Monica Arenas-Hernandez

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 Earles 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.

Further experience with the use of 6-thioguanine in patients with Crohn's disease

Background: 6‐Thioguanine (6‐TG) is efficacious in patients with Crohns Disease (CD) failing conventional immunosuppression but there are reports of hepatotoxicity. We report our experience of the safety and efficacy of 6‐TG in a series of patients with CD. Methods: A retrospective study of patients with CD who failed thiopurines ± methotrexate between 2001 and 2006 was performed. Indications for 6‐TG were; active disease, to allow infliximab withdrawal, steroid sparing, or fistula closure. Patients underwent regular review and those treated longer than 1 year were advised to have liver magnetic resonance imaging (MRI) and liver biopsy. Results: All 30 patients treated with 6‐TG during the period were included. The median dose and duration of 6‐TG was 40 mg daily and 21.5 months, respectively. Initial clinical response was achieved in 18/30 (60%). Eleven of 29 (38%) (1 unrelated death) remained in remission at a median 44 months follow‐up. Seven of 30 (23%) discontinued 6‐TG due to adverse effects; 7/30 (23%) patients developed abnormal liver function tests (LFTs) during treatment, mostly transient and mild. One patient developed a portal hypertensive syndrome resolving on cessation of 6‐TG. Of 11 liver biopsies, none showed nodular regenerative hyperplasia (NRH). The median red blood cell 6‐thioguanine nucleotide (6‐TGN) level was 807 pmol/108. Conclusions: 6‐TG has good clinical efficacy for third‐line immunosuppression in CD but hepatotoxicity remains a concern. However, previous reports of NRH in 6‐TG‐treated inflammatory bowel disease patients have not been substantiated by this cohort.

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

Summary Background  Methotrexate is activated by the sequential addition of glutamic acid residues to form methotrexate polyglutamates (MTXPG1–5). MTXPG1–5 inhibit enzymes of the folate–purine–pyrimidine pathways, and longer‐chain MTXPG3–5 species are more active.

The pharmacogenetic basis of individual variation in thiopurine metabolism

Thiopurines are an important class of immunosuppressive therapy, which have been used in clinical practice for over 50 years. Despite this extensive experience many of the pharmacodynamic and pharmacokinetic properties of these drugs remain unknown. As a consequence there is often no clear explanation for the individual variation in response to treatment, both in terms of efficacy or adverse drug reactions. This review, which emphasizes practice in gastroenterology, summarizes the current understanding of thiopurine drug metabolism and highlights the role of nongenetic and genetic factors other than TPMT, which should be a focus for future research. Correlation of polymorphic variations in these genes with clinical outcomes is expected to clarify the basis for interindividual differences in thiopurine metabolism and enable a more personalized approach to therapy.

Getting the best out of thiopurine therapy: thiopurine S-methyltransferase and beyond.

Thiopurines are the cornerstone of treatment for a wide variety of medical disorders, ranging from pediatric leukemia to inflammatory bowel disease. Because of their complex metabolism and potential toxicities, the use of biomarkers to predict risk and response is paramount. Thiopurine S-methyltransferase and thiopurine metabolite levels have emerged as companion diagnostics with crucial roles in facilitating safe and effective treatment. This review serves to update the reader on how these tools are being developed and implemented in clinical practice. A useful paradigm in thiopurine therapeutic strategy is presented, along with fresh insights into the mechanisms underlying these approaches. We elaborate on potential future developments in the optimization of thiopurine therapy.

A practical guide to thiopurine prescribing and monitoring in IBD

Thiopurines are often the mainstay of treatment for many patients with inflammatory bowel disease. As such, a general understanding of the evidence behind their use and of their metabolism is extremely useful in clinical practice. This review gives a practical overview of thiopurine metabolism, the importance of thiopurine S-methyltransferase testing prior to the start of therapy and the monitoring of thioguanine nucleotide levels while on treatment, guiding a personalised approach to optimising thiopurine therapy.

Adenine phosphoribosyltransferase deficiency in the United Kingdom: two novel mutations and a cross-sectional survey

Background Adenine phosphoribosyltransferase deficiency is an inborn error of metabolism that can cause kidney disease from crystalline nephropathy or kidney stones. Methods We present three cases from a single centre with varied presentations to illustrate how increasing awareness led to better patient identification. We then undertook a cross-sectional survey of all the patients identified from the Purine Research Laboratory in the UK since 1974. Results Our index case presented with recurrent nephrolithiasis and was diagnosed on stone analysis, the second case presented with acute kidney injury and the third case was identified from a biopsy undertaken for acute on chronic kidney injury. Genetic studies identified two novel mutations. Twenty patients were retrospectively identified. The mean age at diagnosis was 25 years (range 2–70); eight were <20 years, seven were 20–40 years and five were >40 years. Five of the 20 patients were deceased, 3 after end-stage renal disease (ESRD). Twelve have normal renal function, one had CKD stage 3, one had severe kidney disease and one was on dialysis. Conclusions Adenine phosphoribosyltransferase deficiency presents in a wide spectrum in all age groups. Patients can be completely asymptomatic and kidney disease may be incorrectly attributed to other conditions. Outcome is poor in late diagnosis and there is a high prevalence of ESRD. Patients with unexplained renal stone disease or deterioration in kidney function should be considered for screening. Identification and surveillance of patients in the UK can improve. There is now a rare disease registry with meetings organized that include patients, families and health care providers to improve awareness.

PWE-013 Should The Target TGN Range Be Different in Those with Intermediate Compared with Normal TPMT Activity?

Introduction Thioguanine nucleotides (TGNs) are the biologically active metabolites of thiopurines. Methylated thiopurine metabolites (MeMPs) formed via thiopurine-S-methyltransferase (TPMT) may also have immunosuppressive properties through inhibition of de novo purine synthesis (DNPS).1 Individuals with intermediate TPMT activity do not produce significant levels of methylated metabolites, with predictably less inhibition of DNPS and therefore may tolerate higher levels of TGNs. If confirmed this suggests that the therapeutic range of TGNs (240–450 pmol/8x108) may differ between patients with intermediate Vs normal TPMT activity. Methods All IBD patients at Guy’s and St Thomas’ Hospitals treated with either azathioprine or mercaptopurine who had TGNs measured from 2002–2013 were included. Patients receiving low dose thiopurine with allopurinol were excluded. Data was collected using the electronic patient record system and statistical analysis was completed in GraphPad Prism version 5.0. Results 2193 TGN measurements were included in the analysis, 340 (15.5%) from patients with intermediate TPMT activity and the remaining 1853 had normal TPMT activity. The average normalised thiopurine doses between groups was 1.13 Vs 1.87 mg/kg/day respectively, reflecting standard dose adjustment in intermediate TPMT patients. The median TGNs levels were significantly higher in patients with intermediate Vs normal TPMT activity; 370 Vs 277 pmol/8x108 (P = <0.00001) and the median MeMP levels were lower in patients with intermediate Vs normal TPMT activity; 104 pmol/8x108 RBC Vs 566 (P = <0.0001). No significant difference was found in the median values of Hb, WBCs, neutrophil or platelet counts between the groups. A lower lymphocyte count (1.3 Vs 1.2 x109/9/ L, P = 0.0010) and trend towards a higher MCV (93 Vs 94, P = 0.0625) was noted in patients with normal TPMT activity. Conclusion Despite significantly higher TGN levels in patients with intermediate as compared to normal TPMT activity, there was no difference in levels of Hb, WBCs, neutrophil or platelets counts. This suggests a greater tolerance to TGNs in patients with intermediate TPMT activity and may indicate that a different TGN therapeutic range is needed in these patients. The likely reason is that methylated metabolites are biologically active and also play a role in overall thiopurine immunosuppression. Reference 1 Tay BS, Lilley RM, Murray AW, et al. Inhibition of phosphoribosyl pyrophosphate amidotransferase from Ehrlich ascites-tumour cells by thiopurine nucleotides. Biochemical Pharmacology. 1969;18(4):936–8. Disclosure of Interest None Declared

PWE-006 The Effects of Smoking on Thiopurine Metabolism: Abstract PWE-006 Table 1

Introduction There is an unpredictable relationship between thiopurine methyl transferase (TPMT) and hypermethylation. It is likely that cytochrome P450 (CYP450) enzymes play a role in the demethylation of methylated mercaptopurine (MMP), altering the MMP:Thioguanine nucleotide (TGN) ratio(Table 1). Smoking has been shown to induce CYP450 enzymes. One study on smoking and olanzapine demonstrated a 6 fold increase in CYP450 activity through smoking.1 This might suggest that smoking could upregulate the demethylation process, reducing MMP. Methods Patients on thiopurines had their smoking status recorded as they attended the inflammatory bowel disease (IBD) clinic. Retrospective data on demographics was collected (Table 1). Only measurable TGNs after 6 weeks of therapy were used for analysis. Metabolites measured whilst on allopurinol were excluded. Average TGNs over the treatment period, MMP and MMP:TGN ratios were compared between smokers and non-smokers. Hypermethylators were defined as any patient with an MMP:TGN ratio of ≥11 during their treatment Results 230 patients were analysed(117 males). Table 1 shows comparison in demographics between smokers and non-smokersAbstract PWE-006 Table 1 Smokers n = 106 Non-smokers n = 124 Male:female 51%:49% 47%:53% Age 37 33 Ethnicity – White:Black:Other 81%:19%:0% 80%:9%:11% Crohn’s:Ulcerative colitis 82%:18% 72%:27% Previous Surgery 45% 27% Mean TPMT 38.4 (median 35) 34.1 (median 35) Hepatotoxicity 15.1% 13.7% Hypermethylation 28.3% 42.7% Mean MMP for smokers was 1683(SD 2565, median 840) versus 2041 in non-smokers(SD 2401, median 1340)(P = 0.713). Mean MeMP:TGN ratio for smokers was 6.15 (SD 9.06, median 2.22) versus 7.81 in non-smokers (SD 8.01, median 4.70)(P = 0.593). Conclusion There were more hypermethylators in the non-smoking cohort than in the smoking cohort (42.7% versus 28.3%(P < 0.05)), however the difference between average MMP and MMP:TGN ratios were not significant although the median differences are compelling. There were significant differences between quantity smoked and the MMP:TGN ratio. Paradoxically, the reduction in methylation if smoking <5 cigarettes/day was reversed when smoking ≥15 cigarettes/day. This may suggest an alternative pathway affected by smoking. Smoking status and quantity should be taken into account when monitoring metabolites. Additionally, polymorphisms for CYP450 (CYP1A2*F and CYP1A2*1 C), known to affect drug metabolism between individuals, may be responsible for variation and require further research. Reference 1 Carrillo JA, Herráiz AG, Ramos SI, Gervasini G, Vizcaíno S, Benítez J. Role of the smoking-induced cytochrome P450 (CYP)1A2 and polymorphic CYP2D6 in steady-state concentration of olanzapine. J Clin Psychopharmacol. 2003 Apr;23(2):119–27. Disclosure of Interest None Declared

Let’s get personal: predicting thiopurine and fluoropyrimidine toxicity

The US FDA now recognizes the need to individualize treatment paradigms using biomarkers that predict response to therapy. In clinical practice the best example of this is TPMT testing, which is used to rationalize the starting dose of azathioprine and mercaptopurine. The more recent addition of drug metabolite monitoring means that thiopurine therapy can now be personalized to unprecedented levels. Of interest, parallels exist between TPMT deficiency as an explanation for thiopurine toxicity and DPD deficiency in fluoropyrimidine toxicity. For these drugs, variations in a single locus predict severe toxicity. However, while TPMT testing has translated into routine clinical practice, DPD testing has not. This article summarizes the recent research investigating interindividual differences in the metabolism of thiopurine and fluoropyrimidine drugs, and explores the attitudes which influence the uptake of pharmacogenetic testing.