TPMT genotype and the use of thiopurines in paediatric inflammatory bowel disease.

BACKGROUND: Thiopurines are used in the treatment of inflammatory bowel disease. They are metabolised via methylation by thiopurine-S-methyltransferase (TPMT), which displays a genetically determined polymorphic activity. Subjects with reduced TPMT activity have a higher concentration of active thiopurine metabolites and may be at increased risk of bone-marrow suppression. AIMS: To evaluate the relevance of TPMT genotyping in the management of thiopurines therapy in inflammatory bowel disease patients. PATIENTS AND METHODS: Adverse effects and clinical response were determined retrospectively and correlated with TPMT genotype in 70 paediatric inflammatory bowel disease patients. RESULTS: Nineteen patients (27.1%) developed adverse effects; of the 51 who did not, 34 (66.7%) responded to treatment. Five patients (7.1%) were heterozygous for a variant TPMT allele; two of these (40%) were intolerant to thiopurines, compared to 17 of the 65 patients (26.2%) with a wild type gene (O.R. 1.88, 95% CI 0.29-12.2, p=0.61); among the 34 responders, the median dosage of the drug required to obtain remission was lower for mutated than for wild type patients (1.6mgkg(-1)day(-1) versus 2.0mgkg(-1)day(-1), p=0.043). CONCLUSIONS: There was no significant association between adverse effects of thiopurines and TPMT heterozygous genotype, but TPMT genotyping could be useful in establishing the most appropriate dose of thiopurines to start treatment.

Biochemical and microscopic evidence for the internalization of drug-containing mast cell granules by macrophages and smooth muscle cells.

During mast cell degranulation the soluble component of the granule is released into extracellular fluid, whereas two neutral proteases and heparin proteoglycans form the extracellular granule remnants. These structures are negatively charged and bind with high affinity LDL and other basic molecules. In this study we show that granule remnants expelled into extracellular fluid are able to bind the aminoglycoside antibiotic gentamicin and the anticancer agent doxorubicin in a dose-dependent manner. In addition, granule remnants loaded with the two basic substances are subsequently phagocytosed by macrophages. Indeed, when cells are incubated for 24 h with 1 mg/ml gentamicin, the intracellular concentration of the drug, which in basal conditions is extremely low, increases significantly in the presence of degranulating mast cells (from 5.1 +/- 1.0 to 25.4 +/- 2.5 microg/mg protein) and a good correlation between histamine release and gentamicin uptake is evident. The antineoplastic agent doxorubicin can penetrate cells by passive diffusion; however, when mast cells are added to macrophage monolayer, incubated for 30 min with 50 microM of the antineoplastic agent, a significant increase in intracellular doxorubicin concentration is observed (from 3.5 +/- 0.2 to 4.7 +/- 0.2 microg/mg protein). Internalization of granule remnants carrying gentamicin or doxorubicin is also evident in smooth muscle cells of the synthetic phenotype. In particular, when smooth muscle cells are incubated for 24 h with 1 mg/ml gentamicin, addition of isolated granules increases the uptake from 2.4 +/- 0.2 to 4.8 +/- 0.4 microg/mg protein. Similar results are obtained in smooth muscle cells incubated for 4 h with doxorubicin 50 microM (from 3.3 +/- 0.2 to 4.8 +/- 0.5 microg/mg protein). Data are confirmed by microscopic experiments by means of fluorescence microscopy and electron microscopic studies. The study demonstrates that basic substances can enter phagocytic cells when loaded to granule remnants. The phenomenon can be of particular interest for substances like the aminoglycosides that do not cross biological membranes; indeed, the storage of these antibiotics in phagocytic cells could have important consequences on their antibacterial activity in vivo. Macrophages and smooth muscle cells can also act as a reservoir for doxorubicin. High concentrations of the antineoplastic agent in these cells could be responsible for toxicity, as well as play an important role in the transport of the drug to tumor cells.