Jaroslav Chládek

Pharmacokinetics and Pharmacodynamics of Methotrexate in Non-Neoplastic Diseases

Low dose pulse methotrexate (LDMTX) therapy has become effective in the treatment of autoimmune and lymphoproliferative diseases. The pharmacokinetics of LDMTX is individually highly variable, resulting in a different systemic exposure to the drug and a variable therapeutic/toxic effect in patients. The improvements and exacerbations of disease activity in relation to the introductions and discontinuations of LDMTX therapy suggest the possible immunosuppresive and anti-inflammatory properties of the drug. Because of a strong correlation between the drug pharmacokinetics and the therapeutic outcomes (pharmacodynamics), it seems to be possible to individualise the LDMTX therapy according to the results of pharmacokinetic/pharmacodynamic analysis. In the case of psoriasis, pharmacokinetic/pharmacodynamic analysis in our local study revealed a highly significant inverse relationship between PASI (expressed as a percent of the initial value) and a steady-state AUCMTX (area under the curve of methotrexate plasma concentrations; r8 = −0.65, p < 0.001). The considerable inter-individual variability and low intra-individual variability in MTX pharmacokinetics, supports a role for therapeutic monitoring and dose individualisation at the start of pharmacotherapy. The results of this study suggest that a steady-state AUCMTX value of 700 nmol · h/L and higher are associated with a significantly better success rate of antipsoriatic therapy than lower values. The preliminary results in our follow-up study suggest the statistically higher incidence of unwanted effects depending on maximum plasma concentration of the drug. Moreover, statistically significant correlation was found between the toxic effects and exposure to the drug regarding methotrexate plasma concentrations and intracellular storage in erythrocytes. However, the data are still in the process of being completed and are not yet published.

Pharmacokinetics of low doses of methotrexate in patients with psoriasis over the early period of treatment

AbstractObjective: The aim of the present study was to investigate the pharmacokinetics and pharmacodynamics of low-dose methotrexate (MTX) in the early phase (3 months) after the start of antipsoriatic therapy. Methods: Ten male and female psoriatic patients who failed to respond to previous conventional therapy were treated with 15 mg oral MTX once per week. The pharmacokinetics in plasma and the urinary excretion of MTX and 7-hydroxymethotrexate (7-OH MTX) were investigated after doses 1, 5 and 13 (corresponding to phases I, II and III, respectively). On the same occasions, MTX accumulation in erythrocytes obtained before MTX administration was investigated. Pharmacodynamics of MTX were evaluated using the psoriasis area and severity index (PASI) score. Results: There were marked intersubject differences (range of coefficients of variation 34.9–76.3%) in the area under the curve (AUC), peak concentration (Cmax) and clearance (CL) of MTX. Total CL was proportional to renal clearance (CLR) (r2 = 0.735, P < 0.0001) which accounted for 73 (19)% of the former. There was a strong linear relationship (r2 = 0.819, P < 0.0001) between CL of MTX and creatinine clearance. Within 48 h of drug administration, the urinary excretion of MTX was 46–99% of the dose, while that of 7-OH MTX was 1.5–8.6%. In 8 of 10 patients, more than 70% of the MTX dose was recovered. No intraindividual variations of MTX kinetic parameters during treatment were observed. MTX concentrations in erythrocytes reached the steady-state concentration in the range 40.7–170 nmol · l−1 after 2 months of therapy. Pharmacodynamic measurement versus pharmacokinetics revealed a significant inverse relationship between PASI score and MTX AUC (rs = −0.912, P < 0.002) and between PASI score and erythrocytic MTX (rs = −0.988, P < 0.002). Conclusion: The relationship between MTX pharmacokinetics (AUC or erythrocytic MTX) and pharmacodynamics (PASI score) may exist. It is likely that the efficacy of psoriasis therapy with MTX could be improved by adjusting the dose according to plasma concentrations obtained after the first MTX administration.

Validation of Nitrite and Nitrate Measurements in Exhaled Breath Condensate

Background: Inflammatory markers in exhaled breath condensate (EBC) are investigated as a non-invasive approach to monitoring of inflammation in the respiratory tract. EBC concentrations of nitrite and nitrate, the stable end products of oxidative metabolism of nitric oxide, are increased in patients with asthma, especially during acute exacerbations. Objectives: To examine methodological aspects of nitrite and nitrate measurements in EBC such as sample collection, storage and analysis. Methods: In a randomized study, EBC was collected twice within 1 h (with and without a nose clip) in 20 healthy adults and 20 patients with well-controlled asthma and no symptoms of allergic rhinitis. Nitrite and nitrate were assayed by ionex chromatography and fluorimetrically after derivatization with diaminonaphthalene. Results: The geometric mean [exp (mean ± SD)] EBC levels of nitrite and nitrate in healthy subjects [4.3 (3.0–6.1) and 11.0 (5.3–22.7) µmol/l] and patients [4.6 (2.6–7.3) and 8.7 (3.2–23.8) µmol/l] did not differ (p = 0.13). Wearing a nose clip (p = 0.3) did not influence nitrite and nitrate concentrations. The mean intra-subject %CVs of EBC concentrations of nitrite were 26 and 21% in healthy subjects and patients, while those of nitrate achieved 49 and 88%, respectively. Conclusions: Ionex chromatography of nitrite and nitrate requires no sample pretreatment and provides comparable results as a more laborious diaminonaphthalene method. EBC samples should be kept cold (8°C) and analyzed for nitrite and nitrate within 24 h of collection or stored in the freezer and thawed preferably only once. Wearing a nose clip during EBC collection has no influence on nitrite and nitrate concentrations. Short-term repeatability of nitrite and nitrate measurements was worse compared to published data on exhaled nitric oxide.

A pilot study of pharmacokinetically guided dosing of oral methotrexate in the initial phase of psoriasis treatment

Background  Clinical studies of low‐dose oral methotrexate (MTX) in the treatment of psoriasis and rheumatoid arthritis document a large interpatient variability in the pharmacokinetics of MTX, including its polyglutamates (MTXPGs) in erythrocytes (RBC). This can be a factor contributing to the variability of therapeutic and toxic effects.

Nitrite in exhaled breath condensate as a marker of nitrossative stress in the airways of patients with asthma, COPD, and idiopathic pulmonary fibrosis

Background: Nitrite and nitrate are exhaled in droplets of an aerosol during breathing and can be assayed in the exhaled breath condensate (EBC) as markers of nitrossative stress in the airways of patients with asthma, COPD, and idiopathic pulmonary fibrosis (IPF). Subjects and methods: Using HPLC with fluorescence detection, nitrite and nitrate were assayed in EBC of 14 atopic patients with mild‐to‐moderate stable asthma, 18 atopic asthmatics with exacerbation, 14 COPD patients without exacerbation, 18 patients with exacerbated COPD, 13 patients with active IPF, and in 29 healthy subjects. Results: The geometric mean [exp(mean±SD)] EBC concentrations of nitrite (micromol/l) in patients with asthma [5.1(2.1–12.3)], exacerbation of asthma [5.1(2.8–9.6)], exacerbation of COPD [5.3(3.2–8.7)], and with IPF [5.5(2.9–10.2)] were higher (P<0.05) compared with those of healthy subjects [2.9(1.6–5.3)] and patients with stable COPD [3.0(1.3–6.7)]. Nitrite concentration increased with decreased lung function of patients with asthma (rs=−0.31, P<0.02). Presumably owing to the contamination of the EBC sample with nitrate during collection, nitrate levels were highly variable among healthy subjects and higher compared with all groups of patients. Conclusion: EBC nitrite is a suitable marker of nitrossative stress in adult patients with lung diseases but cannot differentiate controlled and exacerbated asthma. Further improvements to the methods of EBC collection and sample handling are warranted. J. Clin. Lab. Anal. 24:317–322, 2010.

677TT Genotype Is Associated with Elevated Risk of Methotrexate (MTX) Toxicity in Juvenile Idiopathic Arthritis: Treatment Outcome, Erythrocyte Concentrations of MTX and Folates, and MTHFR Polymorphisms

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/m2/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/m2/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.

An improved high-performance liquid chromatography method for quantification of methotrexate polyglutamates in red blood cells of children with juvenile idiopathic arthritis

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 γ‐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. Copyright

High-performance liquid chromatographic assay for the determination of 5-methyltetrahydrofolate in human plasma.

An isocratic high-performance liquid chromatographic method for the determination of 5-methyltetrahydrofolate (5-MTHF) in human plasma is described. The method involves solid-phase extraction of 5-MTHF and p-aminoacetophenon (an internal standard) using Sep-Pak C18 cartridges. Separation was achieved with an ODS column using acetonitrile and phosphate buffer supplemented with octanesulfonic acid (an ion-pairing agent). The pH of the mobile phase (2.5) was optimal with respect to the mode of detection (fluorescence). The method was validated in the range of 5-MTHF concentrations from 0.0625 micromol/l to 4.0 micromol/l. Within-day and inter-day precision expressed by the relative standard deviation was less than 8.1% and inaccuracy did not exceed 8.7%. The method is specific, accurate and sensitive enough to be used in pharmacokinetic studies for the assessment of the systemic availability of 5-MTHF after leucovorin administration to patients as a rescue after high-dose therapy with methotrexate. The limit of detection was 0.17 pmol which corresponds to a plasma concentration of 1.7 nmol/l. Thus, the assay could potentially be used for the measurement of 5-MTHF in the range of physiological concentrations in plasma (5-20 nmol/l).

Intra‐individual variability and influence of urine collection period on dextromethorphan metabolic ratios in healthy subjects

Abstract— The aim of the study was to evaluate intra‐individual variability in metabolic ratios (MRs) of dextromethorphan (DM) in healthy volunteers and to compare the MRs in urine collected 0–4, 0–8 and 0–24 h post‐dose. Urinary molar ratios of DM to dextrorphan (MR1) and of DM to methoxymorphinan (MR2) were obtained after a single oral 27.5 mg dose of DM hydrobromide to ten healthy male and four female Caucasians (ten extensive metabolizers (EM) and four poor metabolizers (PM) of DM) to probe activities of CYP2D6 and CYP3A, respectively. Seven EM and one PM received DM on three additional occasions within 2 months. For the seven EM, the intra‐individual variability (CVW) in the MRs obtained in the three urine collections ranged from 11 to 93% (MR1) and from 8 to 77% (MR2). The mean CVW estimated separately for the 4, 8 and 24 h urines by two‐way analysis of variance reached 58, 57 and 44% for the MR1 and 50, 42 and 31% for the MR2, respectively. For all 14 subjects, the log‐transformed ratios (MR1) obtained in the 24 h urines were highly correlated with those in either the 8 h (rs = 0.967, P <0.0001) or 4 h urines (rs = 0.946, P <0.0001). Correlation between the log‐transformed MR2s were weaker (24 h vs. 8 h: rs = 0.829, P <0.0001, 24 h vs. 4 h: rs = 0.831, P <0.0001). The MRIs in 4 h and 8 h urines were only 2 and 9% less than those in 24 h urines (median differences) and varied from 48 and 47% below to 85 and 55% above (95% –CI for the differences). However, the MR2s in the 4 h and 8 h urines were shifted towards higher values by 49 and 23% and the corresponding 95% –CI limits were: 16–164% (4 h vs. 24 h) and 30–119% (8 h vs. 24 h). In conclusion, MR1 values in the 4 h urine collection agree well with those in longer collections and their use in epidemiological studies can be recommended. The intra‐individual variability of approximately 50% in the MR1 has to be taken into account in clinical studies with within‐subject design. Accurate determination of the MR2 requires at least a 24 h period of urine collection.

Urinary N-acetyl-beta-D-glucosaminidase activity in healthy children.

Aim:  The principal aim was to establish paediatric reference data for the urinary N‐acetyl‐β‐D‐glucosaminidase  (U‐NAG)  activity.