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Background and Objective: High-dose methotrexate (HD-MTX) is the mainstream therapy of current acute lymphoblastic leukemia (ALL) regimens, but frequent intra- and interindividual differences in the clinical response to HD-MTX lead to chemotherapeutic interruption or discontinuation. The exact mechanism of transport across the cell membrane and the disposition of active methotrexate metabolites-methotrexate polyglutamates (MTXPGs)-are not well described in the literature. The aim of this study was to gain more insight into the plasma distribution of methotrexate and MTXPGs in pediatric patients with ALL and to clarify the obscure pathways of MTXPGs., Methods: We prospectively measured the concentrations of MTXPG 1-7 in plasma samples from three male pediatric patients treated with HD-MTX and leucovorin rescue according to the IC-BFM 2009 protocol using liquid chromatography-mass spectrometry (LC-MS). Blood samples were obtained at 24, 36, 42, and 48 h after the start of HD-MTX treatment., Results: Noticeable plasma concentrations of MTXPGs with a 2.2-fold interpatient variability were detected. The highest interindividual variability in total plasma MTXPG concentration was observed at 36 h, and ranged from 13.78 to 30.82 μmol/L. Among all patients, the predominant polyglutamate types in relation to the total plasma MTXPG concentration at each time point were MTXPG 3 (16.71-30.02%) and MTXPG 5 (26.23-38.60%), while MTXPG 7 was the least abundant MTXPG (3.22-5.02%)., Conclusion: The presence of MTXPGs in plasma of patients with ALL could be related to the action of ABC efflux transporters on blood cells and hepatocytes resulting from the administration of high doses of methotrexate. This study may not draw definitive conclusions, but it does reduce uncertainty about the dynamics of methotrexate and its active metabolites, which may be of vital importance for achieving a clinical response., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Objectives: There are no head-to-head trials of different dose escalation strategies of methotrexate (MTX) in RA. We compared the efficacy, safety and tolerability of 'usual' (5 mg every 4 weeks) versus 'fast' (5 mg every 2 weeks) escalation of oral MTX., Methods: This multicentre, open-label (assessor blinded) RCT included patients 18-55 years of age having active RA with disease duration <5 years, and not on DMARDs. Patients were randomized 1:1 into usual or fast escalation groups, both groups starting MTX at 15 mg/week till a maximum of 25 mg/week. Primary outcome was EULAR good response at 16 weeks, secondary outcomes were ΔDAS28 and adverse effects (AE). Analyses were intention-to-treat., Results: 178 patients with mean DAS28-CRP of 5.4(1.1) were randomized to usual (n=89) or fast escalation groups (n=89). At 16 weeks, there was no difference in good EULAR response in the usual (28.1%) or fast escalation (22.5%) groups (p=0.8). There was no difference in mean ΔDAS28-CRP at 8 weeks (-0.9, -0.8, p=0.72) or 16 weeks (-1.3, -1.3, p=0.98). Even at 24 weeks (extended follow-up), responses were similar. There were no inter-group differences in ΔHAQ, or MTX-polyglutamates 1-3 levels at 8 or 16 weeks. Gastrointestinal AE were higher in the fast escalation group over initial 8 weeks (27%, 40%, p=0.048), but not over 16 weeks. There was no difference in cytopenias, transaminitis, or drug discontinuation/dose reduction between the groups. No serious AE were seen., Conclusion: A faster MTX escalation strategy in RA was not more efficacious over 16-24 weeks, and did not significantly increase AE, except higher gastrointestinal AE initially., Trial Registration Number: CTRI/2018/12/016549., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Methotrexate is the gold standard treatment in rheumatoid arthritis. Once absorbed, it is internalized in cells, where glutamate residues are added to produce polyglutamated forms, which are responsible for the effect of methotrexate. The aim of the current study is to determine the relationship between methotrexate triglutamate concentrations and the clinical evolution in rheumatoid arthritis patients, as well as to characterize the variability in both features to propose strategies for low-dose methotrexate optimization. The quantification of methotrexate triglutamate concentration in red blood cells was performed through ultra-performance liquid chromatography coupled with mass spectrometry. Polymorphisms of genes involved in the formation of polyglutamates were determined by real-time polymerase chain reaction. A multivariate regression was performed to determine the covariates involved in the variability of methotrexate triglutamate concentrations and a population pharmacokinetics model was developed through nonlinear mixed-effects modeling. Disease activity score changed according to methotrexate triglutamate concentrations; patients with good response to treatment had higher concentrations than moderate or nonresponding patients. The methotrexate triglutamate concentrations were related to time under treatment, dose, red blood cells, and body mass index. A 1-compartment open model was selected to estimate the pharmacokinetic parameters; the typical total clearance (L/day) was determined as 1.45 * (body mass index/28 kg/m 2 ) * (red blood cells/4.6 × 10 6 cells/μL) and the volume of distribution was 52.4 L, with an absorption rate of 0.0346/day and a fraction metabolized of 1.03%. Through the application of the model, the initial dose of methotrexate is proposed on the basis of stochastic simulations and considering methotrexate triglutamate concentrations found in responders patients., (© 2021, The American College of Clinical Pharmacology.)

High-dose methotrexate (HDMTX) combined with leucovorin (LV) is the first-line drug therapy for many kinds of malignant tumors. However, the specific treatment plans, such as dosage and duration of administration, are usually formulated according to the clinician's experience and therapeutic drug monitoring (TDM) of methotrexate in patients' plasma, which are responsible for strong individual differences of drug usage. A large number of studies have shown that methotrexate targets the inside of the cell. The key cytotoxic component is the methotrexate polyglutamates (MTXPGs) in the cell. The concentration of methotrexate in plasma does not reflect the efficacy and side effects well. Based on mass spectrometry technology, we developed and validated an accurate, sensitive, and stable method to quantify the intracellular MTX (MTXPG 1 ) and its metabolites MTXPG 2-7 simultaneously. The lower limit of quantification was 0.100 ng/ml, and the run time was only 3 min. Moreover, our team has already developed two LC-MS/MS-based methods to respectively quantify methotrexate in plasma samples and two key proteins (γ-glutamyl hydrolase [GGH] and folylpolyglutamate synthetase [FPGS]) in peripheral blood mononuclear cells (PBMC). Through these highly sensitive and accurate approaches, we have gained a deep understanding of the whole pharmacokinetic process of MTX and explored the key factors affecting the accumulation process of intracellular active components (MTXPGs). Based on this research, it is possible to find a more effective way to provide an accurate reference for clinical drug use than traditional therapeutic drug monitoring (TDM).

Aims: We compared the incidence of adverse events between single and divided-dose regimens of methotrexate (MTX) by using a multicenter randomized controlled trial., Methods: Eighty-nine patients with insufficient control on MTX 8 mg/wk were randomly assigned into single-dose (39 patients) or triple dose (39 patients) groups. The MTX dose for all patients was gradually increased to 16 mg/wk. The primary endpoint was the occurrence of liver dysfunction during the observation period (20 weeks)., Results: There were no differences in baseline data and MTX dose at Week 20 between groups. There was no significant difference in the incidence of liver dysfunction between groups (single dose, 3 [7.7%] patients vs. triple dose, 5 [13.2%] patients; P = .455). The incidence of adverse event increased in triple dose (single dose, 12 [30.8%] patients vs. triple dose, 20 [51.3%]), but the difference was not significant (P = .066). There was no significant difference in disease activity between groups, although MTX-triglutamate (PG3), MTX-PG4, and MTX-PG5 were significantly higher in the single dose group., Conclusions: Weekly split dosing reduced the polyglutamation of MTX. There was no significant difference in efficacy and safety between the 2 groups., (© 2020 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.)

Inadequate adherence to maintenance therapy is a major cause of relapse in patients with acute lymphoblastic leukemia (ALL). Therapeutic monitoring of mercaptopurine (thiopurine) red cell metabolites to assess adherence has been available for many years. Recently a clinical laboratory improvement amendments of 1988-approved test for methotrexate with three polyglutamate residues (MTXPG3) measured in peripheral blood red cells was approved. MTXPG3 is the primary intracellular metabolite of methotrexate, and like thiopurine metabolites, is retained for the life of the red cell giving an estimate of drug exposure over time. Normative values for MTXPG3 are available for adults and children with rheumatoid arthritis on methotrexate monotherapy, which are not applicable for patients with ALL on maintenance. Older literature on the MTXPG3 fraction in children with ALL is limited. We examined the MTXPG3 levels from 123 samples in 76 patients with ALL on maintenance oral methotrexate and mercaptopurine that were collected for clinical care. Male individuals had significantly higher MTXPG3 levels than female individuals which was unrelated to absolute neutrophil count, age, serum creatinine, and average doses of methotrexate or mercaptopurine. The MTXPG3 5th, 10th, 90th, and 95th percentile values are 0, 8.4, 53, and 64, respectively with a median of 24.7 nmol/L. The low 5th percentile MTXPG3 reflects 6 samples from 3 patients, age 16 to 21 years that were considered poorly adherent before collecting the specimen. As with red cell thiopurine (mercaptopurine) metabolites, MTXPG3 normative values may provide useful information to monitor for poor patient adherence or methotrexate toxicity during maintenance chemotherapy in ALL.

Most drugs are metabolized in the human body. Therefore, it is essential for therapeutic drug monitoring studies to also take into account the concentrations of drug metabolites. One of the possible metabolic activities on drugs such as pemetrexed or methotrexate is (poly)glutamation. Here, we report on a series of experiments that we performed to investigate the stability of polyglutamate metabolites in plasma. Removal of glutamate residues from pemetrexed polyglutamate by most likely proteases in human plasma is influenced by temperature as it is observed at 25°C and even more strongly at 37°C, but not at 4°C. The observed protease activity is highly variable among patients; in approximately 15-20% of the patients tested it is not observed, whereas in other individuals the activity is so extensive that after 10min, more than 50% of spiked polyglutamated pemetrexed is degraded at room temperature (5-10% of the tested individuals). Similar observations also pertain to methotrexate polyglutamates. These observations do not extend to pemetrexed and methotrexate themselves which are unaffected by this activity. Due to the considerable and, among individuals, variable protease activities on polyglutamated drug metabolites in plasma, these metabolites are virtually impossible to quantify if no precautions are taken., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objectives: The objective of this study is to evaluate the pharmacokinetics and pharmacodynamics of methotrexate-polyglutamates (MTX-PGs) in erythrocytes in patients with rheumatoid arthritis and correlate them with the efficacy., Methods: MTX-PG concentrations in erythrocytes were measured in 42 MTX-naïve patients repeatedly for 24 weeks by high-performance liquid chromatography. In 56 patients receiving stable MTX doses for at least 12 weeks, the correlation between MTX doses and MTX-PG concentrations was examined. The efficacy was measured by the change of DAS28CRP (ΔDAS28CRP)., Results: There were moderate correlations between MTX dose and MTX-PG 3, 4, and 5. At 24 weeks, MTX-PG2, 3, 4, and 1-5 were higher in patients with ΔDAS28CRP >1.2 than in those with ≤1.2. The cutoff value of MTX-PG1-5 to discriminate ΔDAS28CRP >1.2 from ≤1.2 at 24 weeks was 68.7 nM. Among 20 patients with MTX-PG1-5 > 50.6 nM at 8 weeks, seven already improved at 8 weeks and additional 11 improved at 24 weeks (p < 0.001). On the contrary, among the nine patients with MTX-PG1-5 ≤ 50.6 nM at 8 weeks, none improved at 8 weeks and only one improved at 24 weeks (p = 0.500)., Conclusions: Erythrocyte MTX-PGs might be a potential indicator and predictor of MTX efficacy.

Methotrexate is an efficacious immunosuppressant for induction and maintenance of remission in Crohn's disease. The goal of this pilot study was to determine whether total or individual methotrexate glutamate levels (MTXGlu n ) in red blood cells correlate with disease activity and adverse events in Crohn's disease. A cross-sectional study was undertaken with 12 patients on a stable dose of 25 mg weekly methotrexate (oral or subcutaneous). Clinical disease activity was assessed by the Harvey-Bradshaw Index (HBI), and biologic disease activity was measured by inflammatory markers. Concentrations of individual MTXGlu n levels were measured in red blood cells (RBCs) using high-performance liquid chromatography-mass spectrometry. No association was observed between RBC individual (MTXGlu n ) or total methotrexate glutamate concentrations and clinical disease activity (HBI score) or inflammatory markers or adverse events. Although Crohn's disease patients in remission appeared to generally have higher RBC total longer-chain methotrexate polyglutamate (MTXGlu 3+4+5 ) concentrations compared with those with active disease, a definitive association between RBC MTXGlu 3+4+5 levels and clinical disease activity could not be established. Larger longitudinal studies in patients with diverse disease activity are needed to establish the value of MTXGlu n levels as indicators of treatment efficacy and clinical outcome., (© 2016, The American College of Clinical Pharmacology.)

Background: It is unclear whether erythrocyte methotrexate polyglutamate levels (MTX-glun) are associated with response or adverse effects to methotrexate in rheumatoid arthritis. This preliminary study evaluated their utility in Asian Indian patients over 24 weeks., Methods: Rheumatoid arthritis patients were started on oral methotrexate at a dose of 15 mg/wk, which was escalated to 25 mg by 12 weeks and continued till 24 weeks. Erythrocyte (RBC) MTX-glu1 to MTX-glu5 levels (nmol/L RBC) were determined at 4, 8, 16, and 24 weeks by using reverse-phase high-performance liquid chromatography. Area under the concentration curve (AUC) of MTX-glu1-5, MTX-glu3-5, and MTX-glu3 levels was compared between groups with regards to response and adverse effects., Results: This study included 117 patients with mean (SD) age of 42.7 (±11.9) years and disease duration of 2.0 (1.7) years. Mean (SD) RBC MTX-glu1-5 levels at 4, 8, 16, and 24 weeks were 93 (±29), 129 (±46), 143 (±49), and 159 (±65) nmol/L RBC; the highest individual polyglutamate was MTX-glu3 (40%). There was significant correlation between MTX-glu1-5 (r = 0.38, P < 0.001) and MTX-glu3 (r = 0.49, P < 0.001) with methotrexate dose. There was no significant difference of AUC MTX-glun between responders and nonresponders. However, AUC MTX-glu3 was significantly (P = 0.03) higher in patients with adverse effects. On logistic regression, AUC of MTX-glu3 [odds ratio = 1.004 (95% confidence interval 1.002-1.007)] and methotrexate dose at 24 weeks were independent predictors of adverse effects., Conclusions: In this preliminary study, higher levels of RBC MTX-glu3 were found to be the independent predictors for adverse effects in rheumatoid arthritis patients.

Background and Purpose: Poly-l-glutamic acid (PG) has been used widely as a carrier to deliver anticancer chemotherapeutics. This study evaluates PG as a selective renal drug carrier., Experimental Approach: 3 H-deoxycytidine-labeled PGs (17 or 41 kDa) and 3 H-deoxycytidine were administered intravenously to normal rats and streptozotocin-induced diabetic rats. The biodistribution of these compounds was determined over 24 h. Accumulation of PG in normal kidneys was also tracked using 5-(aminoacetamido) fluorescein (fluoresceinyl glycine amide)-labeled PG (PG-AF). To evaluate the potential of PGs in ferrying renal protective anti-oxidative stress compounds, the model drug 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) was conjugated to 41 kDa PG to form PG-AEBSF. PG-AEBSF was then characterized and evaluated for intracellular anti-oxidative stress efficacy (relative to free AEBSF)., Results: In the normal rat kidneys, 17 kDa radiolabeled PG (PG-Tr) presents a 7-fold higher, while 41 kDa PG-Tr shows a 15-fold higher renal accumulation than the free radiolabel after 24 h post injection. The accumulation of PG-AF was primarily found in the renal tubular tissues at 2 and 6 h after an intravenous administration. In the diabetic (oxidative stress-induced) kidneys, 41 kDa PG-Tr showed the greatest renal accumulation of 8-fold higher than the free compound 24 h post dose. Meanwhile, the synthesized PG-AEBSF was found to inhibit intracellular nicotinamide adenine dinucleotide phosphate oxidase (a reactive oxygen species generator) at an efficiency that is comparable to that of free AEBSF. This indicates the preservation of the anti-oxidative stress properties of AEBSF in the conjugated state., Conclusion/implications: The favorable accumulation property of 41 kDa PG in normal and oxidative stress-induced kidneys, along with its capabilities in conserving the pharmacological properties of the conjugated renal protective drugs, supports its role as a potential renal targeting drug carrier., Competing Interests: The authors report no conflicts of interest in this work.

Currently no quantification method exists for potentially therapeutically relevant polyglutamate metabolites of the drug pemetrexed which is used for the treatment of lung carcinoma patients. We developed and tested an LC-MS/MS-based analytical assay that uses isotope-labeled internal standards to quantify pemetrexed and its (poly)glutamate metabolites in clinical human plasma samples of lung carcinoma patients. UHPLC chromatography and triple quadrupole mass spectrometry showed an LLOQ of 0.2nmol/L for pemetrexed and an LLOQ of 0.5nmol/L for the two metabolites (one glutamate and two glutamate moieties covalently bound to the pemetrexed molecule, for which no other quantification methods have previously been published). The recoveries for PMTX and its metabolites ranged between 30% and 67%. Precision and accuracy at a concentration of 20nmol/L for all four analytes was well below 15% CV. The precision (RSD) in the biological replicates of the separate days (within-run precision) as well as the reproducibility over several days (between-run precision), tested in the range of 5-250nmol/L, were all below 15%. Autosampler, benchtop and freeze-thaw cycle stability of the analytes was also demonstrated. To illustrate the new assay in a relevant biological context, concentrations of pemetrexed and the two metabolites were quantified in plasma samples of lung carcinoma patients treated with pemetrexed. The assay is straightforward, relatively easy to perform, and has potential for use in therapeutic drug monitoring in non-small cell lung carcinoma patients., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Introduction: Methotrexate (MTX) is a cornerstone of treatment in a wide variety of inflammatory conditions, including juvenile idiopathic arthritis (JIA) and juvenile dermatomyositis (JDM). However, owing to its narrow therapeutic index and the considerable interpatient variability in clinical response, monitoring of adherence to MTX is important. The present study demonstrates the feasibility of using methotrexate polyglutamates (MTXPGs) as a biomarker to measure adherence to MTX treatment in children with JIA and JDM., Methods: Data were collected prospectively from a cohort of 48 children (median age 11.5 years) who received oral or subcutaneous (SC) MTX therapy for JIA or JDM. Dried blood spot samples were obtained from children by finger pick at the clinic or via self- or parent-led sampling at home, and they were analysed to determine the variability in MTXPG concentrations and assess adherence to MTX therapy., Results: Wide fluctuations in MTXPG total concentrations (>2.0-fold variations) were found in 17 patients receiving stable weekly doses of MTX, which is indicative of nonadherence or partial adherence to MTX therapy. Age (P = 0.026) and route of administration (P = 0.005) were the most important predictors of nonadherence to MTX treatment. In addition, the study showed that MTX dose and route of administration were significantly associated with variations in the distribution of MTXPG subtypes. Higher doses and SC administration of MTX produced higher levels of total MTXPGs and selective accumulation of longer-chain MTXPGs (P < 0.001 and P < 0.0001, respectively)., Conclusions: Nonadherence to MTX therapy is a significant problem in children with JIA and JDM. The present study suggests that patients with inadequate adherence and/or intolerance to oral MTX may benefit from SC administration of the drug. The clinical utility of MTXPG levels to monitor and optimise adherence to MTX in children has been demonstrated., Trial Registration: ISRCTN Registry identifier: ISRCTN93945409 . Registered 2 December 2011.

We report the application of a fully automated surface-enhanced Raman scattering (SERS)-based solenoid-embedded microfluidic device to the quantitative and sensitive detection of anthrax biomarker poly-γ-D-glutamic acid (PGA) in solution. Analysis is based on the competitive reaction between PGA and PGA-conjugated gold nanoparticles with anti-PGA-immobilized magnetic beads within a microfluidic environment. Magnetic immunocomplexes are trapped by yoke-type solenoids embedded within the device, and their SERS signals were directly measured and analyzed. To improve the accuracy of measurement process, external standard values for PGA-free serum were also measured through use of a control channel. This additional measurement greatly improves the reliability of the assay by minimizing the influence of extraneous experimental variables. The limit of detection (LOD) of PGA in serum, determined by our SERS-based microfluidic sensor, is estimated to be 100 pg/mL. We believe that the defined method represents a valuable analytical tool for the detection of anthrax-related aqueous samples., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Development of high-concentration antibody formulations for subcutaneous administration remains challenging. Recently, a precipitation-redissolution method was proposed to prepare suspensions or precipitates of salt-dissociable protein-poly(amino acid) complexes. To elucidate the utility of this method for protein therapy, we investigated the feasibility of a precipitation-redissolution method using poly(amino acid) for high-concentration antibody formulation. Omalizumab and adalimumab formulations of 150 mg/mL could be prepared using poly-l-glutamic acid (polyE) from low-concentration stock solutions. Enzyme-linked immunosorbent assay, circular dichroism, and size-exclusion chromatography revealed that the formation of antibody-polyE complex and precipitation-redissolution process did not significantly affect the immunoreactivity or secondary structure of the antibodies. The precipitation-redissolution method was less time-consuming and more effective than lyophilization-redissolution, evaporation-redissolution, and ultrafiltration from the viewpoint of final yield. Scalability was confirmed from 400 μL to 1.0 L. The general toxicity and pharmacokinetic profiles of the antibody-polyE complex formulations were similar to those of conventional antibody formulations. These results suggested that the precipitation-redissolution method using poly(amino acid) has great potential as a concentration method for antibody formulation and medicinal use., (© 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Inhalational anthrax is a serious biothreat. Effective antibiotic treatment of inhalational anthrax requires early diagnosis; the further the disease has progressed, the less the likelihood for cure. Current means for diagnosis such as blood culture require several days to a result and require advanced laboratory infrastructure. An alternative approach to diagnosis is detection of a Bacillus anthracis antigen that is shed into blood and can be detected by rapid immunoassay. The goal of the study was to evaluate detection of poly-γ-D-glutamic acid (PGA), the capsular antigen of B. anthracis, as a biomarker surrogate for blood culture in a rabbit model of inhalational anthrax. The mean time to a positive blood culture was 26 ± 5.7 h (mean ± standard deviation), whereas the mean time to a positive ELISA was 22 ± 4.2 h; P = 0.005 in comparison with blood culture. A lateral flow immunoassay was constructed for detection of PGA in plasma at concentrations of less than 1 ng PGA/ml. Use of the lateral flow immunoassay for detection of PGA in the rabbit model found that antigen was detected somewhat earlier than the earliest time point at which the blood culture became positive. The low cost, ease of use, and rapid time to result of the lateral flow immunoassay format make an immunoassay for PGA a viable surrogate for blood culture for detection of infection in individuals who have a likelihood of exposure to B. anthracis.

Background: Methotrexate (MTX) is the cornerstone disease-modifying anti-rheumatic drug in juvenile idiopathic arthritis (JIA). In JIA, it is important to start effective treatment early to avoid long-term sequelae, such as joint damage. To accomplish this goal, it is crucial to know beforehand who is going to respond well to MTX. In addition, MTX adverse effects such as MTX intolerance occur frequently, potentially hindering its efficacy. To avoid inefficacy of an otherwise effective drug, the physician should be timely aware of these adverse events. Consequently, to optimise treatment of JIA patients with MTX, predictors for efficacy and adverse events should be used in daily clinical practice. The aim of this study was to summarise the existing knowledge about such predictors., Methods: A systematic literature search was performed in PubMed, Embase and The Cochrane Library, and 1,331 articles were identified. These were selected based on their relevance to the topic and critically appraised according to pre-defined criteria. Predictors for MTX efficacy and adverse events were extracted from the literature and tabulated., Results: Twenty articles were selected. The overall quality of the studies was good. For MTX efficacy, candidate predictors were antinuclear antibody positivity, the childhood health assessment questionnaire score, the myeloid-related protein 8/14 level, long-chain MTX polyglutamates, bilateral wrist involvement and some single nucleotide polymorphisms (SNPs) in the adenosine triphosphate binding cassette and solute carrier transporter gene families. For MTX adverse events, potential predictors were alanine aminotransferase and thrombocyte level and two SNPs in the γ-glutamyl hydrolase and methylenetetrahydrofolate reductase genes. However, validation of most predictors in independent cohorts was still lacking., Conclusions: Interesting candidate predictors were found, especially for MTX efficacy. However, most of these were not validated. This should be the goal of future efforts. A clinically relevant way to validate the predictors is by means of creating a clinical prediction model.

Background: The folate antagonist methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis. The therapeutic effects of MTX are attributed to the intracellular levels of MTX, present in the cell as polyglutamates (MTX-PGs). We aimed to validate an immunoassay for the measurement of MTX-PG in erythrocytes., Methods: Samples were analyzed by an adapted fluorescence polarization immune assay (FPIA) method on the FLx analyzer (Abbott). Cross-reactivity was determined in both plasma and erythrocyte pellet. In erythrocyte pellet, the imprecision, linearity, and lower limit of quantitation were determined. The method was compared with our in-house liquid chromatography tandem mass spectrometry (LC-MS/MS) method for total MTX-PG., Results: For the adapted FPIA method, a linear range of 25-1000 nmol/L (R = 0.993) was obtained for total MTX-PG in erythrocytes. A coefficient of variation of <17% for interday and <8% for intraday imprecision was found and average recovery was 91%. Lower limit of quantitation was determined at 50 nmol/L total MTX-PG with a coefficient of variation of 15%. There was no significant proportional bias of the FPIA assay compared with our in-house LC-MS/MS method, but a (nonsignificant) constant positive bias was present [FPIA = 1.00 (95% confidence interval: 0.60-1.95) × LC-MS/MS + 31.00 nmol/L (95% confidence interval: -11.83 to 61.00)]. Results could be very different for individual patients as reflected in the poor R of 0.419., Conclusions: The FPIA method can be used to measure total MTX-PG in erythrocytes. Although there was no significant bias detected compared with the LC-MS/MS method, the FPIA method showed constant positive bias, probably because of interference from folates and MTX metabolites 2,4-diamino-N10-methylpteroic acid and 7-hydroxy-MTX. The correlation between both methods was average and resulted in large differences in individual patients, most likely because of problems during sample preparation.

Objective: Low-dose methotrexate (MTX) is the anchor drug in the treatment for rheumatoid arthritis (RA). Response to MTX is related to the intracellular MTX-polyglutamate (MTX-PG) levels and little is known about its determinants. We aimed to define the determinants of erythrocyte MTX-PG concentrations in 2 prospective cohorts of patients with RA., Methods: Patients with RA treated with MTX from 2 longitudinal cohorts were included: 93 from the MTX-R study (Rotterdam, the Netherlands derivation cohort), and 247 from the treatment in Rotterdam Early Arthritis Cohort study (validation cohort). MTX-PG concentrations were measured at 3 months of treatment using liquid chromatography/mass spectrometry. The MTX-PG were used as outcome measure. Various sociodemographic, clinical, biochemical, and genetic factors were assessed at baseline. Associations with MTX-PG levels were analyzed using multivariate regression analysis., Results: Age was positively associated with MTX-PG1 (stβ 0.23, p=0.033) and total MTX-PG (stβ 0.23, p=0.018) in the derivation cohort, and with all MTX-PG in the validation cohort (MTX-PG1: stβ 0.13, p=0.04; MTX-PG2: stβ 0.21, p=0.001; MTX-PG3: stβ 0.22, p<0.001; MTX-PG4+5: stβ 0.25, p<0.001; and total MTX-PG: stβ 0.32, p<0.001). Erythrocyte folate levels were positively associated with MTX-PG3 (stβ 0.3, p=0.021) and total MTX-PG levels (stβ 0.32, p=0.022) in the derivation cohort, which was replicated for MTX-PG3 (stβ 0.15, p=0.04) in the validation cohort. Patients with the folylpolyglutamate synthase (FPGS) rs4451422 wild-type genotype had higher concentrations of MTX-PG3 (p<0.05), MTX-PG4+5 (p<0.05), and total MTX-PG (p<0.05) in both cohorts. In the combined cohort, MTX dose was positively associated with levels of MTX-PG3 (stβ 0.23, p<0.001), MTX-PG4+5 (stβ 0.30, p<0.001), and total MTX-PG (stβ 0.20, p=0.002), but negatively associated with MTX-PG2 levels (stβ -0.22, p<0.001)., Conclusion: Our prospective study shows that higher age, higher MTX dose, higher erythrocyte folate status, and the FPGS rs4451422 wild-type genotype are associated with higher MTX-PG concentrations. While only up to 21% of interpatient variability can be explained by these determinants, this knowledge may aid in the development of personalized treatment in RA.

Recently we reported for the first time a new type of nanocapsules consisting of an oily core and a polymer shell made of a polyglutamic acid-polyethylene glycol (PEG-PGA) grafted copolymer with a 24% w/w PEG content. The goal of the work presented here has been to develop a new version of these nanocapsules, in which the shell is made of a di-block PEG-PGA copolymer with a 57% w/w PEG content and to evaluate their potential for improving the biodistribution and pharmacokinetics of the anticancer drug docetaxel (DCX). A comparative analysis of the biodistribution of fluorescently labeled PGA-PEG nanocapsules versus PGA nanocapsules or a control nanoemulsion (containing the same oil than the nanocapsules) showed that the nanocapsules, and in particular PEGylated nanocapsules, have significantly higher half-life, MRT (Mean Residence Time) and AUC (Area under the Curve) than the nanoemulsion. On a separate set of experiments, PGA-PEG nanocapsules were loaded with DCX and their antitumor efficacy was evaluated in a xenograft U87MG glioma mouse model. The results showed that the survival rate for mice treated with DCX-loaded nanocapsules was significantly increased over the control Taxotere®, while the antitumoral effect of both formulations was comparable (60% tumor growth inhibition with respect to the untreated mice). These results highlight the potential use of these novel nanocapsules as a new drug delivery platform in cancer therapy., (Copyright © 2014. Published by Elsevier B.V.)

Objective: Development and validation of a selective and sensitive LCMS method for the determination of methotrexate polyglutamates in dried blood spots (DBS)., Methods: DBS samples [spiked or patient samples] were prepared by applying blood to Guthrie cards which was then dried at room temperature. The method utilised 6-mm disks punched from the DBS samples (equivalent to approximately 12 µl of whole blood). The simple treatment procedure was based on protein precipitation using perchloric acid followed by solid phase extraction using MAX cartridges. The extracted sample was chromatographed using a reversed phase system involving an Atlantis T3-C18 column (3 µm, 2.1 × 150 mm) preceded by Atlantis guard column of matching chemistry. Analytes were subjected to LCMS analysis using positive electrospray ionization., Key Results: The method was linear over the range 5-400 nmol/L. The limits of detection and quantification were 1.6 and 5 nmol/L for individual polyglutamates and 1.5 and 4.5 nmol/L for total polyglutamates, respectively. The method has been applied successfully to the determination of DBS finger-prick samples from 47 paediatric patients and results confirmed with concentrations measured in matched RBC samples using conventional HPLC-UV technique., Conclusions and Clinical Relevance: The methodology has a potential for application in a range of clinical studies (e.g. pharmacokinetic evaluations or medication adherence assessment) since it is minimally invasive and easy to perform, potentially allowing parents to take blood samples at home. The feasibility of using DBS sampling can be of major value for future clinical trials or clinical care in paediatric rheumatology.

Background: The methotrexate (MTX) polyglutamate assay has been validated in adults with arthritis., Objective: We sought to assess clinical response rates and the value of the methotrexate polyglutamate assay in MTX-treated children with inflammatory skin diseases., Methods: In this retrospective review, 46 MTX-treated children with a diagnosis of atopic dermatitis, psoriasis, or psoriasis-eczema overlap were serially assessed with the methotrexate polyglutamate assay., Results: In all, 38 children (83%) achieved good to excellent response: 27 (59%) within 12 weeks and 11 (24%) after dose-adjustment. Good to excellent responses were highest for psoriasis/overlap: 15 of 16 (94%), compared with 23 of 30 (77%) with atopic dermatitis. Mean maximum polyglutamate levels were 31.5 nmol/L for responders versus 18.1 nmol/L for nonresponders (P = .035). This difference was also significant for the subset with atopic dermatitis, but not for those with psoriasis/overlap. After dose modification, late responders ultimately achieved a significantly higher mean maximum methotrexate polyglutamate assay (41.9 nmol/L) compared with nonresponders (P = .002)., Limitations: Retrospective design and small sample size were limitations., Conclusions: MTX is an effective treatment for the majority of children with inflammatory skin diseases, but a subset requires dose modification to achieve good to excellent response. Methotrexate polyglutamate assay levels reflect response to treatment, but are most useful to support dose modification among children who fail to respond within 12 weeks., (Copyright © 2013 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.)

Background: Measurement of intracellular concentrations of methotrexate (MTX) and its polyglutamated metabolites (MTXGlu(2-5)) in red blood cells (RBCs) has been suggested as a potential means of monitoring low-dose MTX treatment of rheumatoid arthritis (RA). However, a possible correlation between RBC MTX and MTXGlu2-5 concentrations and clinical outcomes of MTX treatment in RA is debated. A better understanding of the dose-concentration-time relationship of MTX and MTXGlu(2-5) in RBCs by population pharmacokinetic modelling is desirable and will facilitate assessing a potential RBC concentration-effect relationship in the future., Aim: The purpose of this analysis was to describe the pharmacokinetics of MTX and MTXGlu(2-5) in RBCs. Secondary objectives included investigation of deglutamation reactions and the loss of MTX and MTXGlu(2-5) from the RBC., Methods: A model was developed using NONMEM(®) version 7.2 based on RBC data obtained from 48 patients with RA receiving once-weekly low-dose MTX treatment. This model was linked to a fixed two-compartment model that was used to describe the pharmacokinetics of MTX in the plasma. A series of five compartments were used to describe the intracellular pharmacokinetics of MTX and MTXGlu(2-5) in RBCs. Biologically plausible covariates were tested for a significant effect on MTX plasma clearance and the intracellular volume of distribution of all MTX species in RBCs ([Formula: see text]). The developed model was used to test hypotheses related to the enzymatic deglutamation of MTXGlu(2-5) and potential loss of MTXGlu(2-5) from RBCs., Results: The final RBC pharmacokinetic model required the intracellular volumes of distribution for the parent and metabolites to be set to the value estimated for the parent drug MTX alone, and the rate constants describing the polyglutamation steps were fixed at literature values. Significant covariates included effect of body surface area-adjusted estimated glomerular filtration rate on renal plasma clearance and effect of allometrically scaled total body weight with a fixed exponent of 0.75 on non-renal plasma clearance of MTX. The only significant covariate with an effect on [Formula: see text] was mean corpuscular volume (MCV). The model supported single deglutamation steps and a single mechanism of MTX and MTXGlu(2-5) loss from RBCs., Conclusions: The developed model enabled acceptable description of the intracellular kinetics of MTX and MTXGlu(2-5) in RBCs. In the future it can form the basis of a full pharmacokinetic-pharmacodynamic model to assess the time-RBC concentration-effect relationship of low-dose MTX treatment in RA.

The folate antagonist methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis. The therapeutic effects of MTX are attributed to the intracellular levels of MTX, present in the cell as polyglutamates (MTXPGn). We developed a new liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS)-based assay to separately quantitate MTXPGn in red blood cells using stable-isotope-labelled internal standards. Samples were analyzed by LC-ESI-MS/MS using a Waters Acquity UPLC BEH C18 column with a 5-100% organic gradient of 10 mM ammonium bicarbonate (pH 10) and methanol. The analysis consisted of simple sample preparation and a 6-min run time. Detection was done using a Waters Acquity UPLC coupled to a Waters Quattro Premier XE with electrospray ionization operating in the positive ionization mode. Assay validation was performed following recent Food and Drug Administration guidelines. The method was linear from 1-1,000 nM for all MTXPGn (R(2) > 0.99). The coefficient of variation ranged from 1-4% for intraday precision and 6-15% for interday precision. Samples were stable for at least 1 month at -80 °C. Recovery ranged from 98-100%, and the relative matrix-effect varied from 95-99%. The lower limit of quantitation was 1 nM for each MTXPGn. Fifty patient samples from the tREACH study were analyzed. The MTXPGn concentration and distribution of these samples were comparable with values reported in literature. The developed LC-ESI-MS/MS method for the quantitative measurement of MTXPGn in red blood cells is both sensitive and precise within the clinically relevant range. The method can be easily applied in clinical laboratories due to the combination of simple pre-treatment with robust LC-ESI-MS/MS.

The aims of the present study were to define inter-individual differences in response to methotrexate (MTX) through MTX polyglutamate (MTX-PG) levels in red blood cells (RBC) and MTX-related gene polymorphisms. A total of 145 rheumatoid arthritis patients were recruited. MTX-PG1-5 concentrations in RBC were measured, and 11 single nucleotide polymorphisms, all in MTX-related genes involved in the folate pathway, were analyzed. Disease activity was also assessed. There was no direct relationship between any MTX-PG concentration and the patient's disease condition, but detectability of MTX-PG5 was extracted as a candidate marker for response to MTX. When disease activity was compared between patients in which MTX-PG5 was detectable and undetectable, all indexes except the visual analog scale (VAS) and C-reactive protein (CRP) were found to be significantly lower in the former patients. Reduced folate carrier 1 (RFC1) 80G>A was significantly associated with the detectability of MTX-PG5; detectability of MTX-PG5 was lower in patients with the A mutant allele. The present study suggests that detectability of MTX-PG5 in RBC is a possible biomarker for response to MTX, and the RFC1 80G>A mutation is associated with low detectability of MTX-PG5. Prospective studies with a sufficient number of patients are needed to confirm the present findings.

Methotrexate (MTX) is currently one of the most widely used drugs for treatment of rheumatoid arthritis (RA) through polyglutamation of methotrexate polyglutamates (MTXPGs), a process attaching sequential γ-linked glutamic residues to MTX. A new and sensitive LC/MS/MS method was developed and validated for determination of whole-blood MTX and total MTX (MTX+MTXPGs), and then concentration of MTXPGs was calculated. To determine whole-blood MTX, whole blood was precipitated with 50% trifluoroacetic acid, and extraction was performed using ethyl acetoacetate. Analytes were subjected to LC/MS/MS analysis using positive electrospray ionization. To determine whole-blood total MTX, whole blood was incubated with ascorbic acid (200 mM) at 37°C for 3h to enzymatically convert the MTXPGs to MTX, and then processed with the same method mentioned above. Recoveries of spiked MTX at ppb (ng/mL) level were between 26.2% and 37.8% with intra- and inter-day precision less than 15.8% and 11.8%, respectively. The lower limit of detection (LLOD) and lower limit of quantitation (LLOQ) were 0.5 ng/mL and 1 ng/mL, respectively. The sensitive LC/MS/MS method was fully validated with high selectivity and acceptable accuracy and precision, which was successfully applied to determine the erythrocyte methotrexate polyglutamates in patients with RA., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Methotrexate is activated by the sequential addition of glutamic acid residues to form methotrexate polyglutamates (MTXPG(1-5)). MTXPG(1-5) inhibit enzymes of the folate-purine-pyrimidine pathways, and longer-chain MTXPG(3-5) species are more active., Objectives: To determine the pattern of erythrocyte MTXPG(1-5) in patients initiated on oral methotrexate for psoriasis, and to investigate the potential utility of MTXPGs as markers of compliance and/or clinical response., Methods: This was a single-centre, prospective study of 55 adult patients with chronic plaque psoriasis initiated on weekly oral methotrexate. Erythrocyte MTXPG(1-5) concentrations were measured (at weeks 4, 8, 12, 24 and 52) using high-performance liquid chromatography. Methotrexate responders achieved ≥ 50% improvement in Psoriasis Area and Severity Index or physician's global score of 'clear'/'nearly clear' at 24 weeks., Results: MTXPG levels were measured in 14-33 patients at each time point. All MTXPG(1-5) species were detected at week 4 of therapy. Steady state for long-chain MTXPG(3-5) and total MTXPG(1-5) was achieved by week 24. MTXPG(3) emerged as the predominant MTXPG species (from week 12 onwards) and reflected overall polyglutamate status (correlating strongly with MTXPG(2-5) , MTXPG(3-5) and MTXPG(4-5) ; R = 0·76-0·95, P < 1·55 × 10(-5)). Age, renal function and sex were not significant determinants of MTXPG(3) concentration. No significant association was identified between MTXPG and adverse events or responder status., Conclusions: This is the first study to demonstrate the prospective accumulation of MTXPG(1-5) in patients with psoriasis. The detection of MTXPGs early in therapy and the establishment of a steady state with continuous treatment may offer measuring of MTXPG as a test to monitor patient compliance with therapy. Larger studies are required to determine the role of MTXPG as a potential biomarker of clinical response., (© 2012 The Authors. BJD © 2012 British Association of Dermatologists.)

Objectives: Methotrexate (MTX) is a cornerstone in the treatment of rheumatoid arthritis (RA). Although in general MTX is very effective, the major drawback is the large inter-patient variability in clinical response. The circulating levels of MTX polyglutamates (MTXPGs) are supposed to correlate with clinical efficacy, therefore having a potential role in drug monitoring. However, there is a controversial discussion about the importance of methotrexate polyglutamates as outcome parameters in the therapy of rheumatoid arthritis. The aim of the present study was to investigate the formation and pharmacokinetics of MTXPGs and to correlate their concentration with clinical response in MTX-naïve patients., Methods: The pharmacokinetics of erythrocyte MTXPGs was determined in samples of nineteen MTX-naïve patients by high pressure liquid chromatography (HPLC) using post-column photo-oxidation and fluorimetric detection. The relationship between erythrocyte concentrations of MTXPGs and the primary outcome parameter DAS-28 was assessed using the Spearman's correlation coefficient., Results: The short-chain polyglutamate MTXPG2 revealed to be a potential marker for clinical outcome in rheumatoid arthritis with a statistically significant positive correlation of MTXPG2 Cmax levels and improvement in DAS-28 (+0.518, p=0.023) over 16 weeks. Furthermore, Cmax levels of MTXPG2 negatively correlated with basophils (-0.478, p=0.038) and eosinophils (-0.531, p=0.019), both pro-inflammatory cells involved in the disease., Conclusions: MTXPG2 seems to be a potential indicator for clinical response and may serve as a marker for drug monitoring.

The in vivo efficacy of doxorubicin (DOX)-loaded poly(γ-benzyl l-glutamate)-block-hyaluronan (PBLG(23)-b-HYA(10))-based polymersomes (PolyDOX) was evaluated. Samples were efficiently labeled with technetium-99m radionuclide with good stability for in vivo studies. PolyDOX enhanced circulation time compared to free DOX. Biodistribution studies revealed selective accumulation of PolyDOX in the Ehrlich ascites tumor (EAT) as a result of passive accumulation and active targeting (CD44-mediated endocytosis) in EAT-bearing mice. Toxicity studies demonstrated PolyDOX is a safe drug carrier, and no hemolysis was observed with PolyDOX equivalent to 200 μg/mL of free DOX. PolyDOX dominantly controlled tumor growth by delaying doubling time of EATs compared to free DOX over 30 days after treatment. PolyDOX also increased life span six times more than free DOX. Hence, it is reasonable to expect that higher DOX levels attributable to PolyDOX improve the therapeutic index and reduce side effects due to site-specific drug accumulation., From the Clinical Editor: In this preclinical project, doxorubicin loaded polymersomes enhanced intracellular uptake of doxorubicin in a murine model of Ehrlich Ascites Tumor (EAT) through CD44 receptor mediated endocytosis, resulting in prolonged Tumor Doubling Time and increase in life span of mice., (2012 Elsevier Inc. All rights reserved.)

Background: Low-dose methotrexate is used for the treatment of rheumatoid arthritis and juvenile idiopathic arthritis, but its effectiveness greatly varies between individuals. Therapeutic drug monitoring of intracellular methotrexate metabolites, the γ-polyglutamates (MTXGlu(n)), in human erythrocytes has shown promise in providing a basis for individualization of therapy., Results: This work presents expedient methodology for the analysis of MTXGlu(1-7) in human erythrocytes by ion-pair UPLC with detection by tandem MS (UPLC-ESI-MS/MS). The use of N,N-dimethylheptylamine as an ion-pair agent was found to be favorable over others. Thermal extraction of erythrocyte lysates provides a simple one-step extraction procedure. The entire chromatographic run time is 6 min and the assay was validated within the therapeutic range of these metabolites, Conclusion: The developed sample preparation procedure in combination with ion-pair UPLC-ESI-MS/MS analysis allowed for expedient quantitation of MTXGlu(1-7) in human erythrocytes. The rapid analysis time would enable therapeutic drug monitoring of MTXGlu(1-7) in the clinic.

Objective: To determine the effects of changing from oral to subcutaneous (SC) methotrexate (MTX) in patients with rheumatoid arthritis (RA) on red blood cell MTX polyglutamate (RBC MTXGlu(n)) concentrations, disease activity, and adverse effects., Methods: Thirty patients were changed from oral to SC MTX. Trough RBC MTXGlu(n) concentrations were measured for 24 weeks and concentrations fitted to a first-order accumulation model. Disease activity was assessed by 28-joint Disease Activity Score (DAS28)., Results: MTXGlu(3), MTXGlu(4), and MTXGlu(5) concentrations, but not MTXGlu(1) and MTXGlu(2), increased significantly over 24 weeks, reaching 90% of new steady-state concentrations by about 40 weeks. A decrease in DAS28 was associated with increased RBC MTXGlu(5) (p = 0.035) and RBC MTXGlu(3-5) (p = 0.032). No change in adverse effect frequency occurred., Conclusion: Changing to SC MTX results in increased long-chain MTXGlu(n). However, it takes at least 6 months for RBC steady-state concentrations to be achieved. Increased long-chain MTXGlu(n) concentrations were significantly associated with reduced disease activity.

Objectives: Methotrexate (MTX) is a cornerstone in the treatment of rheumatoid arthritis (RA). Among its anti-proliferative activity, the anti-inflammatory mechanisms of MTX seem to play a major role in the treatment of RA. MTX reduces the production of pro-inflammatory cytokines such as interleukin (IL)-1, IL-2, IL-6 and interferon (INF)-γ, while the gene expression of anti-inflammatory Th2 cytokines like IL-4 and IL-10 is increased - altogether resulting in the anti-inflammatory effect. As little is known about the impact of MTX on other cytokines involved in the pathogenesis of RA, the present trial investigated the effect of MTX on IL-12A and IL-18 gene expression by peripheral blood mononuclear cells (PBMCs). For comparison, the effect on IL-6 and tumour necrosis factor (TNF) was analysed., Methods: Using real-time PCR, mRNA concentrations of pro-inflammatory cytokines were determined in PBMCs from 17 patients before and during MTX therapy. Furthermore, gene expression was correlated with clinical and pharmacokinetic parameters such as methotrexate polyglutamate concentrations (Spearman's correlation coefficient). To eliminate concomitant corticosteroids as confounding factor, a subgroup analysis for methotrexate without corticosteroids was performed in 6 patients., Results: MTX statistically significantly reduced the mRNA expression of IL-12A by PBMCs in rheumatoid arthritis patients (Wilcoxon-test for paired samples, p<0.046). Consistent with other reports, IL-6 was reduced under MTX treatment. Although the combination of MTX and corticosteroids significantly reduced the gene expression of IL-18, this key molecule was unaffected by MTX without corticosteroids. Our results were further supported by a negative correlation of methotrexate polyglutamate concentrations and the mRNA expression of the pro-inflammatory cytokines IL-6 and IL-12A., Conclusions: We describe a novel effect of MTX reducing the gene expression of IL-12A independently of corticosteroid application in patients. This impact was further enhanced by a reduction of IL-12A-producing lymphocytes and neutrophils under MTX treatment. These results expand the understanding of the mechanism of action of the most widely used drug in RA.

A new ultrafast quantitative and high-throughput mass spectrometric method using matrix-assisted laser desorption/ionization triple quadrupole tandem mass spectrometry has been developed and validated for determination of intracellular erythrocyte concentrations of the antifolate drug methotrexate (MTX) and its polyglutamate metabolites. The method consists of a solid-phase extraction of MTX and MTX-polyglutamate metabolites from deproteinized erythrocyte lysates spiked with aminopterin as internal standard. The newly developed method was validated according to the most recent FDA guidelines on linearity, recovery, within-run and between-run accuracy and precision and stability of the analytes. The low limit of quantification (LLOQ) was 10 nmol/L for all analytes while the limit of detection (LOD) determined at a signal-to-noise (S/N) ratio = 3:1 in drug- free erythrocyte lysate was on average 0.3 nmol/L. After validation, the new method was used in the measurement of intracellular erythrocyte concentrations of MTX and MTX-polyglutamate metabolites (MTXPG2 to MTXPG7) in packed human erythrocyte samples collected from patients with rheumatoid arthritis receiving low-dose oral methotrexate therapy. Mean (SD) intracellular erythrocyte concentrations observed in patient samples were 12.8 (12.6), 12.4 (9.4), 44.4 (30.0), 33.6 (35.9) and 9.4 (8.2) nmol/L for MTX to MTXPG5, respectively, in 10(6) erythrocytes. The highest observed glutamylation degree of MTX was MTXPG5, the very long chain MTX-polyglutamate metabolites MTXPG6 and MTXPG7 were not detected in the packed erythrocyte pellets collected from rheumatoid arthritis patients., (Copyright © 2011 John Wiley & Sons, Ltd.)

Methotrexate (MTX) is widely used for the treatment of childhood acute lymphoblastic leukemia (ALL). The accumulation of MTX and its active metabolites, methotrexate polyglutamates (MTXPG), in ALL cells is an important determinant of its antileukemic effects. We studied 194 of 356 patients enrolled on St. Jude Total XV protocol for newly diagnosed ALL with the goal of characterizing the intracellular pharmacokinetics of MTXPG in leukemia cells; relating these pharmacokinetics to ALL lineage, ploidy and molecular subtype; and using a folate pathway model to simulate optimal treatment strategies. Serial MTX concentrations were measured in plasma and intracellular MTXPG concentrations were measured in circulating leukemia cells. A pharmacokinetic model was developed which accounted for the plasma disposition of MTX along with the transport and metabolism of MTXPG. In addition, a folate pathway model was adapted to simulate the effects of treatment strategies on the inhibition of de novo purine synthesis (DNPS). The intracellular MTXPG pharmacokinetic model parameters differed significantly by lineage, ploidy, and molecular subtypes of ALL. Folylpolyglutamate synthetase (FPGS) activity was higher in B vs T lineage ALL (p<0.005), MTX influx and FPGS activity were higher in hyperdiploid vs non-hyperdiploid ALL (p<0.03), MTX influx and FPGS activity were lower in the t(12;21) (ETV6-RUNX1) subtype (p<0.05), and the ratio of FPGS to γ-glutamyl hydrolase (GGH) activity was lower in the t(1;19) (TCF3-PBX1) subtype (p<0.03) than other genetic subtypes. In addition, the folate pathway model showed differential inhibition of DNPS relative to MTXPG accumulation, MTX dose, and schedule. This study has provided new insights into the intracellular disposition of MTX in leukemia cells and how it affects treatment efficacy.

Objective: MTX is a prodrug producing anti-arthritic effects through a folylpolyglutamate synthase-mediated activation to MTX polyglutamates (MTXPGs). Our objective was to characterize the pharmacokinetics of intracellular MTXPGs and the factors associated with their accumulation in adult RA patients treated with MTX weekly., Methods: MTX pharmacokinetics were evaluated in 47 MTX-naïve patients enrolled in an MTX dose-escalation study for an average of 20 weeks and 223 patients enrolled in a cross-sectional study under long-term MTX therapy. Short-chain (MTXPG1-2), long-chain (MTXPG3) and very long-chain (MTXPG4-5) concentrations were measured in circulating red blood cells using liquid chromatography. Statistical analyses consisted of non-linear mixed models, multivariate regression analyses and Wilcoxon signed-rank test., Results: The accumulation of MTXPG1-5 was sigmoidal and steady-state concentrations were achieved after 7 weeks of therapy. However, additional exposure and MTX dosage escalation produced a selective redistribution towards longer chain MTXPGs at the expense of shorter chain MTXPGs. Age, glomerular filtration rate and route of MTX administration were the most important predictors of MTXPG accumulation. In 10 patients, a switch from oral to parenteral MTX was associated with a 37% increase in long-chain MTXPGs, a 132% increase in very long-chain MTXPGs and a concomitant 31% reduction in disease activity (P<0.02)., Conclusion: The selective emergence of long-chain MTXPGs is function of dose, time of exposure and hence dosage intensity. Switching from oral to parenteral MTX produces a selective accumulation of longer chain MTXPGs that are known to be more potent inhibitors of de novo purine biosynthesis than shorter chain MTXPGs.

Methotrexate (MTX) is one of the most commonly prescribed and most effective drugs for the treatment of rheumatoid arthritis (RA). Given the partial response of many patients and the side effect profile of the drug, there is considerable interest in identifying biomarkers to guide MTX therapy in RA. Upon entering cells, MTX is polyglutamated. Measuring MTX polyglutamate (MTX PG) levels in circulating red blood cells has been proposed as an objective measure to help optimize MTX therapy in RA. Data are conflicting with regard to the clinical utility of MTX PG measurements as a predictor of the efficacy or toxicity of low-dose MTX effects in RA. Should large, randomized clinical trials of this assay show consistent, reproducible, long-term correlations between MTX PG levels and efficacy or toxicity, this test could become a prominent tool for clinicians to optimize the use of MTX in treating RA.

Methotrexate is the most commonly used drug in the treatment of rheumatoid arthritis, although 30-40% of patients fail to adequately respond. An accurate method for measuring methotrexate polyglutamates, the stable active metabolite of methotrexate, has recently been described. The objective of this review article is to determine if clinical use of this measurement would improve methotrexate efficacy, or decrease adverse reactions. Additionally the pharmacologic rationale for this test is discussed. Although methotrexate response improves at higher methotrexate polyglutamate levels, there is no absolute correlation of level with effect. Moreover, overlapping methotrexate polyglutamate levels between clinical responders and nonresponders limits the clinical utility of this measurement.

Objective: Intracellular methotrexate (MTX) polyglutamates (MTXGlu) have been shown to be potentially useful biomarkers of clinical response in adult patients with rheumatoid arthritis. The present study was undertaken to measure intracellular MTXGlu concentrations in a cohort of patients with juvenile idiopathic arthritis (JIA) to determine the predictors of MTXGlu variability in these patients., Methods: Blood samples were obtained from patients with JIA who were being treated with a stable dose of MTX for >or=3 months. Clinical data were collected by chart review. Concentrations of MTXGlu(1-7) in red blood cell lysates were quantitated using an innovative ion-pairing chromatography procedure, with detection by mass spectrometry., Results: Patients with JIA from a single center (n = 99; mean +/- SD age 117.8 +/- 56.5 months, 69 female) were included in the analysis. The mean +/- SD dose of MTX was 0.51 +/- 0.25 mg/kg per week, with a median treatment duration of 18 months (interquartile range 3-156 months). MTX was administered subcutaneously in 66 patients (67%). Fifty-six patients (57%) had active arthritis at the time of the clinic visit. Total intracellular MTXGlu (MTXGlu(TOT)) concentrations varied 40-fold, with a mean +/- SD total concentration of 85.8 +/- 48.4 nmoles/liter. Concentrations of each MTXGlu subtype (MTXGlu(1-7)) were measured individually and as a percentage of MTXGlu(TOT) in each patient. MTXGlu(3) was the most prominent subtype identified, comprising 42% of MTXGlu(TOT), and the interindividual variability in the concentration of MTXGlu(3) was the most highly correlated with that of MTXGlu(TOT) (r = 0.96). The route of MTX administration was significantly associated with MTXGlu(1-5) subtypes; higher concentrations of MTXGlu(1 + 2) were observed in patients receiving oral doses of MTX, whereas higher concentrations of MTXGlu(3-5) were observed in patients receiving subcutaneous doses of MTX (P < 0.0001)., Conclusion: In this cohort of patients with JIA, the MTXGlu(TOT) concentration varied 40-fold. Individual MTXGlu metabolites (MTXGlu(1-7)), which have, until now, not been previously reported in patients with JIA, were detected. The route of MTX administration contributed to the variability in concentrations of MTXGlu(1-5).

To enhance the stability of the anticancer drug gemcitabine (2'-deoxy-2',2'-difluorocytidine), it was conjugated to poly-l-glutamic acid (PG-H) via a carbodiimide reaction. The synthesised poly-l-glutamic acid-gemcitabine (PG-G) was purified and characterised by using SDS-PAGE to estimate its molecular weight, HPLC to determine its purity and degree of drug loading, and NMR to elucidate the structure. In vitro aqueous hydrolytic studies showed that the gemcitabine release from the polymeric drug conjugate was pH dependent, and that the conjugation to PG-H improved its stability in human plasma. The release of the bound gemcitabine from PG-G in plasma was mediated by a hydrolytic process. It began with a lag phase, followed by linear release between 12 and 48h, and reached equilibrium at 72h with 51% of the gemcitabine released. In vitro cytotoxicity studies using MCF-7 and MDA-MB-231 human mammary cancer cells, as well as human dermal fibroblasts (HDF), showed that PG-G displayed a lower dose dependent cytotoxic effect with respect to the parent drug gemcitabine. On the other hand, in 4T1 mouse mammary tumour cells, PG-G and gemcitabine showed similar toxicities. Gemcitabine was more than likely released hydrolytically from PG-G and taken up by MCF-7, MDA-MB-231 and HDF, whereas both released gemcitabine and PG-G were taken up by 4T1 to mediate the observed cytotoxicities. The improved stability and extended sustained release profile may render PG-G a potential anticancer prodrug., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

A specific and sensitive liquid chromatography-tandem mass spectrometric method for quantitative determination of paclitaxel in rat plasma was developed and validated using docetaxel as an internal standard. Liquid-liquid extraction using tert-butyl methyl ether was used to extract the drug and the internal standard from plasma. The separation of paclitaxel was performed on a C(18) column with a mobile phase of acetonitrile:water:formic acid (65:35:0.1, v/v/v) over 5min. The assay was based on the selected reaction monitoring transitions at m/z of the precursor-product ion transitions m/z 854.2-->286.1 for paclitaxel and 808.3-->527.2 for internal standard. The lower limit of quantification was 0.5ng/mL based on 100microL of plasma. Intra- and inter-day assay variations were less than 15%, and the accuracy values were between 95.4 and 105.4%. The extraction recoveries ranged from 96.7 to 103.7% across the calibration curve range. The method was successfully applied to measurement of low concentrations of paclitaxel or regenerated paclitaxel in plasma after intravenous administration of a single dose (10mg/kg) of a poly(l-glutamic acid)-alanine-paclitaxel conjugate to rats., (Copyright 2009 Elsevier B.V. All rights reserved.)

The folate antagonist methotrexate is commonly used in low dose for treatment of rheumatoid arthritis and juvenile idiopathic arthritis. Therapeutic effects are attributed to intracellular levels of various methotrexate polyglutamates. The present methodology, combining a simple preparation step with ion-pairing reversed-phase liquid chromatography and electrospray ionization mass spectrometry, is suitable for the measurement of methotrexate and its polyglutamates(2-7), in human red blood cells. Sample preparation consists of perchloric acid protein precipitation followed by solid-phase extraction. Baseline separation of all analytes was achieved within 10 min using a Phenomenex Synergy C18 column together with a gradient solvent program obtained from blending acetonitrile with pH 7.5, 5 mM aqueous dimethylhexylamine. Seven methotrexate polyglutamates were detected using multiple reaction monitoring, with the mass spectrometer operating in positive ion mode. Using 20 microL injection volumes, limits of detection were 2.5 nM for individual methotrexate polyglutamates, while large volume (100 microL) injections led to detection limits of 0.5 nM and linear calibration from 0.5 to 100 nM for individual analytes. Finally, the presented methodology was applied for the analysis of methotrexate and its polyglutamates in red blood cells obtained from patients being treated for juvenile idiopathic arthritis with methotrexate. Significantly, the methodology proved suitable for determination of long-chain methotrexate polyglutamates(5-7) and further, appears to be superior with respect to sensitivity, selectivity and speed as compared to all previously described approaches., (Copyright 2009 John Wiley & Sons, Ltd.)

Methotrexate (MTX), i.e., MTX-polygluatmate 1 (MTX-PG(1)), exerts its antirheumatic effects mainly by < or = 6 (MTX-PG(2-7)) via folypolyglutamyl synthase in cells. The authors developed a new method using fluorescence polarization immunoassay to determine MTX-PG(1-7) concentrations in erythrocytes (RBC). MTX-PG(2-7) in RBC of rheumatoid arthritis (RA) patients receiving MTX was converted to MTX in the presence of plasma gamma-glutamyl hydrolase and mercaptoethanol at 37 degrees C. The MTX in RBC was extracted in a perchloric acid deproteinization step then on a solid-phase extraction column. The concentration of MTX was measured by TDX analyzer. The mean MTX recovery rate was 76.1% (n=8). The intraday and interday coefficients of variation were <11.3% (n=8) and <12.4% (n=3), respectively, at low and high concentrations (30-300 nmol/l). The calibration curve was linear over the range 30-300 nmol/l. The total concentration of MTX-PGs (mean+/-S.D.) in RBC obtained from 95 Japanese RA patient blood samples was 97.3+/-8.1 nmol/l for the MTX dose of 0.13+/-0.05 mg/week/kg. This newly developed method for the quantification of MTX-PGs in RBC is sensitive and accurate and can be applied for routine monitoring of MTX therapy in RA patients.

Systemic anthrax manifests as toxemia, rapidly disseminating septicemia, immune collapse, and death. Virulence factors include the anti-phagocytic gamma-linked poly-d-glutamic acid (PGA) capsule and two binary toxins, complexes of protective antigen (PA) with lethal factor (LF) and edema factor. We report the characterization of LF, PA, and PGA levels during the course of inhalation anthrax in five rhesus macaques. We describe bacteremia, blood differentials, and detection of the PA gene (pagA) by PCR analysis of the blood as confirmation of infection. For four of five animals tested, LF exhibited a triphasic kinetic profile. LF levels (mean +/- standard error [SE] between animals) were low at 24 h postchallenge (0.03 +/- 1.82 ng/ml), increased at 48 h to 39.53 +/- 0.12 ng/ml (phase 1), declined at 72 h to 13.31 +/- 0.24 ng/ml (phase 2), and increased at 96 h (82.78 +/- 2.01 ng/ml) and 120 h (185.12 +/- 5.68 ng/ml; phase 3). The fifth animal had an extended phase 2. PGA levels were triphasic; they were nondetectable at 24 h, increased at 48 h (2,037 +/- 2 ng/ml), declined at 72 h (14 +/- 0.2 ng/ml), and then increased at 96 h (3,401 +/- 8 ng/ml) and 120 h (6,004 +/- 187 ng/ml). Bacteremia was also triphasic: positive at 48 h, negative at 72 h, and positive at euthanasia. Blood neutrophils increased from preexposure (34.4% +/- 0.13%) to 48 h (75.6% +/- 0.08%) and declined at 72 h (62.4% +/- 0.05%). The 72-h declines may establish a "go/no go" turning point in infection, after which systemic bacteremia ensues and the host's condition deteriorates. This study emphasizes the value of LF detection as a tool for early diagnosis of inhalation anthrax before the onset of fulminant systemic infection.

To identify the major factors predicting the response to Methotrexate (MTX) therapy in rheumatoid arthritis (RA) patients, we evaluated the relationship between the response to MTX and factors such as the concentration of MTX-polyglutamates (MTX-PGs) in erythrocytes (RBCs), genotypes of thymidylate synthase (TYMS) 5'-UTR (2R/3R) and 3'-UTR (-6/+6), 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, and other patient-related factors. Thirty-six Japanese RA patients were enrolled in this cohort study. The concentrations of MTX-PGs in RBCs were measured, and polymorphisms were determined using PCR-RFLP method. As an indicator of the accumulated capacity of MTX-PGs in the RBCs of each patient, the MTX dose/MTX-PGs (AC-MPG, l/week) was calculated. The response to MTX therapy was assessed using the MTX dose for a>or=50% decrease in CRP level (MTX dose for 50%CRP, mg/week), and the relationships between MTX dose for 50%CRP and various other factors were evaluated using multiple linear regression analysis. The MTX dose was 6.9+/-0.3 mg/week and the MTX-PGs concentration in RBCs was 97.3+/-8.1 nmol/l (n=36, blood samples=95, mean+/-S.D.). The range of MTX dose for 50%CRP was 2.0-13.0 mg/week. Most individual AC-MPG levels showed no change during the evaluation period (coefficient of variation=5.9%). Based on the results of multiple linear regression analysis, AC-MPG, TYMS 3'-UTR (-6/+6), and ESR at the start of MTX therapy were associated with the MTX dose for 50%CRP. AC-MPG, TYMS 3'-UTR (-6/+6), and ESR might be the major predictive factors for the response to MTX therapy in Japanese RA patients.

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.

Objective: There is evidence supporting a therapeutic range for methotrexate polyglutamate (MTXGlu) concentrations in the treatment of rheumatoid arthritis (RA). Knowledge of the pharmacokinetics of MTXGlu1-5 is required for optimal timing of blood sampling. The aim of this study was to determine the time to steady state and the half-life of accumulation of red blood cell (RBC) MTXGlu1-5 in patients with RA commencing oral MTX, and the time for RBC MTXGlu1-5 to become undetectable and the half-life of elimination of RBC MTXGlu1-5 in patients ceasing treatment with oral MTX., Methods: Ten patients beginning treatment and 10 patients stopping treatment with low-dose oral MTX were recruited. Blood samples were initially collected weekly, with gradual extension to monthly collection over the study period. RBC MTXGlu1-5 concentrations were assayed by high-performance liquid chromatography. Results were analyzed using a first-order exponential method., Results: The median times to reach steady state in RBCs (defined as 90% of the maximum concentration) were 6.2, 10.6, 41.2, 149, and 139.8 weeks, respectively, for MTXGlu1, MTXGlu2, MTXGlu3, MTXGlu4, and MTXGlu5. The median half-life of accumulation for RBC MTXGlu1-5 ranged from 1.9 weeks to 45.2 weeks. The median times for MTXGlus to become undetectable in RBCs were 4.5, 5.5, 10, 6, and 4 weeks, respectively, for MTXGlu1, MTXGlu2, MTXGlu3, MTXGlu4, and MTXGlu5. The median half-life of elimination for RBC MTXGlu1-5 ranged from 1.2 weeks to 4.3 weeks., Conclusion: There is wide interpatient variability of RBC MTXGlu1-5 accumulation and elimination in adults with RA. These data also suggest that after a dose change, >6 months are required for RBC MTXGlu1-5 to reach steady state. Such delays in achieving steady state suggest that more rapid dose escalation or subcutaneous administration from the outset should be considered.

Objective: We assessed the effect of folic acid (FA) on the pharmacokinetics and pharmacodynamics of low-dose oral methotrexate (MTX) during the remission-induction phase of psoriasis treatment., Methods: In a 32-week, open-label, two-way cross-over study, patients (n=20, seven men, aged 35-70 years) with moderate-to-severe plaque psoriasis were randomly assigned to receive MTX plus FA (20 mg/week) for 16 weeks followed by MTX monotherapy (three doses of MTX separated by 12-h intervals once a week) for an additional 16 weeks (treatment arm A, n=10) or to receive the opposite sequence of treatments (arm B, n=10). Dosing of MTX was individualised with the help of pre-study evaluation of plasma MTX pharmacokinetics. The Psoriasis Area and Severity Index (PASI), biochemistry and haematology tests and erythrocyte concentration of MTX polyglutamates (MTXPG) were evaluated throughout the study., Results: In arms A and B, the mean (range) concentrations of MTXPG (nmol/L) were comparable [week 16: 96.2 (32.0-157) vs. 111 (73.7-175), P=0.32; week 32: 103 (55.8-173) vs. 83.6 (27.4-129), P=0.24]. After 16 weeks, the mean+/-SEM PASI decreased from 20.1+/-2.1 to 8.8+/-1.3 in arm A, while a greater reduction from 27.2+/-2.1 to 5.1+/-1.0 occurred in arm B (P<0.001). Positive correlations were found between the percent improvement in PASI at week 16 and the ratios of the concentration of MTXPG to plasma folate (rho=0.59, P=0.008) or RBC folate concentration (rho=0.56, P=0.013). Due to an accelerated decline in PASI in arm A and a trend to its worsening in arm B after crossing over of treatments, the mean absolute PASI scores in both arms were comparable at week 32., Conclusion: The antipsoriatic effect of MTX during the remission-induction phase of treatment is influenced by folate status and may be significantly less if combined treatment with FA is used, irrespective of pre-treatment folate levels. The individual tailoring of MTX dosing needs further attention because the mean percent PASI improvement from baseline was 83% and the inter-patient variability in response was low after 16 weeks of monotherapy with MTX.

Methotrexate (MTX) has been widely used at low dose for the treatment of different diseases including rheumatoid arthritis. MTX might be present in plasma in free form, and in blood cells in methotrexate polyglutamate (MTXPG). A rapid and sensitive HPLC method was developed for the determination of plasma MTX level, whole-blood MTX level, and whole-blood total MTX (MTX+MTXPG) level. To determine plasma MTX level or whole-blood MTX level, a 0.2-ml aliquot of plasma or whole blood (after a freeze-thaw cycle to break blood cells) was well mixed with 0.8 ml methanol and centrifuged. To determine whole-blood total MTX level, a 0.1-ml aliquot of whole blood (after a freeze-thaw cycle) was mixed with 80 microl ascorbic acid (114 mM) and incubated at 37 degrees C for 2h to enzymatically convert the MTXPG to MTX. Then 20 microl NaOH solution (0.5M) and 0.8 ml methanol were added and mixed well. After centrifugation, a 0.5-ml aliquot of the supernatant was evaporated to dryness and re-dissolved in 0.2 ml hydrochloric acid (10mM). Methylene chloride (0.2 ml) was added and mixed well. After centrifugation, the top aqueous layer was injected to HPLC for analysis. After the MTX was eluted from the HPLC column, it was electrochemically oxidized and detected by a fluorescence detector. Recoveries of spiked MTX at ppb (ng/ml) level were between 87.9 and 118% with within-day relative standard deviation less than 5.2% and day-to-day relative standard deviation less than 9.8%. The limit of detection (LOD) and limit of quantitation (LOQ) of the described method were 1.2 and 2.6 ng/ml, respectively.