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Woolf R.T.,King's College London | West S.L.,King's College London | Arenas-Hernandez M.,Purine Research Laboratory | Hare N.,King's College London | And 5 more authors.
British Journal of Dermatology | Year: 2012

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 3-5species 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. © 2012 The Authors BJD © 2012 British Association of Dermatologists 2012.


Gaspar H.B.,University College London | Gaspar H.B.,Great Ormond Street Hospital NHS Trust | Cooray S.,University College London | Cooray S.,Great Ormond Street Hospital NHS Trust | And 20 more authors.
Science Translational Medicine | Year: 2011

Genetic defects in the purine salvage enzyme adenosine deaminase (ADA) lead to severe combined immunodeficiency (SCID) with profound depletion of T, B, and natural killer cell lineages. Human leukocyte antigen-matched allogeneic hematopoietic stem cell transplantation (HSCT) offers a successful treatment option. However, individuals who lack a matched donor must receive mismatched transplants, which are associated with considerable morbidity and mortality. Enzyme replacement therapy (ERT) for ADA-SCID is available, but the associated suboptimal correction of immunological defects leaves patients susceptible to infection. Here, six children were treated with autologous CD34-positive hematopoietic bone marrow stem and progenitor cells transduced with a conventional gammaretroviral vector encoding the human ADA gene. All patients stopped ERT and received mild chemotherapy before infusion of gene-modified cells. All patients survived, with a median follow-up of 43 months (range, 24 to 84 months). Four of the six patients recovered immune function as a result of engraftment of genecorrected cells. In two patients, treatment failed because of disease-specific and technical reasons: Both restarted ERT and remain well. Of the four reconstituted patients, three remained off enzyme replacement. Moreover, three of these four patients discontinued immunoglobulin replacement, and all showed effective metabolic detoxification. All patients remained free of infection, and two cleared problematic persistent cytomegalovirus infection. There were no adverse leukemic side effects. Thus, gene therapy for ADA-SCID is safe, with effective immunological and metabolic correction, and may offer a viable alternative to conventional unrelated donor HSCT.


Fairbanks L.D.,Purine Research Laboratory | Levene M.,St George's, University of London | Bax B.E.,St George's, University of London
Journal of Pharmaceutical and Biomedical Analysis | Year: 2013

A sensitive and simple reverse-phase high performance liquid chromatographic (HPLC) assay has been validated for the determination of thymine as a measure of thymidine phosphorylase activity encapsulated in erythrocytes (EE-TP), a formulation which is under clinical development as an enzyme replacement therapy for the treatment of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Diluted erythrocyte lysates were incubated in 100mM sodium phosphate buffer and 10mM thymidine at 37°C for 10min and the reaction stopped with 40% trichloroacetic acid. Following centrifugation, the supernatant was washed with water saturated diethyl ether, and injected onto a Spherisorb C18 column (125mm×4.6mm, 5μm), with a mobile phase (40mM ammonium acetate, 5mM tetrabutyl ammonium hydrogen sulphate, pH 2.70) delivered at a flow rate of 1.0ml/min and run time of 8min. Ultraviolet detection (UV) was employed at 254nm. The method was linear in the range of 5-500nmol/ml (r2=0.992), specific with intra- and inter-day precisions of <9.6 and accuracies within ±20%. Limits of detection and quantification were 1.2nmol/ml and 10nmol/ml, respectively. The method was applied to quantify thymidine phosphorylase activity in samples of in-process controls and batches of EE-TP manufactured for clinical use. © 2012 Elsevier B.V.


Booth C.,Institute of Child Health | Booth C.,Great Ormond Street Hospital National Health Service Trust | Algar V.E.,Great Ormond Street Hospital National Health Service Trust | Xu-Bayford J.,Great Ormond Street Hospital National Health Service Trust | And 4 more authors.
Journal of Clinical Immunology | Year: 2012

Adenosine deaminase deficiency is a disorder of purine metabolism manifesting severe combined immunodeficiency (ADA-SCID) and systemic abnormalities. Increased levels of the substrate deoxyadenosine triphosphate (dATP) lead to immunodeficiency and are associated in a murine model with pulmonary insufficiency. We compared a cohort of patients with ADA-SCID and X-linked SCID and found that despite similar radiological and respiratory findings, positive microbiology is significantly less frequent in ADA-SCID patients (p<0.0005), suggesting a metabolic pathogenesis for the lung disease. Clinicians should be aware of this possibility and correct metabolic abnormalities either through enzyme replacement or haematopoietic stem cell transplant, in addition to treating infectious complications. © 2012 Springer Science+Business Media, LLC.


Smith M.,Thomas College | Blaker P.,Thomas College | Patel C.,Kingston Hospital NHS Trust | Marinaki A.,Purine Research Laboratory | And 5 more authors.
International Journal of Clinical Practice | Year: 2013

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. © 2012 Blackwell Publishing Ltd.


Smith M.A.,Thomas College | Marinaki A.M.,Purine Research Laboratory | Sanderson J.D.,Thomas College
Pharmacogenomics | Year: 2010

In recent years, the benefits of early aggressive treatment paradigms for inflammatory bowel disease have emerged. Symptomatic improvement is no longer considered adequate; instead, the aim of treatment has become mucosal healing and altered natural history. Nonetheless, we still fail to achieve these end points in a large number of our patients. There are many reasons why patients fail to respond or develop toxicity when exposed to drugs used for inflammatory bowel disease, but genetic variation is likely to account for a significant proportion of this. Some examples, notably thiopurine methyltransferase polymorphism in thiopurine treatment, are already established in clinical practice. We present a review of the expanding literature in this field, highlighting many interesting developments in pharmacogenomics applied to inflammatory bowel disease and, where possible, providing guidance on the translation of these developments into clinical practice. © 2010 Future Medicine Ltd.


Smith M.A.,Guys And St Thomas Nhs Foundation Trust | Blaker P.,Guys And St Thomas Nhs Foundation Trust | Marinaki A.M.,Purine Research Laboratory | Anderson S.H.,Guys And St Thomas Nhs Foundation Trust | And 2 more authors.
Journal of Crohn's and Colitis | Year: 2012

Background and aims: Azathioprine and mercaptopurine remain first line immunomodulatory treatments for inflammatory bowel disease. Toxicity and non-response are significant issues. Co-prescription of allopurinol with reduced-dose (25-33%) azathioprine or mercaptopurine may overcome these problems. We present the outcome of co-prescription in a large single-centre cohort. Method: Patients on thiopurine/allopurinol co-prescription were identified. Indication for and outcome on combination treatment were established. Blood parameters and metabolite results were compared on single agent and combination treatment. Toxicity associated with combination treatment was sought. Results: 110 patients on combination treatment were identified. Clinical remission was achieved in 60/79 (76%) of patients in whom the effect of thiopurine could be studied in isolation. 20/25 patients with hepatotoxicity tolerated combination treatment and normalised their liver function tests. 24/28 patients with atypical side effects tolerated co-therapy. 13/20 non-responders responded to combination treatment. In patients started on combination treatment as first line therapy, 15/23 achieved clinical remission. Thioguanine nucleotides were significantly higher and methylated metabolites significantly lower on combination therapy. Mean cell volume was higher and total white cell and neutrophil counts lower on combination treatment. 13 adverse events occurred, including 6 specific to co-therapy (3 rash, 2 abnormal liver function tests, 1 dosing error). All were minor and self-limiting. Conclusion: This is the largest published experience of the use of allopurinol to optimise outcomes on thiopurine treatment. Combination therapy permitted successful treatment of a significant number of patients who would otherwise have been labelled as thiopurine failures. A few self-limiting side effects were encountered. © 2012 European Crohn's and Colitis Organisation.


Smith M.A.,Guys And St Thomas Nhs Foundation Trust | Irving P.M.,Guys And St Thomas Nhs Foundation Trust | Marinaki A.M.,Purine Research Laboratory | Sanderson J.D.,Guys And St Thomas Nhs Foundation Trust
Alimentary Pharmacology and Therapeutics | Year: 2010

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. © 2010 Blackwell Publishing Ltd.

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