Insights into the cellular pharmacokinetics and pharmacodynamics of thiopurine antimetabolites in a model of human intestinal cells.

Thiopurines, immunomodulating drugs used in the management of different chronic autoimmune conditions and as anti-leukemic agents, may exert in some cases gastrointestinal toxicity. Moreover, since these agents are administered orally, they are absorbed across the gastrointestinal tract epithelium. On these premises, cellular and molecular events occurring in intestinal cells may be important to understand thiopurine effects. However, quantitative information on the biotransformation of thiopurines in intestinal tissues is still limited. To shed light on biotransformation processes specific of the intestinal tissue, in this study thiopurine metabolites concentrations were analyzed by an in vitro model of human healthy colon, the HCEC cell line, upon exposure to cytotoxic concentrations of azathioprine or mercaptopurine; the investigation was carried out using an innovative mass spectrometry method, that allowed the simultaneous quantification of 11 mono-, di-, and triphosphate thionucleotides. Among the 11 metabolites evaluated, TIMP, TGMP, TGDP, TGTP, MeTIMP, MeTIDP and MeTITP were detectable in HCEC cells treated with azathioprine or mercaptopurine, considering two different incubation times before the addition of the drugs (4 and 48 h). Different associations between metabolites concentrations and cytotoxicity were detected. In particular, the cytotoxicity was dependent on the TGMP, TGDP, TGTP and MeTITP concentrations after the 4 h incubation before the addition of thiopurines. This may be an indication that, to study the association between thiopurine metabolite concentrations and the cytotoxicity activity in vitro, short growth times before treatment should be used. Moreover, for the first time our findings highlight the strong correlation between cytotoxicity and thiopurine pharmacokinetics in HCEC intestinal cells in vitro suggesting that these cells could be a suitable in vitro model for studying thiopurine intestinal cytotoxicity.

Phosphorylation of pyrimidine L-deoxynucleoside analog diphosphates. Kinetics of phosphorylation and dephosphorylation of nucleoside analog diphosphates and triphosphates by 3-phosphoglycerate kinase.

Anticancer and antiviral D- and L-nucleoside analogs are phosphorylated stepwise in the cells to the pharmacologically active triphosphate metabolites. We recently reported that in the last step, L-deoxynucleoside analog diphosphates are phosphorylated by 3-phosphoglycerate kinase (PGK). To explain the preference of PGK for L- over D-deoxynucleoside analog diphosphates, the kinetics of their phosphorylation were compared with the dephosphorylation of the respective triphosphates using recombinant human PGK. The results attributed favorable phosphorylation of L-deoxynucleoside analog diphosphates by PGK to differences in k(cat), which were consequences of varied orientations of the sugar and diphosphates in the catalytic site of PGK. The amino acids involved in the catalytic reaction of PGK (including Glu(344), Lys(220), and Asn(337)) were therefore mutated. The impact of mutations on the phosphorylation of L- and D-deoxynucleoside analog diphosphates was different from those on dephosphorylation of the respective triphosphates. This suggested that the interactions of the nucleoside analogs with amino acids during the transition state are different in the phosphorylation and dephosphorylation reactions. Thus, reversible action of the enzyme may not involve the same configuration of the active site. Furthermore, the amino acid determinants of the action of PGK for L-deoxynucleotides were not the same as for the D-deoxynucleotides. This study also suggests the potential impact of nucleoside analog diphosphates and triphosphates on the multiple cellular functions of PGK, which may contribute to the action of the analogs.