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Inflammation bowel disease I
A biochemical mechanism for the role of allopurinol in TMPT inhibition
  1. P A Blaker *1,
  2. M Arenas2,
  3. L Fairbanks2,
  4. P Irving1,
  5. A M Marinaki2,
  6. J Sanderson1
  1. 1Department of Gastroenterology, Guy's and St. Thomas' Hospital, London, UK
  2. 2Department of Chemical Pathology, Guy's and St. Thomas' Hospital, London, UK

Abstract

Introduction Hypermethylation of thiopurines has been associated with drug toxicity and non-response to treatment. In such patients the use of low dose thiopurines with concomitant allopurinol is advocated1 2. Allopurinol is observed to cause a reduction in methylated metabolites of thiopurines; however the biochemical mechanisms remain incompletely understood. Using an intact erythrocyte model we propose a novel pathway of allopurinol mediated thiopurine-S-methyltransferase (TPMT) inhibition, through the production of 2-hydroxy-6-thiopurine (2OH6MP).

Methods EDTA whole blood was obtained from healthy volunteers; the plasma and top 5th were removed and the red cells washed with 0.9% saline. 150 μL of Earl's balanced salt solution was added to 100 μL of packed red cells. Cells were incubated with 250 μM of 6-mercaptopurine (6-MP) for 0, 2, 4 and 6 h at 37°C. In the second experiment cells were pre-incubated for 2 h with 280 μM of 2OH6MP prior to the addition of 250 μM 6-MP for either 2 or 4 h. At the end of the incubation period, the media was removed and the red cells lysed with 15% perchloric acid after the addition of dithiotreitol. Methylated thiopurine-metabolites were reduced to the base by boiling at 100°C for 1 h. 75 μL of the red cell lysates and supernatant media were separated by reverse phase HPLC to detect the methylated metabolites of 6-MP.

Results In packed red cells there was an increase in the concentration of 6-methylmercaptopurine (6-MeMP) detected at each time point. However, the rate of 6-MeMP production remained constant (mean 0.825 pmol L−1 h−1, SEM ± 0.038). The concentration of 6-MeMP observed in the media was up to 7-fold lower than the concentration in red cells (mean 0.133 pmol L−1 h−1, SEM ± 0.009). In red cells pre-incubated with 2OH6MP prior to the addition of 6-MP there was a significant reduction in the rate of 6-MeMP production at both 2 (0.878 pmol L−1 h−1 vs 0.135 pmol L−1 h−1, p <0.0001, two-sided T-test) and 4 h (0.732 pmol L−1 h−1 vs 0.096 pmol L−1 h−1, p<0.0001, two-sided T-test).

Conclusion The data suggests that 6-MP enters red blood cells, where it undergoes methylation to 6-MeMP. The presence of 2OH6MP leads to a reduction in the rate of 6-MeMP production, most likely through direct inhibition of TPMT. We propose that 6-MP undergoes preferential oxidation via aldehyde oxidase, producing 2OH6MP, which leads to feed-back inhibition of TMPT and thereby a reduction in methylated thiopurine-metabolites. This mechanism may explain why patients treated with a combination of thiopurine and allopurinol have dramatically decreased methylated metabolites.

  • Allopurinol
  • Thiopurine

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Footnotes

  • Competing interests None.

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