. 936 patients (10%) had potential TPMT deficiency, and 58 patients (1%) had potential TPMT absence and were at risk for leukopenia. 140 patients (2%) had too high a dose.. Measurement of AZA/MP metabolites can be used by practising gastroenterologists to identify potential reasons for nonresponse to AZA or MP, and to identify patients at risk for certain drug-related toxicities.

Topics: Azathioprine; Humans; Inflammatory Bowel Diseases; Mercaptopurine; Patient Compliance; Risk Factors; Thioguanine

Monitoring 6-thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) activity is especially important when patients are treated with 6-thiopurine drugs, since severe bone marrow toxicity may be induced if patients have deficient TPMT activity.. We have developed a method based on high-performance liquid chromatography (HPLC) for the measurement of TPMT activity in various cell types: erythrocytes (RBC), human peripheral blood mononuclear cells (pMNC) and human malignant lymphoblasts (Molt-F4). The enzymatic activity is measured by the amount of 6-methylmercaptopurine formed, using 6-mercaptopurine (6MP) as substrate and S-adenosylmethionine as co-substrate.. The K(m) values calculated for 6MP were 0.54 (RBC), 0.85 (pMNC) and 0.65 (Molt-F4 cells) mmol/L. The K(m) values for S-adenosylmethionine were 11.9 (RBC), 16.4 (pMNC) and 6.65 (Molt-F4 cells) micro mol/L. The assay variation was 8.2-17%. TPMT activity was determined in a control group of 103 children and young adults (44 female, 59 male). The values observed were (mean +/- standard deviation): female children and young adults, 15.1 +/- 4.8 pmol/10(7) cells per h (n = 44); male children and young adults, 15.8 +/- 6.4 pmol/10(7) cells per h (n = 59). No gender or age differences were found.. The HPLC-based method enables the rapid screening of TPMT activities in large groups of patients treated with 6-thiopurines.

Topics: Bone Marrow; Child; Chromatography, High Pressure Liquid; Erythrocytes; Humans; Kinetics; Leukocytes, Mononuclear; Mercaptopurine; Methyltransferases; Reference Values; Reproducibility of Results; Time Factors

The aim of the present study was to develop a real-time reverse-transcription polymerase chain reaction (RT-PCR) methodology for the quantification of thiopurine methyltransferase (TPMT) gene expression in whole blood and compare it with the TPMT enzyme activity measured in red blood cells.. TPMT gene expression was quantified relative to the housekeeping gene cyclophilin (huCYC) and expressed as a TPMT/huCYC ratio. TPMT activity in red blood cells was determined by measuring the formation rate of 6-(14)C-methylmercaptopurine from 6-MP using S-adenosyl-L-((14)C-methyl)-methionine as methyl donor. Thirty-nine individuals were included in the study. A cut-off value of 9 U/ml pRBC was used to distinguish intermediate TPMT enzyme activity from high TPMT enzyme activity.. Sequencing of the real-time RT-PCR amplicon proved that the method was specific for the TPMT cDNA, without co-amplification of the highly similar TPMT processed pseudogene. The intra-assay coefficients of variation (CVs), as determined by the threshold cycle, were 0.7% for TPMT and 0.5% for huCYC. The interassay CVs were 1.5% for TPMT and 4.0% for huCYC. The intra- and interassay CVs, as determined by the TPMT/huCYC ratio, were 8.6% and 25%, respectively. There was a statistically significant correlation between TPMT enzyme activity and mRNA level in blood cells from individuals with an enzyme activity above 9 U/ml pRBC (r(s)=0.66, P=0.0001). However, we did not find any statistically significant correlation in individuals with lower enzyme activity or when analysing the whole population.. We present a specific and robust real-time RT-PCR method for quantifying TPMT gene expression. The method may be used for studies on TPMT gene regulation.

Topics: Erythrocytes; Gene Expression; Humans; Mercaptopurine; Methyltransferases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

To develop a non-high-performance liquid chromatography method for the determination of thiopurine- S-methyltransferase (TPMT) phenotype using thin-layer chromatography and quantitative scanning.. TPMT reaction was performed using a radiochemical assay. The reaction product [(14)C]-6-methylmercaptopurine was separated using thin-layer chromatography and quantified by means of radioactive scanning. Day-to-day variance was determined to validate results.. Determination of TPMT phenotype using thin-layer chromatography and quantitative scanning is reliable (day-to-day variance 8.5+/-1.7%, mean+/-SEM). Mean TPMT activity in 314 randomly selected patient samples was 11.8+/-3.3 units/ml red blood cells (mean+/-SD, range 3.5-25 units/ml).. We developed a new assay variant for the determination of TPMT phenotype that is easy to perform, reliable and reduces production of radioactive waste. This may lead to more frequent pretreatment determination of TPMT phenotype and increase drug safety and efficacy by individualising thiopurine doses.

Topics: Carbon Radioisotopes; Chromatography, Thin Layer; Humans; Indicators and Reagents; Mercaptopurine; Methyltransferases; Phenotype

6-Mercaptopurine (6-MP) and its prodrug azathioprine (AZA) have proven efficacy in the treatment of Crohn disease (CD). The immunosuppressive properties of AZA/6-MP are mediated by the intracellular metabolism of 6-MP into its active metabolites, 6-thioguanine nucleotides (6TGN) and 6methylmercaptopurine (6-MMP). Preliminary studies have suggested that the red blood cell concentration of 6TGN (RBC 6TGN) is a potential guide to therapy. The aims of the study were to evaluate the RBC 6TGN concentrations in adult patients with CD under long-term AZA/6-MP therapy and to correlate it with response to treatment and haematological parameters.. Twenty-eight CD patients treated for at least 3 months with AZA/6-MP were prospectively studied. Patients were separated into three main groups: group 1 (n = 19), corresponding to quiescent CD receiving AZA (dose: 2.05 +/- 0.4 mg/kg/day for a mean of 28.6 +/- 25 months) or 6-MP (dose: 1.4 +/- 01 mg/kg/day for a mean of 7.5 +/- 3.5 months) alone; group 2 (n = 6), corresponding to quiescent CD treated by AZA (dose: 2.14 +/- 0.5 mg/kg/day for a mean of 29.5 +/- 22 months) with oral steroids; and group 3 (n = 3), corresponding to active CD on AZA (dose: 1.94 +/- 0.6 mg/kg/day for a mean of 31.3 +/- 35 months) as the only treatment. An assessment was also made by merging groups 1 and 2 forming a larger group of patients (n = 25) defined by clinical remission and groups 2 and 3 forming a larger group of patients (n = 9), non-complete responders with AZA/6-MP alone. Crohn disease index activity (CDAI), blood samples for full blood count and differential white cell count and measurement of RBC 6TGN and 6-MMP concentrations were evaluated at inclusion and at 6 months (n = 17). RBC 6TGN were measured using high performance liquid chromatography (HPLC) on heparinized blood.. The baseline characteristics of the three groups of patients were similar. There was no significant difference among the three groups of patients regarding the dose and the duration of immunosuppressive treatment. There was no significant difference between groups according to various parameters tested. Particularly, the median RBC 6TGN concentration at inclusion was similar in the three groups of patients (166 (105-688), 183 (90-261) and 160 (52-194) pmol/8 x 10(8) RBC, respectively). The majority of patients had no detectable level of 6-MMP metabolite, except for 3 patients. There was also no difference between merging groups. Furthermore, there was no significant correlation between RBC 6TGN concentrations and the various biological parameters tested except for the mean erythrocyte volume. At 6 months, all patients of group 1 remained in remission and median RBC 6TGN concentration remained stable. No side effects were observed.. There is, contrary to preliminary studies, a broad overlap in RBC 6TGN levels as well as for haematological parameters in patients in remission or not and responders or not to AZA/6-MP therapy. This suggests, beside a variability in the metabolism of these drugs, the existence of complex mechanisms of action. Nevertheless, beside the use of RBC 6TGN determination to confirm compliance to therapy, this dosage could be useful in non-responding patients, allowing, in absence of leukopenia, to increase the dose of AZA/6-MP safely.

Topics: Adult; Azathioprine; Case-Control Studies; Crohn Disease; Drug Monitoring; Drug Therapy, Combination; Erythrocytes; Female; Follow-Up Studies; Humans; Immunosuppressive Agents; Male; Mercaptopurine; Prospective Studies; Thioguanine; Time Factors

The conversion of the cytotoxic and immunosuppressive 6-mercaptopurine (6MP) to the active 6-thioguanine nucleotides (6TGN) is necessary for clinical efficacy of 6MP and its prodrug azathioprine. Another metabolite, 6-methylmercaptopurine nucleotide (6MMPN), is formed via a competing pathway by thiopurine methyl transferase. The concentrations of 6TGN and 6MMPN are measured in washed erythrocytes as a surrogate to the intracellular levels of these metabolites in the target tissues. Analysis of 6TGN and 6MMPN in multi-center clinical studies is more complicated because of the requirement to wash erythrocytes. In this investigation, we found no differences in the concentrations of 6TGN and 6MMPN in blood versus washed erythrocytes in samples obtained from patients taking therapeutic doses of oral 6MP or azathioprine for inflammatory bowel disease. We concluded that whole blood could be used for the analysis of these analytes, thus saving sample preparation time. We also found that the erythrocyte 6TGN concentration in blood at ambient temperature declined 2-4% per day, a loss that can be avoided by shipping blood samples frozen. The loss of 6TGN in blood stored at approximately -80 degrees C was 1% after 1 week and 12% after 24 weeks, indicating the analyte was moderately stable. 6MMPN in blood did not significantly change after 24 weeks of storage at approximately -80 degrees C. In addition, the sensitivity of the 6TGN assay was improved by modifying the HPLC conditions, which made the method more suitable for quantifying low levels of 6TGN in human intestinal biopsy samples and blood.

Topics: Chromatography, High Pressure Liquid; Humans; Inflammatory Bowel Diseases; Mercaptopurine; Reproducibility of Results; Spectrometry, Fluorescence; Thioguanine

Thioguanine and mercaptopurine are prodrugs requiring conversion into thiopurine nucleotides to exert cytotoxicity. Thiopurine S-methyltransferase (TPMT), an enzyme subject to genetic polymorphism, catabolizes thiopurines into inactive methylated bases, but also produces methylthioguanine nucleotides and methylmercaptopurine nucleotides from thioguanine and mercaptopurine nucleotides, respectively. To study the effect of TPMT on activation versus inactivation of mercaptopurine and thioguanine, we used a retroviral gene transfer technique to develop human CCRF-CEM cell lines that did (TPMT+) and did not (MOCK) overexpress TPMT. After transduction, TPMT activities were 14-fold higher in the TPMT+ versus the MOCK cell lines (P < 0.001). TPMT+ cells were less sensitive to thioguanine than MOCK cells (IC(50) = 1.10+/- 0.12 microM versus 0.55 +/- 0.19 microM; P = 0.02); in contrast, TPMT+ cells were more sensitive to mercaptopurine than MOCK cells (IC(50) = 0.52 +/- 0.20 microM versus 1.50 +/- 0.23 microM; P < 0.01). The lower sensitivity of TPMT+ versus MOCK cells to thioguanine was associated with lower thioguanine nucleotide concentrations (917 +/- 282 versus 1515 +/- 183 pmol/5 x 10(6) cells; P = 0.01), higher methylthioguanine nucleotide concentrations (252 +/- 34 versus 27 +/- 10 pmol/5 x 10(6) cells; P = 0.01), less inhibition of de novo purine synthesis (13 versus 95%; P < 0.01), and lower deoxythioguanosine incorporation into DNA (2.0 +/- 0.6% versus 7.2 +/- 2.0%; P < 0.001). The higher sensitivity of TPMT+ cells to mercaptopurine was associated with higher concentrations of methylmercaptopurine nucleotide (2601 +/- 1055 versus 174 +/- 77 pmol/5 x 10(6) cells; P = 0.01) and greater inhibition of de novo purine synthesis (>99% versus 74%; P < 0.01) compared with MOCK cells. We conclude that methylation of mercaptopurine contributes to the antiproliferative properties of the drug, probably through inhibition of de novo purine synthesis by methylmercaptopurine nucleotides, whereas thioguanine is inactivated primarily by TPMT.

Topics: 3T3 Cells; Animals; Antimetabolites, Antineoplastic; Biotransformation; Cytosol; Deoxyguanosine; DNA, Neoplasm; Gene Transfer Techniques; HeLa Cells; Humans; Leukemia-Lymphoma, Adult T-Cell; Mercaptopurine; Methyltransferases; Mice; Purine Nucleotides; Purines; Retroviridae; Thioguanine; Thionucleosides; Thionucleotides; Tumor Cells, Cultured

The current article describes a new assay to measure thiopurine methyltransferase (TPMT) activity from red blood cells. This method is based on the measurement of the reaction product 6-methylmercaptopurine (6-MMP) by high-performance liquid chromatography (HPLC). 6-MMP is extracted by ethyl acetate with recoveries of 85%, 80%, 80%, and 92% for 50, 250, 500, and 1,000 ng/100 microL packed red blood cells, respectively. 6-MMP was identified and measured by a Zorbax CN column installed in an HPLC system. The chromatograms were resolved using a mobile phase consisting of 40 mmol/L sodium phosphate buffer (pH 3) and methanol in a gradient from 1% to 20% of methanol. Under these conditions 6-MMP is well resolved from substrates (6-mercaptopurine and S-adenosyl-L-methionine) and endogenous peaks. When the TPMT activity from 20 patients was measured by the HPLC-linked assay and the classic radiochemical method, a linear correlation was obtained between both procedures ( y = 0.99x + 0.33; x-axis, radiochemical assay; y-axis, HPLC-linked assay; r = 0.98). In conclusion, the current report describes a new, reliable, safe, and nonradioactive method to measure TPMT activity that is shorter and simpler than the previously described ones.

Topics: Antimetabolites, Antineoplastic; Azathioprine; Chromatography, High Pressure Liquid; Drug Monitoring; Erythrocytes; Humans; Linear Models; Mercaptopurine; Methyltransferases; Radiochemistry; S-Adenosylmethionine; Thioguanine

The metabolism of azathioprine (AZA) was studied by monitoring the concentrations of red blood cell (RBC) 6-thioguanine nucleotides (6-TGN) and of 6-methylmercaptopurine metabolites (6-mMP) in 27 renal transplantation recipients and in 10 patient subjects with glomerulonephritis (GN). Concentrations of 6-TGNs and 6-mMP metabolites were measured using high-performance liquid chromatography (HPLC). Six patients from the group of renal transplantation recipients were also administered allopurinol. Median values of RBC 6-TGN and of 6-mMP metabolites concentrations in 21 renal transplantation recipients (without allopurinol) were 122 pmol/8x10(8) RBCs (range, <60-298) and 280 pmol/8x10(8) RBC (range, <150-1330), respectively; there was no correlation between concentrations of 6-TGN and of 6-mMP metabolites. The group of 21 renal transplantation recipients received different AZA doses (100 or 50 mg/d) related to clinical symptoms of AZA intolerance. The median values of 6-TGN concentrations in these subgroups were 131 and 122 pmol/8x10(8) RBCs and were not significantly different. Median values of 6-TGN concentrations in patients given allopurinol were significantly higher, despite AZA dose reduction, compared with the group without allopurinol and were equal to 363 and 122 pmol/8x10(8) RBC, p < 0.004, respectively. No significant differences were found between the concentrations of 6-mMP metabolites in either group. In the group of renal transplantation recipients, a significant correlation between white blood cell (WBC) count and 6-TGN concentration was established (r(s) = -0.59, p < 0.005). In the group of GN patients, the median values of 6-TGN and of 6-mMP metabolites concentrations were 108 pmol/8x10(8) RBCs (range, 0-297) and 420 pmol/8x10(8) RBC (range, 0-1440), respectively. There were no significant correlations between either the WBC count and 6-TGN concentrations or between 6-TGN concentrations and 6-mMP metabolites. We expect the results of our study to provide indications for better individualization of AZA therapy.

Topics: Adult; Allopurinol; Antimetabolites; Azathioprine; Erythrocytes; Female; Glomerulonephritis; Humans; Immunosuppressive Agents; Kidney Transplantation; Male; Mercaptopurine; Middle Aged; Thioguanine

Angiogenesis has been identified as an important target for antineoplastic therapy. The use of purine analogue antimetabolites in combination chemotherapy of solid tumors has been proposed. To assess the possibility that selected purine analogues may affect tumor neovascularization, 6-methylmercaptopurine riboside (6-MMPR), 6-methylmercaptopurine, 2-aminopurine, and adenosine were evaluated for the capacity to inhibit angiogenesis in vitro and in vivo. 6-MMPR inhibited fibroblast growth factor-2 (FGF2)-induced proliferation and delayed the repair of mechanically wounded monolayer in endothelial GM 7373 cell cultures. 6-MMPR also inhibited the formation of solid sprouts within fibrin gel by FGF2-treated murine brain microvascular endothelial cells and the formation of capillary-like structures on Matrigel by murine aortic endothelial cells transfected with FGF2 cDNA. 6-MMPR affected FGF2-induced intracellular signaling in murine aortic endothelial cells by inhibiting the phosphorylation of extracellular signal-regulated kinase-2. The other molecules were ineffective in all of the assays. In vivo, 6-MMPR inhibited vascularization in the chick embryo chorioallantoic membrane and prevented blood vessel formation induced by human endometrial adenocarcinoma specimens grafted onto the chorioallantoic membrane. Also, topical administration of 6-MMPR caused the regression of newly formed blood vessels in the rabbit cornea. Thus, 6-MMPR specifically inhibits both the early and the late phases of the angiogenesis process in vitro and exerts a potent anti-angiogenic activity in vivo. These results provide a new rationale for the use of selected purine analogues in combination therapy of solid cancer.

Topics: 2-Aminopurine; Adenocarcinoma; Adenosine; Allantois; Animals; Antimetabolites, Antineoplastic; Antineoplastic Agents; Aorta; Brain; Calcium-Calmodulin-Dependent Protein Kinases; Cattle; Cells, Cultured; Chick Embryo; Chorion; Cornea; Depression, Chemical; Endometrial Neoplasms; Endothelium, Vascular; Female; Fibroblast Growth Factor 2; Humans; Mercaptopurine; Methylthioinosine; Mice; Mitogen-Activated Protein Kinase 1; Neoplasm Transplantation; Neovascularization, Physiologic; Phosphorylation; Protein Processing, Post-Translational; Rabbits; Recombinant Fusion Proteins; Signal Transduction; Transfection

Methods of assaying 6-mercaptopurine (6MP) and its methylated and non-methylated metabolites are essential for the therapeutic dose in treating patients with acute lymphoblastic leukemia. However, previous methods are technically complicated and unsuitable for clinical use. Thus, we have now developed a method utilizing reversed-phase high-performance liquid chromatography (HPLC) in order to quantify these compounds in human red blood cells (RBCs) in a single sample to serve as an index of cytotoxic activity. The agents 6MP, 6-thioguanine (6TG) and 6-methylmercaptopurine (6MMP) were well separated by this assay. Linear relationships were observed between the peak areas and the RBC concentrations of 6MP, 6TG and 6MMP over the range of 20-2000, 18-1800 and 18-1800 pmol per 25 mg hemoglobin (Hb), respectively. The limit of quantitation of the assay is 20, 18 and 18 pmol per 25 mg Hb, respectively. This assay system is suitable for routine clinical use.

Topics: Antimetabolites, Antineoplastic; Chromatography, High Pressure Liquid; Erythrocytes; Hemoglobins; Humans; Mercaptopurine; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Quality Control; Reproducibility of Results; Thioguanine

6-Thioguanine and 6-methylmercaptopurine (Me6-MP) nucleotides are the two major thiopurine metabolites of azathioprine found in erythrocytes. During the acid hydrolysis required for the conversion of thiopurine nucleotides into their free bases, Me6-MP was converted into a compound that could be analyzed on a Purospher RP18-e column with dihydrogen phosphate-methanol buffer as eluent. The pH of the acid extract strongly influenced the conversion of Me6-MP into its derivative. The Me6-MP derivative was identified using liquid chromatography-mass spectrometry and infrared and nuclear magnetic resonance spectrometric methods. During the acid hydrolysis of thiopurine nucleotides in erythrocytes, Me6-MP undergoes degradation, leading to 4-amino-5-(methylthio)carbonyl imidazole.

Topics: Azathioprine; Chromatography, Liquid; Erythrocytes; Humans; Hydrolysis; Imidazoles; Immunosuppressive Agents; In Vitro Techniques; Magnetic Resonance Spectroscopy; Mass Spectrometry; Mercaptopurine; Perchlorates; Spectrophotometry, Infrared; Thionucleotides

A non-radioactive method that uses reverse-phase high performance liquid chromatography is described for the determination of thiopurine methyltransferase (E.C. 2.1.1.67) activity in human erythrocytes. The method is based on the direct quantitation of 6-methyl-mercaptopurine produced from 6-mercaptopurine by crude erythrocyte lysates. The method is accurate and reliable and suitable for diagnostic use. Activity values in control adults ranged from 5 to 32 pmol/h/mg haemoglobin. The activity in the erythrocytes of adult males was significantly higher compared to females (21 +/- 5 and 15 +/- 8 pmol/h/mg haemoglobin, respectively). The activity measured in the erythrocytes of children (22 +/- 5 pmol/h/mg haemoglobin) did not show any significant difference compared to adults. Thiopurine methyltransferase activity was measured in a female patient with systemic sclerosis who developed severe bone marrow depression after treatment with azathioprine and allopurinol. Activity (6.3 +/- 0.5 pmol/h/mg haemoglobin) was found in the lowest range of controls thus supporting the hypothesis that it could be responsible for increased azathioprine cytotoxicity.

Topics: Adolescent; Adult; Child; Child, Preschool; Chromatography, High Pressure Liquid; Erythrocytes; Female; Humans; Male; Mercaptopurine; Methyltransferases

Measurement of red cell 6-mercaptopurine (MP) derived 6-thioguanine nucleotide (TGN) and methylmercaptopurine metabolites (MeMPs) can be used to monitor therapy in children who are administered MP for childhood lymphoblastic leukemia. Red cell TGNs are not influenced by the time of blood sampling in relation to the last MP dose. The purpose of this study was to find out whether the same is true for the MeMPs. Plasma MP and red cell MP metabolite pharmacokinetics were studied in seven children immediately before and for 4 hours after a protocol standardized dose of MP. Duplicate blood samples were taken, one was processed immediately whereas one was left at an ambient temperature for 24 hours. The variation in TGN and MeMP metabolites over the 0- to 4-hour period (10 time points per child) was within the error of the assays used. The coefficients of variation for the TGNs ranged from 2.7% to 7% and for the MeMPs, 4% to 10.7%. There was no difference in the TGN and MeMP concentrations measured when the blood samples were left for 24 hours. If a child takes a MP tablet immediately before a clinic appointment, it has no major influence on MeMP measurements.

Topics: Administration, Oral; Antimetabolites, Antineoplastic; Child; Child, Preschool; Erythrocytes; Female; Guanine Nucleotides; Humans; Male; Mercaptopurine; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Thionucleotides; Time Factors

The drug combination N-(phosphonacetyl)-L-aspartic acid (PALA), methylmercaptopurine riboside (MMPR) and 6-aminonicotinamide (6AN), referred to as PMA, induces regressions of advanced CD8F1 murine mammary carcinomas in vivo. We demonstrated that CD8F1 tumor regressions were preceded by the appearance of apoptotic bodies, as observed by microscopic examination of morphology and TUNEL endlabeling, and fragmentation of DNA into nucleosomal "ladder" patterns. These indications of apoptosis were present as early as 6 h after simultaneous administration of MMPR and 6AN and further increased by over fivefold during the next 3 to 6 h, then remained at 7 to 12.8% (0.6 to 2.4% in saline-treated controls) of the cell population for at least 24 h after MMPR + 6AN administration. The 5'-phosphate derivative of MMRP, MMPR-5P, which inhibits de novo purine biosynthesis, was present at a "steady-state" level, and significant (40%) depletion of ATP had occurred by 3 h and both of these events preceded the onset of apoptosis. In addition, MMPR-5P was retained in CD8F1 tumors at a high level over a prolonged period (> 96 h) even as tumors were undergoing regression. The prolonged presence of MMPR-5P was important for optimal chemotherapeutic effect, since treatment with iodotubercidin (IodoT), an inhibitor of MMPR/adenosine kinase, 6 h after MMPR+6AN administration prevented the prolonged accumulation of MMPR-5P and reversed the regression of CD8F1 tumors. In addition, compared to the PMA-treated group, there was a significant restoration of ATP levels after treatment with IodoT. In individual PMA-treated CD8F1 tumors the degree of ATP depletion was found to correlate with the degree of tumor shrinkage at 24 h, after tumors had sufficient time to respond to treatment. These results define the time-course of drug-induced apoptosis in CD8F1 tumors, show that ATP depletion occurs prior to apoptosis and demonstrate that prolonged retention of MMPR-5P is associated with optimal chemotherapy. Collectively, these results suggest that the depletion of ATP by PMA treatment may be a component of the biochemical apoptotic cascade in the CD8F1 tumor.

Topics: 6-Aminonicotinamide; Adenosine Triphosphate; Animals; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Aspartic Acid; Female; Mammary Neoplasms, Experimental; Mercaptopurine; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Phosphonoacetic Acid; Time Factors

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.

Topics: Chromatography, High Pressure Liquid; Erythrocytes; Humans; Mercaptopurine; Methyltransferases

A three-drug combination, PMA, consisting of (phosphonacetyl)-L-aspartic acid + 6-methylmercaptopurine riboside + 5-aminonicotinamide, preceding either 5-fluorouracil (5-FU) or adriamycin (Adr), produced tumor-regressing activity in a murine advanced breast tumor model not attainable with either 5-FU or Adr as single agents, or with any lesser combination of these drugs administered at maximally tolerated doses. Marked tumor-regressing activity was further increased significantly by using 5-FU and Adr together in conjunction with the modulatory biochemical conditioning (particularly ATP depletion) provided by pretreatment with PMA.

Topics: Animals; Antibiotics, Antineoplastic; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Aspartic Acid; Doxorubicin; Fluorouracil; Mammary Neoplasms, Animal; Mercaptopurine; Mice; Niacinamide; Phosphonoacetic Acid

The reaction of 16 alpha,17 alpha-epoxy-3 beta-hydroxy-5-pregnen-20-one with 6-methyl thiopurine activated with sodium hydride leads to the coupling of the purine base with the carbonyl group at C-20 to give a steroidal nucleoside analog, which is termed "nucleosteroid." In the presence of an excess of purine, a parallel reaction occurs in which the oxirane ring is opened, presumably by nucleophilic attack of an intermediate C-20 oxyanion, and yields as the main product of reaction an oligomeric mixture of nucleosteroid units linked together by ether linkages. Analogous reactions conducted with 3 beta-hydroxy-5-pregnen-20-one and with 3 beta,17 alpha-dihydroxy-5-pregnen-20-one gave minor amounts or only traces of the corresponding coupling adduct, and oligomerization did not occur. This behavior is interpreted in terms of the conformational differences showed by the different steroids to the attack by the purine.

Topics: Acetylation; Magnetic Resonance Spectroscopy; Mass Spectrometry; Mercaptopurine; Molecular Structure; Pregnenes; Purines; Steroids

6-Methylmercaptopurine ribonucleoside-5'-phosphate (MeSPuRMP), the sole metabolite of 6-methylmercaptopurine ribonucleoside (MeSPuRib), is a strong inhibitor of purine de novo synthesis, inducing depletion of intracellular purine nucleotides and subsequent cell death in several tumor cell lines. In this study prevention of MeSPuRib cytotoxicity by compounds of the purine salvage pathway was studied in Molt F4 human malignant T-lymphoblasts. Adenosine, adenine and inosine were able to prevent depletion of the adenine nucleotide pool when used in combination with 0.5 microM MeSPuRib, but had virtually no effect on depletion of guanine nucleotides. Nevertheless, these three purine compounds were able to reduce the cytotoxic effects induced by MeSPuRib. Addition of guanosine to cells treated with 0.5 microM MeSPuRib normalized the guanine nucleotide pool, but adenine nucleotides remained depleted. Under these conditions, inhibition of cell growth was significantly decreased. With the combination of guanosine and 10 microM MeSPuRib, cytotoxicity was increased compared to 10 microM MeSPuRib alone, associated with a depletion of adenine nucleotides to 9% of untreated cells. Since cell growth and cell viability of Molt F4 cells are less inhibited by MeSPuRib under conditions where adenine nucleotide depletion is prevented by purine compounds (and where the other nucleotides are depleted) we conclude that depletion of adenine nucleotides is an important factor in MeSPuRib cytotoxicity.

Topics: Adenine; Adenosine; Antineoplastic Agents; Cell Division; Cell Survival; Cells, Cultured; Guanosine; Humans; Inosine; Mercaptopurine; Methylthioinosine; Nucleosides; Nucleotides; Purine Nucleosides; Ribonucleosides; T-Lymphocytes; Thioinosine; Thionucleotides

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).

Topics: Adult; Chromatography, High Pressure Liquid; Female; France; Gallic Acid; Humans; Male; Mercaptopurine; Methyltransferases; Phenotype; Polymorphism, Genetic

The combination of N-(phosphonacetyl)-L-aspartate, 6-methylmercaptopurine, and 6-aminonicotinamide has been shown to be an effective antineoplastic regimen and also to enhance the effects of other chemotherapeutic agents. The mechanism of action of this combination of drugs is not known definitively, but one possible mechanism is biochemical modulation of energy metabolism and inhibition of production of tumor ATP. Tumor-bearing mice were treated with N-(phosphonacetyl)-L-aspartate, followed 17 h later by 6-methylmercaptopurine and 6-aminonicotinamide. 31P nuclear magnetic resonance spectroscopic studies demonstrated a significant depletion of high energy phosphates at 10 h post-6-methylmercaptopurine and 6-aminonicotinamide. The addition of radiation at this time was shown to induce a significantly longer tumor growth delay and a greater number of regressions (including durable complete regressions) than either chemotherapy or radiation alone. The combination of chemotherapy and radiation was found to be supra-additive compared to the antineoplastic effects of either modality administered separately, without a measurable increase in host toxicity.

Topics: 6-Aminonicotinamide; Animals; Antineoplastic Combined Chemotherapy Protocols; Aspartic Acid; Combined Modality Therapy; Female; Mercaptopurine; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Neoplasm Transplantation; Neoplasms, Experimental; Phosphonoacetic Acid

6-Chloropurine (CP) has antitumor activity against animal and human neoplasms, but the mechanism is unclear. Recently, we have shown that S-(6-purinyl)glutathione (PG), a putative metabolite of CP, is metabolized in vivo to yield the antitumor drug, 6-mercaptopurine (6-MP). In this study, CP metabolism to PG and 6-MP was investigated in an effort to provide further insights into the mechanism of CP antitumor activity. Rat hepatic and renal glutathione S-transferases metabolized CP to PG; Vmax values for liver and kidney cytosol were 166 and 24 nmol/mg of protein/min, respectively. PG was isolated and characterized by fast atom bombardment mass spectrometry from the bile of rats given CP. When rats were given CP (14 mumol/kg), PG excretion was linear with time for up to 1 hr; nearly 80% of the PG excreted at 2 hr was excreted at 1 hr. Rats given CP (10-1200 mumol/kg) excreted at 1 hr into bile nearly 18% of the dose as PG; rats given CP (400-1200 mumol/kg) excreted at 24 hr into urine nearly 4% of the dose as 6-MP and its further metabolites, 6-methylthiopurine and 6-thiouric acid. CP, PG, 6-MP, 6-methylthiopurine and 6-thiouric acid were also detected in plasma, liver and kidney of rats given CP (1200 mumol/kg); in these tissues, maximum CP concentrations were observed at 30 min, as compared to 60 to 180 min, and plasma CP concentrations were higher than those detected in liver or kidney. Liver or kidney CP metabolite concentrations at 30 to 120 min were, however, higher than those detected in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bile; Biliary Tract; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Glutathione; Glutathione Transferase; Kidney; Liver; Male; Mercaptopurine; Microsomes; Proteins; Purines; Rats; Rats, Sprague-Dawley; Time Factors; Uric Acid

A reversed-phase high-performance liquid chromatographic (HPLC) procedure was developed to quantify intracellular lymphocyte 6-thioguanine, methylmercaptopurine and methylthioguanine. The free base of each metabolite was obtained by acid hydrolysis, which allowed for a total determination of thiopurine metabolites. 6-Thioguanine was analyzed on an octadecylsilane column using acetonitrile-10 mM sodium phosphate (11:89), pH 7, containing 0.06% tetrabutylammonium chloride. 6-Thioguanine was oxidized with potassium permanganate, and fluorescence was measured at 330 nm excitation and 410 nm emission. Methylmercaptopurine and methylthioguanine were separated on a cyanopropylsilane column using methanol-40 mM sodium phosphate (22:78), pH 2.7, and detected by ultraviolet absorbance at 314 and 290 nm, respectively.

Topics: Chromatography, High Pressure Liquid; Fluorescence; Humans; Lymphocytes; Mercaptopurine; Thioguanine

Topics: Bromosuccinimide; Coenzymes; Mercaptopurine; Nucleosides; Oxidation-Reduction; Pyrrolidinones