Properties of small rRNA methyltransferase RsmD: mutational and kinetic study.

Ribosomal RNA modification is accomplished by a variety of enzymes acting on all stages of ribosome assembly. Among rRNA methyltransferases of Escherichia coli, RsmD deserves special attention. Despite its minimalistic domain architecture, it is able to recognize a single target nucleotide G966 of the 16S rRNA. RsmD acts late in the assembly process and is able to modify a completely assembled 30S subunit. Here, we show that it possesses superior binding properties toward the unmodified 30S subunit but is unable to bind a 30S subunit modified at G966. RsmD is unusual in its ability to withstand multiple amino acid substitutions of the active site. Such efficiency of RsmD may be useful to complete the modification of a 30S subunit ahead of the 30S subunit's involvement in translation.

Population pharmacokinetic and pharmacogenetic analysis of 6-mercaptopurine in paediatric patients with acute lymphoblastic leukaemia.

AIMS: To investigate the population pharmacokinetics of 6-mercaptopurine (6-MP) active metabolites in paediatric patients with acute lymphoblastic leukaemia (ALL) and examine the effects of various genetic polymorphisms on the disposition of these metabolites. METHODS: Data were collected prospectively from 19 paediatric patients with ALL (n = 75 samples, 150 concentrations) who received 6-MP maintenance chemotherapy (titrated to a target dose of 75 mg m(-2) day(-1)). All patients were genotyped for polymorphisms in three enzymes involved in 6-MP metabolism. Population pharmacokinetic analysis was performed with the nonlinear mixed effects modelling program (nonmem) to determine the population mean parameter estimate of clearance for the active metabolites. RESULTS: The developed model revealed considerable interindividual variability (IIV) in the clearance of 6-MP active metabolites [6-thioguanine nucleotides (6-TGNs) and 6-methylmercaptopurine nucleotides (6-mMPNs)]. Body surface area explained a significant part of 6-TGNs clearance IIV when incorporated in the model (IIV reduced from 69.9 to 29.3%). The most influential covariate examined, however, was thiopurine methyltransferase (TPMT) genotype, which resulted in the greatest reduction in the model's objective function (P < 0.005) when incorporated as a covariate affecting the fractional metabolic transformation of 6-MP into 6-TGNs. The other genetic covariates tested were not statistically significant and therefore were not included in the final model. CONCLUSIONS: The developed pharmacokinetic model (if successful at external validation) would offer a more rational dosing approach for 6-MP than the traditional empirical method since it combines the current practice of using body surface area in 6-MP dosing with a pharmacogenetically guided dosing based on TPMT genotype.

Drug Insight: pharmacology and toxicity of thiopurine therapy in patients with IBD.

Thiopurines are frequently used for the treatment of IBD. The complex pharmacology, metabolism, mechanism of action and toxicity profile of these immunosuppressive drugs have now been partly elucidated. The activity of thiopurines is partly mediated by the metabolite 6-thioguanosine 5'-triphosphate, which inhibits the function of the small GTPase Rac1, leading to apoptosis of activated T cells, and influences the conjugation of T cells with antigen-presenting cells. The activity of the enzyme thiopurine S-methyltransferase has a major influence on the bioavailability and toxicity of thiopurines, and several thiopurine metabolites might have adverse effects in patients. Myelotoxicity can be caused by grossly elevated levels of 6-thioguanine nucleotides, and elevated levels of 6-methylmercaptopurine ribonucleotides have been associated with hepatotoxicity. The sensitivity and specificity of these methylated metabolites for predicting thiopurine-induced liver enzyme abnormalities are, however, poor. 6-Thioguanine has been suggested as an alternative to the classical thiopurines azathioprine and 6-mercaptopurine for the treatment of IBD, but there are concerns about its toxicity profile, especially with regard to the induction of nodular regenerative hyperplasia of the liver. Data now suggest that the induction of nodular regenerative hyperplasia of the liver during 6-thioguanine therapy might be dose-dependent or dependent on the level of 6-thioguanine nucleotides.

Actividad de la tiopurina metiltransferasa en la enfermedad inflamatoria intestinal. Un estudio en 7.046 pacientes españoles / Thiopurine methyltransferase activity in the inflammatory bowel disease. A study on 7046 Spanish patients

Fundamento y objetivo: El objetivo del presente estudio es describir la actividad enzimática de la tiopurina metiltransferasa (TPMT) en un grupo muy numeroso de pacientes españoles con enfermedad inflamatoria intestinal (EII), evaluar el efecto de algunas variables sobre dicha actividad y conocer la proporción de pacientes con baja actividad y, por tanto, mayor riesgo de mielotoxicidad por azatioprina/6-mercaptopurina. Pacientes y método: Se determinó la actividad de TPMT mediante un método radioquímico en pacientes con EII. La asociación entre diversas variables y la actividad de TPMT se estudió mediante regresión lineal múltiple. Resultados: Se incluyó a 7.046 pacientes, el 70% con enfermedad de Crohn, el 22% con colitis ulcerosa y un 8% con colitis indeterminada. El valor medio de TPMT fue de 20 U/ml (extremos: 0-46). La distribución de actividad de TPMT fue: un 0,5% con valores bajos (= 13,8). Los valores de TPMT no siguieron una distribución normal (p < 0,001). En el estudio univariante se demostraron diferencias estadísticamente significativas (p < 0,001), aunque de dudosa relevancia clínica al ser mínimos, en los valores de TPMT en función de la edad, el sexo, el tipo de enfermedad y el tratamiento con azatioprina/6-mercaptopurina. En el estudio multivariante, el sexo y el tratamiento con 5-aminosalicilatos, glucocorticoides y azatioprina/6-mercaptopurina se asoció significativamente con la actividad de TPMT. Conclusiones: El 0,5% de los pacientes españoles con EII tiene una baja actividad enzimática de TPMT (< 5 U/ml, lo que indica un mayor riesgo de mielotoxicidad por azatioprina/6-mercaptopurina), una cifra similar a la descrita en otros países. Los diversos fármacos empleados en el tratamiento de la EII, como los 5-aminosalicilatos o la azatioprina/6-mercaptopurina, no parecen modificar de forma clínicamente relevante dicha actividad enzimática

Background and objective: Our objective was to assess the activity of thiopurine methyltransferase (TPMT) in a very large number of Spanish patients with inflammatory bowel disease (IBD), to evaluate the influence of several variables (including azathioprine or 6-mercaptopurine) on that activity, and to know the proportion of patients with low TPMT activity and therefore high risk of myelotoxicity when treated with these drugs. Patients and method: TPMT activity in red blood cells (RBCs) was measured by a radiochemical method. The association between several variables and TPMT values was assessed by multiple lineal regression. Results: 7046 patients were included (mean age: 37 years; 53% males): 70% with Crohn's disease, 22% with ulcerative colitis, and 8% with indeterminate colitis. Mean TPMT value was 20 (6) U/ml RBCs (minimum 0 and maximum 46). TPMT activity distribution was as follows: low levels (= 13.8), 88.4%. TPMT values did not follow a normal distribution (p < 0.001). In the univariate study, statistically significant differences (p < 0.001), yet of doubtly clinical significance because its minimal magnitude, were demonstrated in TPMT values depending on age, sex, type of disease, and treatment with azathioprine/6-mercaptopurine. In the multivariate study, the variables associated with TPMT activity were: sex, treatment with 5-aminosalicylates, steroids and azathioprine/6-mercaptopurine. Conclusions: This study shows that 0.5% of the Spanish patients with IBD have low TPMT activity (< 5 U/ml RBCs), a figure similar to that reported in other countries, these patients being at higher risk of myelotoxicity when treated with azathioprine or 6-mercaptopurine. The drugs usually prescribed for the treatment of IBD, including 5-aminosalicylates and azathioprine/6-ercaptopurine, do not seem to modify in a clinically relevant manner TPMT activity

Actividad eritrocitaria de tiopurina metiltransferasa y tratamiento con tiopurinas en la enfermedad inflamatoria intestinal / Erythrocyte activity of TPMT and treatment with thiopurines in inflammatory bowel disease

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