A practical guide to the use of thiopurines in oral medicine.

Thiopurines are widely used as first-line immunosuppressive therapies in the management of chronic inflammatory oral disease. However, despite over half a century of clinical experience, the evidence base for their use is limited. The aims of this paper were to review the evidence for the use of thiopurines in oral medicine and provide a contemporary model of thiopurine metabolism and mechanism of action and a rationale for clinical use and safe practice.

Pharmacogenetics of pemetrexed combination therapy in lung cancer: pathway analysis reveals novel toxicity associations.

Identification of polymorphisms that influence pemetrexed tolerability could lead to individualised treatment regimens and improve quality of life. Twenty-eight polymorphisms within eleven candidate genes were genotyped using the Illumina Human Exome v1.1 BeadChip and tested for their association with the clinical outcomes of non-small cell lung cancer and mesothelioma patients receiving pemetrexed/platinum doublet chemotherapy (n=136). GGH rs11545078 was associated with a reduced incidence of grade ⩾3 toxicity within the first four cycles of therapy (odds ratio (OR) 0.25, P=0.018), as well as reduced grade ⩾3 haematological toxicity (OR 0.13, P=0.048). DHFR rs1650697 conferred an increased risk of grade ⩾3 toxicity (OR 2.14, P=0.034). Furthermore, FOLR3 rs61734430 was associated with an increased likelihood of disease progression at mid-treatment radiological evaluation (OR 4.05, P=0.023). Polymorphisms within SLC19A1 (rs3788189, rs1051298 and rs914232) were associated with overall survival. This study confirms previous pharmacogenetic associations and identifies novel markers of pemetrexed toxicity.

Mechanism of allopurinol induced TPMT inhibition.

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.

Pharmacogenetic variants in the DPYD, TYMS, CDA and MTHFR genes are clinically significant predictors of fluoropyrimidine toxicity.

BACKGROUND: Fluoropyrimidine drugs are extensively used for the treatment of solid cancers. However, adverse drug reactions are a major clinical problem, often necessitating treatment discontinuation. The aim of this study was to identify pharmacogenetic markers predicting fluoropyrimidine toxicity. METHODS: Toxicity in the first four cycles of 5-fluorouracil or capecitabine-based chemotherapy were recorded for a series of 430 patients. The association between demographic variables, DPYD, DPYS, TYMS, MTHFR, CDA genotypes, and toxicity were analysed using logistic regression models. RESULTS: Four DPYD sequence variants (c.1905+1G>A, c.2846A>T, c.1601G>A and c.1679T>G) were found in 6% of the cohort and were significantly associated with grade 3-4 toxicity (P<0.0001). The TYMS 3'-untranslated region del/del genotype substantially increased the risk of severe toxicity (P=0.0123, odds ratio (OR)=3.08, 95% confidence interval (CI): 1.38-6.87). For patients treated with capecitabine, a MTHFR c.1298CC homozygous variant genotype predicted hand-foot syndrome (P=4.1 × 10⁻6, OR=9.99, 95% CI: 3.84-27.8). The linked CDA c.-92A>G and CDA c.-451C>T variants predicted grade 2-4 diarrhoea (P=0.0055, OR=2.3, 95% CI: 1.3-4.2 and P=0.0082, OR=2.3, 95% CI: 1.3-4.2, respectively). CONCLUSION: We have identified a panel of clinically useful pharmacogenetic markers predicting toxicity to fluoropyrimidine therapy. Dose reduction should be considered in patients carrying these sequence variants.

The impact of introducing thioguanine nucleotide monitoring into an inflammatory bowel disease clinic.

BACKGROUND: Thioguanine nucleotides (TGNs) are the active product of thiopurine metabolism. Levels have been correlated with effective clinical response. Nonetheless, the value of TGN monitoring in clinical practice is debated. We report the influence of introducing TGN monitoring into a large adult inflammatory bowel disease (IBD) clinic. PATIENTS AND METHODS: Patients with IBD undergoing TGN monitoring were identified from Purine Research Laboratory records. Whole blood TGNs and methylated mercaptopurine nucleotides were hydrolysed to the base and measured using HPLC. Clinical and laboratory data were obtained retrospectively. RESULTS: One hundred and eighty-nine patients with 608 available TGN results were identified. In non-responders, TGNs directed treatment change in 39/53 patients. When treatment was changed as directed by TGN, 18/20 (90%) improved vs. 7/21 (33%) where the treatment decision was not TGN-directed, p < 0.001. Where treatment change was directed at optimisation of thiopurine therapy, 14/20 achieved steroid-free remission at 6 months vs. 3/10 where the TGN was ignored, (p = 0.037). Six per cent of patients were non-adherent, 25% under-dosed and 29% over-dosed by TGN. Twelve per cent of patients predominantly methylated thiopurines, this group had low TGN levels and high risk of hepatotoxicity. In responders, adherence and dosing issues were identified and TGN-guided dose-reduction was possible without precipitating relapse. Mean cell volume (MCV), white blood cell count (WBC) and lymphocyte counts were not adequate surrogate markers. MCV/WBC ratio correlated with clinical response, but was less useful than TGN for guiding clinical decisions. CONCLUSIONS: Monitoring TGNs enables thiopurine therapy to be optimised and individualised, guiding effective treatment decisions and improving clinical outcomes.

Methotrexate polyglutamates as a marker of patient compliance and clinical response in psoriasis: a single-centre prospective study.

BACKGROUND: Methotrexate is activated by the sequential addition of glutamic acid residues to form methotrexate polyglutamates (MTXPG(1-5)). MTXPG(1-5) inhibit enzymes of the folate-purine-pyrimidine pathways, and longer-chain MTXPG(3-5) species are more active. OBJECTIVES: To determine the pattern of erythrocyte MTXPG(1-5) in patients initiated on oral methotrexate for psoriasis, and to investigate the potential utility of MTXPGs as markers of compliance and/or clinical response. METHODS: This was a single-centre, prospective study of 55 adult patients with chronic plaque psoriasis initiated on weekly oral methotrexate. Erythrocyte MTXPG(1-5) concentrations were measured (at weeks 4, 8, 12, 24 and 52) using high-performance liquid chromatography. Methotrexate responders achieved ≥ 50% improvement in Psoriasis Area and Severity Index or physician's global score of 'clear'/'nearly clear' at 24 weeks. RESULTS: MTXPG levels were measured in 14-33 patients at each time point. All MTXPG(1-5) species were detected at week 4 of therapy. Steady state for long-chain MTXPG(3-5) and total MTXPG(1-5) was achieved by week 24. MTXPG(3) emerged as the predominant MTXPG species (from week 12 onwards) and reflected overall polyglutamate status (correlating strongly with MTXPG(2-5) , MTXPG(3-5) and MTXPG(4-5) ; R = 0·76-0·95, P < 1·55 × 10(-5)). Age, renal function and sex were not significant determinants of MTXPG(3) concentration. No significant association was identified between MTXPG and adverse events or responder status. CONCLUSIONS: This is the first study to demonstrate the prospective accumulation of MTXPG(1-5) in patients with psoriasis. The detection of MTXPGs early in therapy and the establishment of a steady state with continuous treatment may offer measuring of MTXPG as a test to monitor patient compliance with therapy. Larger studies are required to determine the role of MTXPG as a potential biomarker of clinical response.

HPRT deficiency: identification of twenty-four novel variants including an unusual deep intronic mutation.

Hypoxanthine phosphoribosyltranferase (HPRT) deficiency is an X-linked disorder of purine salvage that ranges phenotypically from hyperuricaemia to Lesch-Nyhan Syndrome. Molecular testing is necessary to identify female carriers within families as a prelude to prenatal diagnosis. During the period 1999-2010 the Purine Research Laboratory studied 106 patients from 68 different families. Genomic sequencing revealed mutations in 88% of these families, 24 of which were novel. In eight patients, exon sequencing was not informative. Copy-DNA analysis in one patient revealed an insertion derived from a deep intronic sequence with a genomic mutation flanking this region, resulting in the creation of a false exon. Carrier testing was performed in 21 mothers of affected patients, out of these, 81% (17) were found to be carriers of the disease-associated mutation. Our results confirm the extraordinary variety and complexity of mutations in HPRT deficiency. A combination of genomic and cDNA sequencing may be necessary to define mutations.

Review article: malignancy on thiopurine treatment with special reference to inflammatory bowel disease.

BACKGROUND: Immunosuppression is a risk factor for carcinogenesis. Thiopurines specifically contribute to this. As thiopurines are used more aggressively in the treatment of IBD, it is likely that we will see more thiopurine-related malignancy. AIM: To review the literature, exploring how immunosuppression, thiopurines specifically, might cause cancer and which malignancies occur in practice, placing specific emphasis on IBD cohorts. METHODS: Search terms included 'malignancy' 'cancer' 'azathioprine' 'mercaptopurine' 'tioguanine (thioguanine)' 'thiopurine' and 'inflammatory bowel disease' 'Crohn's disease' 'ulcerative colitis'. We also searched for specific cancers (lymphoma, colorectal cancer, skin cancer, cervical cancer) and reviewed the reference lists of the articles detected. RESULTS: Immunosuppression is associated with an increased risk of cancer. Thiopurines are associated with specific additional risks. In IBD cohorts, very few thiopurine-related malignancies have been reported. However, studies suggest a relative risk of 4-5 for lymphoma. This still translates into a low actual risk, (one extra lymphoma in every 300-1400 years of thiopurine treatment). CONCLUSIONS: Whilst we must be aware of this risk and counsel our patients appropriately, thiopurines remain a mainstay of IBD therapy. We present practical advice aimed at minimizing our patients' risk of developing malignancy, whilst optimizing the benefits that thiopurines can provide.

Novel pharmacogenetic markers for treatment outcome in azathioprine-treated inflammatory bowel disease.

BACKGROUND: Azathioprine (AZA) pharmacogenetics are complex and much studied. Genetic polymorphism in TPMT is known to influence treatment outcome. Xanthine oxidase/dehydrogenase (XDH) and aldehyde oxidase (AO) compete with TPMT to inactivate AZA. AIM: To assess whether genetic polymorphism in AOX1, XDH and MOCOS (the product of which activates the essential cofactor for AO and XDH) is associated with AZA treatment outcome in IBD. METHODS: Real-time PCR was conducted for a panel of single nucleotide polymorphism (SNPs) in AOX1, XDH and MOCOS using TaqMan SNP genotyping assays in a prospective cohort of 192 patients receiving AZA for IBD. RESULTS: Single nucleotide polymorphism AOX1 c.3404A > G (Asn1135Ser, rs55754655) predicted lack of AZA response (P = 0.035, OR 2.54, 95%CI 1.06-6.13) and when combined with TPMT activity, this information allowed stratification of a patient's chance of AZA response, ranging from 86% in patients where both markers were favourable to 33% where they were unfavourable (P < 0.0001). We also demonstrated a weak protective effect against adverse drug reactions (ADRs) from SNPs XDH c.837C > T (P = 0.048, OR 0.23, 95% CI 0.05-1.05) and MOCOS c.2107A > C, (P = 0.058 in recessive model, OR 0.64, 95%CI 0.36-1.15), which was stronger where they coincided (P = 0.019). CONCLUSION: These findings have important implications for clinical practice and our understanding of AZA metabolism.

An unusual genetic variant in the MOCS1 gene leads to complete missplicing of an alternatively spliced exon in a patient with molybdenum cofactor deficiency.

Molybdenum cofactor deficiency (MOCOD) is a rare inherited metabolic disorder resulting in the combined deficiency of aldehyde oxidase (AO, EC 1.2.3.1), xanthine dehydrogenase (XDH, EC 1.1.1.204), and sulfite oxidase (SUOX, EC 1.8.3.1). The majority of patients typically present soon after birth with intractable seizures, developmental delay and lens dislocation and do not survive early childhood. Milder cases have been reported. We report an unusual mutation in the MOCS1 gene associated with a relatively mild clinical phenotype, in a patient who presented with normal uric acid (UA) levels in plasma. We also report a new MOCS1 mRNA splice variant in the 5' region of the gene. MOCS1 genomic DNA and cDNA from peripheral blood leukocytes were sequenced. MOCS1 mRNA splice variants were amplified with fluorescently labelled primers and quantitated. A novel homozygous mutation MOCS1c.1165+6T > C in intron 9 resulting in miss-splicing of exon 9 was found. Multiple alternatively spliced MOCS1 transcripts have been previously reported. A new MOCS1 transcript in the 5' - exon 1 region was identified in both patient and controls. This new transcript derived from the Larin variant and lacked exon 1 d.

Prospective evaluation of the pharmacogenetics of azathioprine in the treatment of inflammatory bowel disease.

AIM: To investigate whether pharmacogenetic loci or metabolite concentrations explain clinical response or side effects to AZA. METHODS: Patients with IBD were given 2 mg/kg of AZA without dose escalation or adjustment. Serial clinical response, thiopurine methyl transferase (TPMT) activity and thioguanine nucleotide (TGN) concentrations were measured over 6 months. All patients were genotyped for inosine triphosphatase (ITPase) and TPMT. Clinical response and side effects were compared to these variables. RESULTS: Two hundred and seven patients were analysed. Thirty-nine per cent withdrew due to adverse effects. Heterozygous TPMT genotype strongly predicted adverse effects (79% heterozygous vs. 35% wild-type TPMT, P < 0.001). The ITPA 94C>A mutation was associated with withdrawal due to flu-like symptoms (P = 0.014). A baseline TPMT activity below 35 pmol/h/mg/Hb was associated with a greater chance of clinical response compared with a TPMT above 35 pmo/h/mg/Hb (81% vs. 43% respectively, P < 0.001). Patients achieving a mean TGN level above 100 were significantly more likely to respond (P = 0.0017). CONCLUSIONS: TPMT testing predicts adverse effects and reduced chance of clinical response (TPMT >35 pmol/h/mg/Hb). ITPase deficiency is a predictor of adverse effects and TGN concentrations above 100 correlate with clinical response.

Influence of xanthine oxidase on thiopurine metabolism in Crohn's disease.

BACKGROUND: The thiopurines, azathioprine (AZA) and mercaptopurine are extensively used in Crohn's discase (CD). Thiopurine bioactivation can be diverted by either thiopurine methyltransferase (TPMT), or by xanthine oxidase/dehydrogenase (XOD) which forms 6-thiouric acid (6TU). AIM: To investigate whether chronic inflammation could influence small intestinal XOD activity using urinary excretion of 6TU as a surrogate marker of XOD activity. METHODS: 6-Thiouric acid excretion was compared between 32 CD patients and nine dermatology patients (control group), on AZA. Six CD patients were interesting: five with low TPMT activity (one deficient, four intermediate), and one receiving AZA/allopurinol co-therapy. RESULTS: There was no statistical difference in 6TU excretion between the CD and control group. CD location, severity or surgery did not affect excretion. The TPMT-deficient patient excreted 89% of daily AZA dose as 6TU, but excretion by TPMT carriers was essentially normal. Concurrent 5-aminosalicylic acid therapy increased 6TU excretion significantly (median 32.9%), consistent with inhibiting TPMT. 6TU was undetectable in the patient on AZA/allopurinol co-therapy. CONCLUSIONS: The results refuted our hypothesis, but fitted a model where most of an oral thiopurine dose effectively escapes first-pass metabolism by gut XOD, but is heavily catabolized by TPMT. Bioavailability of thiopurines may be competitively inhibited by dietary purines.

The effect of pre-operative anxiety on induction of anaesthesia with propofol.

In this prospective study, we investigated the effects of anxiety on the induction dose of propofol and subsequent cardiovascular changes in 197 patients. Pre-operative state and trait anxiety scores were measured using the State Trait Anxiety Inventory. Propofol was administered at 40 mg x kg(-1) x h(-1). Propofol dose was recorded at loss of verbal response and when EEG Bispectral Index decreased to 50. Thereafter, propofol infusion rate was reduced to 8 mg x kg(-1) x h(-1). Cardiovascular data were collected for 15 min after starting induction. Maximum percentage decreases in heart rate and mean arterial pressure, and the point at which the latter occurred, were recorded. On multivariate analysis, anxiety scores did not significantly affect propofol dose or cardiovascular end-points, although Bispectral Index at loss of verbal response decreased with increasing trait anxiety (p = 0.02). Anxiety, measured using State Trait Anxiety Inventory, does not appear independently to affect the induction characteristics of propofol.

Assessment of thiopurine methyltransferase enzyme activity is superior to genotype in predicting myelosuppression following azathioprine therapy in patients with inflammatory bowel disease.

BACKGROUND: Myelosuppression occurs in 2-7% of inflammatory bowel disease (IBD) patients treated with azathioprine, and can be associated with reduced activity of thiopurine methyltransferase (TPMT) in some patients. It has been proposed that pretreatment assessment of TPMT status reduces the incidence of toxicity and is cost-effective. AIMS: To determine if screening for TPMT status predicts side-effects to azathioprine in patients with IBD and to ascertain whether screening by TPMT enzyme activity or genotype is superior. METHODS: Sequential IBD patients were identified and azathioprine tolerance recorded. Blood was collected for measurement of TPMT activity and TPMT*3C, TPMT*3A and TPMT*2 genotypes. RESULTS: Of 130 patients, 25% stopped azathioprine because of toxicity. Four patients experienced severe myelosuppression (WCC < 2). Eleven of 17 patients with reduced TPMT activity were heterozygotes, including one patient with marked TPMT deficiency who experienced severe myelosuppression. There was no association between intermediate TPMT deficiency and any side-effect. CONCLUSIONS: Moderate reduction of TPMT activity in heterozygotes was not associated with toxicity, but very low TPMT activity caused severe myelosuppression in one patient. This would have been predicted by measuring TPMT activity but not by genotyping. Measurement of TPMT activity may therefore be superior to genotype in predicting severe myelosuppression.

Azathioprine and breastfeeding: is it safe?

Traditionally, women receiving azathioprine have been discouraged from breastfeeding because of theoretical potential risks of neonatal bone marrow suppression, susceptibility to infection, and pancreatitis. The aims of this study were to measure the concentration of 6-mercaptopurine (6-MP) in breast milk of mothers receiving azathioprine and in the blood of their babies and to investigate any immunosuppressive effects on the babies. Women receiving azathioprine, who after appropriate counselling wished to breastfeed their babies, were approached for inclusion in the study. Breast milk samples were obtained from recruited women, and 6-MP levels were measured in each breast milk sample. Haemoglobin level, white cell and platelet counts, and 6-MP and 6-thioguanine nucleotides (6-TGN) levels were measured in the respective neonatal blood samples. Clinical signs of immunosuppression in the neonates were noted. Thirty-one breast milk samples were collected from ten women. Low concentrations of 6-MP (1.2 and 7.6 nanograms/ml, compared with therapeutic immunosuppressant level of 50 nanograms/ml in serum) were detected in two breast milk samples obtained from one woman. 6-MP was not detected in any of the other 29 samples. 6-MP and 6-TGN were undetectable in the neonatal blood. There were no clinical or haematological signs of immunosuppression in any of the ten neonates. We conclude that breastfeeding should not be withheld in infants of mothers receiving azathioprine.

4-pyridone-3-carboxamide ribonucleoside triphosphate accumulating in erythrocytes in end stage renal failure originates from tryptophan metabolism.

We recently identified an erythrocyte nucleotide accumulating in end-stage renal disease as 4-pyridone-3-carboxamide ribonucleotide triphosphate (4PYTP), a nucleotide never described previously. Plasma tryptophan concentration has been previously reported to be reduced in patients in chronic renal failure that is in turn associated with elevated precursors of tryptophan metabolism, including L -kynurenine and quinolinic acid, both of which have been implicated in the neurotoxic manifestations of chronic renal failure. Here we compare mean erythrocyte 4PYTP, and plasma tryptophan concentrations, in controls and four patient groups with renal impairment (10 per group) and confirmed a reduction in plasma tryptophan in patients on dialysis that corrected with renal transplantation. We found: An inverse correlation between plasma tryptophan and red cell 4PYTP concentrations (R(2)=0.44, P<0.001) when all patients were grouped together. Restoration of both tryptophan and 4PYTP concentrations to control values was only achieved following renal transplantation. 4PYTP was absent from erythrocytes in Molybdenum cofactor (MoCF) deficiency implicating aldehyde oxidase/dehydrogenase, a Molybdenum requiring enzyme. High 4PYTP erythrocyte concentrations in adenine or hypoxanthine-phosphoribosyltransferase deficient patients in severe uremia (113 microM and 103 microM), confirmed the lack of involvement of either enzyme in 4PYTP formation. We propose that 4PYTP is formed by a novel route involving the oxidation of the intermediates of NAD turnover from quinolinic acid by aldehyde oxidase.