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Objective: To investigate whether methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms and erythrocyte concentration of methotrexate (EMTX) could serve as predictors of methotrexate (MTX) efficacy and toxicity in patients with juvenile idiopathic arthritis (JIA)., Methods: Genetic analyses and EMTX and folate assessment were performed in 69 patients with JIA aged 2.5-19.6 years (30 male) treated with MTX using a dose-escalation protocol and classified as full responders (disease inactivity; n = 51) or nonresponders (< 30% improvement in pediatric American College of Rheumatology-30 criteria while receiving ≥ 15 mg/m(2)/week parenteral MTX for at least 3 months; n = 18)., Results: Nonresponders were treated with the higher median MTX dose (17.2 vs 12.6 mg/m(2)/week; p < 0.0001) and accumulated more EMTX (217 vs 106 nmol/l; p < 0.02) and erythrocyte folates (763 vs 592 nmol/l; p = 0.052) than responders. Analysis of MTHFR allele and genotype frequencies in relation to response failed to detect association. The frequency of any adverse effect was 29.4% in responders and 33.3% in nonresponders (p = 0.77). The frequency of 677T allele was elevated in patients with adverse effects (52.4% vs 20.9%; OR 3.88, 95% CI 1.8-8.6, p < 0.002). The probability of any adverse effect was significantly higher in patients with 677TT compared to the 677CC genotype (OR 55.5, 95% CI 2.9-1080, p < 0.001)., Conclusion: MTHFR genotyping may have a predictive value for the risk of MTX-associated toxicity in patients with JIA. Despite the lack of therapeutic effect, nonresponders accumulated adequate concentrations of EMTX.

Objective: To compare the bioavailability of oral and subcutaneous methotrexate (MTX) in children with juvenile idiopathic arthritis (JIA)., Methods: Seventeen JIA patients were administered oral (6.1-22.5 mg/m(2)) or subcutaneous (8.8-28.6 mg/m(2)) MTX. Blood samples were drawn pre-dose, and at 1, 2, and 4 hours after administration. Plasma MTX was determined by high-performance liquid chromatography. Non-compartmental pharmacokinetic analysis included the maximum concentration of plasma MTX (C(max)) and the area under the plasma concentration-time curve in the interval of 0-4h (AUC(0-4h))., Results: The slopes of the regression lines of the dose-corrected parameters Cmax and AUC(0-4h) plotted against the dose were negative for oral administration indicating non-linearity in pharmacokinetics, while they did not differ from zero for subcutaneous MTX. In two groups dosed orally with < or = 10 or >10 mg/m(2) (the average doses: 7.8 vs. 13.8 mg/m(2), p<0.002), the C(max) and AUC(0-4h) were comparable (p > or = 0.32). In four patients switched from oral to subcutaneous administration of the same dose, the bioavailability of oral MTX tended to be 11-15% lower when compared to subcutaneous route., Conclusion: The differences in the pharmacokinetic measures of early systemic exposure between oral and subcutaneous routes support the view that lower and saturable intestinal absorption of oral MTX limits its bioavailability and efficacy within the range of standard doses used to treat children with JIA. In light of this evidence it can be recommended to use parenteral route of administration when MTX dose around and above 10-15 mg/m(2) is needed to achieve sufficient response.

Methotrexate is used widely in the pharmacotherapy of juvenile idiopathic arthritis. Polyglutamates of methotrexate are active metabolites which accumulate in cells including erythrocytes. Their intracellular concentration may reflect methotrexate bioavailability and, at the same time, may serve as a bioindicator for optimization of methotrexate therapy and drug monitoring. Therefore, a simple and selective isocratic reversed phase chromatographic method with fluorescence detection (excitation/emission wavelengths of 370/463 nm) was developed which quantifies the sum of all methotrexate polyglutamates in erythrocytes as methotrexate after their enzymatic conversion with gamma-glutamylhydrolase. Separation was carried out on a Phenomenex GEMINI C18 column using a mobile phase flowing at a rate of 0.6 ml/min and consisting of a mixture (110:890:0.25 v/v) of acetonitrile, ammonium acetate buffer (0.05 M, pH=5.5) and hydrogen peroxide 30% (w/w). The method was found linear over the concentration range of 25-400 nmol/l. Its intra- and inter-day precision and accuracy were characterized by coefficients of variation and relative errors less than 20%. The limits of detection and quantification achieved 10.9 and 32.9 nmol/l, respectively. The method was proved suitable for monitoring the concentration of methotrexate polyglutamates in erythrocytes of patients with juvenile idiopathic arthritis.