Abstract
Combination treatment with low-dose thiopurine and allopurinol (AP) has successfully been used in patients with inflammatory bowel disease with a so called skewed thiopurine metabolite profile. In red blood cells in vivo, it reduces the concentration of methylated metabolites and increases the concentration of the phosphorylated ones, which is associated with improved therapeutic efficacy. This study aimed to investigate the largely unknown mechanism of AP on thiopurine metabolism in cells with an active thiopurine metabolic pathway using HepG2 and HEK293 cells. Cells were treated with 6-mercaptopurine (6MP) and AP or its metabolite oxypurinol. The expression of genes known to be associated with thiopurine metabolism, and the concentration of thiopurine metabolites were analyzed. Gene expression levels were only affected by AP in the presence of 6MP. The addition of AP to 6MP affected the expression of in total 19 genes in the two cell lines. In both cell lines the expression of the transporter SLC29A2 was reduced by the combined treatment. Six regulated genes in HepG2 cells and 8 regulated genes in HEK293 cells were connected to networks with 18 and 35 genes, respectively, present at known susceptibility loci for inflammatory bowel disease, when analyzed using a protein-protein interaction database. The genes identified as regulated as well as the disease associated interacting genes represent new candidates for further investigation in the context of combination therapy with thiopurines and AP. However, no differences in absolute metabolite concentrations were observed between 6MP+AP or 6MP+oxypurinol vs. 6MP alone in either of the two cell lines. In conclusion; the effect of AP on gene expression levels requires the presence of 6MP, at least in vitro. Previously described AP-effects on metabolite concentrations observed in red blood cells in vivo could not be reproduced in our cell lines in vitro. AP’s effects in relation to thiopurine metabolism are complex. The network-identified susceptibility genes represented biological processes mainly associated with purine nucleotide biosynthetic processes, lymphocyte proliferation, NF-KB activation, JAK-STAT signaling, and apoptotic signaling at oxidative stress.
Highlights
Azathioprine and 6-mercaptopurine (6MP) are important drugs in the treatment of inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD) [1,2,3].Following oral administration the first pass metabolism of thiopurines is extensive (S1 Fig)
In HepG2 cells transfected to express xanthine oxidase (XO), no genes were regulated by AP, 6MP or the combination treatment
We investigated the effects of the addition of AP to 6MP in cell-based models (HepG2 and HEK293 cells) with active thiopurine metabolic pathways and in which both gene expression levels and metabolite concentrations were studied following exposure to clinically relevant drug concentrations
Summary
Azathioprine and 6-mercaptopurine (6MP) are important drugs in the treatment of inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD) [1,2,3].Following oral administration the first pass metabolism of thiopurines is extensive (S1 Fig). A skewed metabolite profile with excessive production of meTIN and subtherapeutic 6TGN levels is found in approximately 15–20% of patients [13, 14] and has been associated with glucocorticosteroid dependency and adverse events [15,16,17]. It seems that the dominant metabolic pathway may differ between patient groups. Combination treatment with allopurinol, a xanthine oxidase (XO) inhibitor, and a reduced dose of thiopurine (~25–33% of original dose) has successfully been used in these patients and switches the metabolism towards predominately 6TGN production and improved therapeutic efficacy [15, 18]. High XO activity per se does not explain the phenotype [19]
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