Adherence to low-dose methotrexate in children with juvenile idiopathic arthritis using a sensitive methotrexate assay.

BACKGROUND: Low-dose weekly methotrexate (MTX) is the mainstay of treatment in juvenile idiopathic arthritis. Unfortunately, a substantial part of patients has insufficient efficacy of MTX. A potential cause of this inadequate response is suboptimal drug adherence. The aim of this study was to assess MTX adherence in juvenile idiopathic arthritis patients by quantification of MTX concentrations in plasma. Secondly, the association between MTX concentrations and either self-reported adherence issues, or concomitant use of biologics was examined. METHODS: This was a retrospective, observational study using plasma samples from juvenile idiopathic arthritis patients. An ultrasensitive liquid chromatography-tandem mass spectrometry method was developed for quantification of MTX and its metabolite 7-hydroxy-MTX in plasma. The determined MTX plasma concentrations in juvenile idiopathic arthritis patients were compared with corresponding adherence limits, categorising them as either adherent or possibly non-adherent to MTX therapy. RESULTS: Plasma samples of 43 patients with juvenile idiopathic arthritis were analysed. Adherence to MTX in this population was 88% shortly after initiation of MTX therapy and decreased to 77% after one year of treatment. Teenagers were more at risk for non-adherence (p = 0.002). We could not find an association between MTX adherence with either self-reported adherence issues, nor with the use of concomitant biological treatment (p = 1.00 and p = 0.27, respectively; Fisher's Exact). CONCLUSIONS: Quantification of MTX in plasma is a feasible and objective method to assess adherence in patients using low-dose weekly MTX. In clinical practice, the use of this method could be a helpful tool for physicians to refute or support suspicion of non-adherence to MTX therapy.

Validation of UV-Vis spectrophotometric and colorimetric methods to quantify methotrexate in plasma and rat skin tissue: Application to in vitro release, ex vivo and in vivo studies from dissolving microarray patch loaded pH-sensitive nanoparticle.

This research transformed MTX into smart nanoparticles that respond to the acidic conditions present in inflammation. These nanoparticles were then incorporated into a patch that dissolves over time, aiding their penetration. A method using UV-Vis spectrophotometry was validated to support the development of this new delivery system. This method was used to measure the quantity of MTX in the prepared patches in various scenarios: in laboratory solutions with pH 7.4 and pH 5.0, in skin tissue, and plasma. This validation was conducted in laboratory studies, tissue samples, and live subjects, adhering to established guidelines. The resulting calibration curve displayed a linear relationship (correlation coefficient 0.999) across these scenarios. The lowest quantity of MTX that could be accurately detected was 0.6 µg/mL in pH 7.4 solutions, 1.46 µg/mL in pH 5.0 solutions, 1.11 µg/mL in skin tissue, and 1.48 µg/mL in plasma. This validated method exhibited precision and accuracy and was not influenced by dilution effects. The method was effectively used to measure MTX levels in the developed patch in controlled lab settings and biological systems (in vitro, ex vivo, and in vivo). This showed consistent drug content in the patches, controlled release patterns over 24 h, and pharmacokinetic profiles spanning 48 h. However, additional analytical approaches were necessary for quantifying MTX in studies focused on the drug's effects on the body's functions.

Optimization and validation of an analytical method for the estimation of methotrexate in rabbit plasma.

Methotrexate (MTX) is an anticancer drug used for the treatment of various cancers and autoimmune diseases. In this study, High Performance Liquid Chromatography (HPLC) method was developed and validated for the estimation of MTX in rabbit plasma with high estimation rate and recovery. Various validation parameters like, sensitivity, sample recovery, accuracy and precision analysis were studied. The pre-saturated reversed C18 end capped HPLC column was used to separate MTX present in rabbit plasma. A solvent mixture of 100mM phosphate buffer pH 7.4 and acetonitrile (92:8 percent v/v) was employed as the mobile phase. Analysis was carried out at ʎ max 303 nm and retention time of MTX was found 5.32 min. During the method development and validation ICHQ2 (R1) guidelines were strictly followed. Developed method was found excellent in terms of recovery of MTX from plasma samples (98.6%). It is obvious from the current study that the developed HPLC method can be utilized to analyze the level of MTX in patients. Furthermore, the cost of the developed method for the determination of MTX would be very low as compared to the previously reported methods.

Plasma Distribution of Methotrexate and Its Polyglutamates in Pediatric Acute Lymphoblastic Leukemia: Preliminary Insights.

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 MTXPG1-7 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 MTXPG3 (16.71-30.02%) and MTXPG5 (26.23-38.60%), while MTXPG7 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.

New Therapeutic Approach to Reduce Methotrexate Toxicity after High-Dose Chemotherapy in a Child with Acute Lymphocytic Leukemia: Efficacy and Safety of Hemoadsorption with HA-230 Adsorber.

BACKGROUND: High-dose methotrexate (HDMTX) is likely to cause a number of side effects and manifest itself as hepatotoxicity, nephrotoxicity, mucositis, and neurotoxicity. A several studies demonstrated the efficacy of extracorporeal detoxification methods such as plasma exchange, hemodialysis (HD), HD filtration, and hemoperfusion for the treatment of MTX delayed clearance. However, none of the existing methods as effective as expected and limited for general implementation due to a procedure-related complication. CASE REPORT: Here, we report a successful implementation of HA-230 hemoadsorption procedure to remove cumulated MTX from the body and reduce its toxicity in a child with ALL after high-dose chemotherapy. RESULTS AND CONCLUSION: Based on our results, single-hemoadsorption procedure with the HA-230 adsorber in case of delayed methotrexate clearance was safe and well-tolerated in a pediatric patient with ALL and would significantly improve the patient's condition. Further studies need to demonstrate its safety and efficacy in a large number of pediatric patients.

Simultaneous measurement of upadacitinib and methotrexate by UPLC-MS/MS and its pharmacokinetic application in rats.

Upadacitinib, as a selective and reversible Janus kinase (JAK) inhibitor, has been widely used in the treatment of atopic dermatitis, ulcerative colitis and other inflammatory bowel diseases and other immune-mediated diseases. The combination of methotrexate and upadacitinib is a common clinical treatment strategy for rheumatoid arthritis (RA) in recent years. In this study, we established an ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for quantitative measurement of upadacitinib and methotrexate, by which we successfully determined pharmacokinetic parameters of them in rat plasma. In order to pretreat the samples, we used acetonitrile as the precipitant, and for the internal standard (IS), we chose tofacitinib. The Acquity BEHC18 (2.1 mm × 50 mm, 1.7 µm) column, with acetonitrile and 0.1% formic acid aqueous solution composed mobile phases, was used to separate upadacitinib, methotrexate and tofacitinib. A Xevo TQ-S triple quadrupole tandem mass spectrometer was used as the detecting instrument in the positive ion mode. For upadacitinib, excellent linearity was shown of this assay in the calibration range with 0.1-200 ng/mL, and as for methotrexate, the range was 0.05-100 ng/mL. As the results indicated, the lower limit of quantification (LLOQ) was respectively 0.1 and 0.05 ng/mL for upadacitinib and methotrexate, the intra- and inter-day precision were ≤ 13.3%, and the accuracy of all the analytes ranged from -4.1% to 12.7%. The recovery of each analyte was > 80.2% in this experiment, and matrix effects we observed were unobvious. The establishment of this method and its successful application in rat plasma can provide a theoretical and technical support for the deeper study of pharmacodynamics and the clinical medication strategies.

Half-dose glucarpidase as efficient rescue for toxic methotrexate levels in patients with acute kidney injury.

PURPOSE: High-dose methotrexate (HDMTX)-associated acute kidney injury with delayed MTX clearance has been linked to an excess in MTX-induced toxicities. Glucarpidase is a recombinant enzyme that rapidly hydrolyzes MTX into non-toxic metabolites. The recommended dose of glucarpidase is 50 U/kg, which has never been formally established in a dose finding study in humans. Few case reports, mostly in children, suggest that lower doses of glucarpidase might be equally effective in lowering MTX levels. METHODS: Seven patients with toxic MTX plasma concentrations following HDMTX therapy were treated with half-dose glucarpidase (mean 25 U/kg, range 17-32 U/kg). MTX levels were measured immunologically as well as by liquid chromatography-mass spectrometry (LC-MS). Toxicities were assessed according to National Cancer Institute-Common Terminology Criteria for Adverse Events (CTCAE) v5.0. RESULTS: All patients experienced HDMTX-associated kidney injury (median increase in creatinine levels within 48 h after HDMTX initiation compared to baseline of 251%, range 80-455%) and showed toxic MTX plasma concentrations (range 3.1-182.4 µmol/L) before glucarpidase injection. The drug was administered 42-70 h after HDMTX initiation. Within one day after glucarpidase injection, MTX plasma concentrations decreased by ≥ 97.7% translating into levels of 0.02-2.03 µmol/L. MTX rebound was detected in plasma 42-73 h after glucarpidase initiation, but concentrations remained consistent at < 10 µmol/L. CONCLUSION: Half-dose glucarpidase seems to be effective in lowering MTX levels to concentrations manageable with continued intensified folinic acid rescue.

Design of a methotrexate-controlled chemical dimerization system and its use in bio-electronic devices.

Natural evolution produced polypeptides that selectively recognize chemical entities and their polymers, ranging from ions to proteins and nucleic acids. Such selective interactions serve as entry points to biological signaling and metabolic pathways. The ability to engineer artificial versions of such entry points is a key goal of synthetic biology, bioengineering and bioelectronics. We set out to map the optimal strategy for developing artificial small molecule:protein complexes that function as chemically induced dimerization (CID) systems. Using several starting points, we evolved CID systems controlled by a therapeutic drug methotrexate. Biophysical and structural analysis of methotrexate-controlled CID system reveals the critical role played by drug-induced conformational change in ligand-controlled protein complex assembly. We demonstrate utility of the developed CID by constructing electrochemical biosensors of methotrexate that enable quantification of methotrexate in human serum. Furthermore, using the methotrexate and functionally related biosensor of rapamycin we developed a multiplexed bioelectronic system that can perform repeated measurements of multiple analytes. The presented results open the door for construction of genetically encoded signaling systems for use in bioelectronics and diagnostics, as well as metabolic and signaling network engineering.

A Validated HPLC-MS/MS Method for Quantification of Methotrexate and Application for Therapeutic Drug Monitoring in Children and Adults with Non-Hodgkin Lymphoma.

PURPOSE: A simple, rapid and reliable method to quantify methotrexate (MTX) in human plasma by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established and validated in two laboratories. PATIENTS AND METHODS: Sample separation was achieved on a Synergi Hydro-RP column (50 mm×2.0 mm, 2.5 µm) with a gradient elution program in 3.5 min after a simple protein precipitation with methanol (MeOH) and acetonitrile (ACN) (1:1). About 5 mM ammonium formate aqueous solution with 0.2% formic acid and ACN were used as mobile phase with a flow rate of 0.5 mL/min at 40 °C. Mass spectrometry detection using AB Sciex Triple Quad 4500 mass spectrometer (4500 QQQ) and Qtrap 5500 mass spectrometer (5500 Q-trap) were both characterized by electrospray ionization (ESI) for positive ions in multiple reaction-monitoring (MRM) mode. Quantitative ion pairs were m/z 455.1→m/z 308.0 for MTX and m/z 248.1→m/z 121.0 for tinidazole (TNZ) used as internal standard (IS). RESULTS: Linear calibration curves were generated over the range of 5-1000 ng/mL (r2> 0.99) on both the 4500 QQQ and 5500 Q-trap, both of the intra- and inter-batch precision were less than 7.67% and accuracy ranged from 96.33% to 108.94%. The recovery and matrix effect were 82.20-93.98% and 102.69-105.28%, respectively. CONCLUSION: An analytical method transfer was achieved by re-verification in two laboratories to ensure stability and reproducibility and this method has been applied for therapeutic drug monitoring (TDM) successfully in children and adults with NHL, and during routine TDM, two delayed elimination of MTX cases were observed and analyzed.

Simultaneous determination of folate and methotrexate metabolites in serum by LC-MS/MS during high-dose methotrexate therapy.

High-dose methotrexate (HDMTX) is a central component in the treatment of acute lymphoblastic leukemia, osteosarcoma, and some lymphomas and brain tumors. MTX is given at lethal doses and then is followed by rescue treatment with folinic acid (FA). Despite FA rescue, many patients suffer severe toxicity. The pharmacokinetics of FA rescue have not been sufficiently studied. However, optimization of FA rescue could potentially increase anti-tumor effects, whilst decreasing organ toxicity. Here, we describe our efforts to establish and optimize a liquid chromatography tandem mass spectrometric (LC-MS/MS) method for the simultaneous determination of five essential components of the folate cycle, as well as MTX and its two metabolites. The method was applied to 6 individual patients receiving HDMTX, with 3 or 4 measurements for each patient. The method allows analysis of samples that were initially frozen. This notion, together with the test results in the 6 pilot patients, shows the feasibility of this method to study MTX and FA pharmacokinetics during HDMTX treatment. The method has the potential to optimize HDMTX and FA rescue treatment in individual patients.

A simple and accurate HFCF-UF method for the analysis of homocysteine, cysteine, cysteinyl-glycine, and glutathione in human blood.

The presence of reduced aminothiols, including homocysteine (Hcy), cysteine (Cys), cysteinyl-glycine (CG), and glutathione (GSH), is significantly increased in the pathological state. However, there have been no reports on the relationship between reduced aminothiols (Hcy, Cys, CG, and GSH) and different genders, ages, and drug combinations in human blood. The accurate quantification of these reduced thiols in biological fluids is important for monitoring some special pathological conditions of humans. However, the published methods typically not only require cumbersome and technically challenging processing procedures to ensure reliable measurements, but are also laborious and time-consuming, which may disturb the initial physiological balance and lead to inaccurate results. We developed a hollow fiber centrifugal ultrafiltration (HFCF-UF) method for sample preparation coupled with a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and used it to determine four reduced aminothiols (Hcy, Cys, CG, and GSH) in human blood for the first time. A total of 96 clinical patients were enrolled in our study. The influence of different genders, ages, and drug combinations on the levels of four reduced thiols in human blood was also discussed by SPSS 24.0. The sample preparation was simplified to a single 5 min centrifugation step in a sealed system that did not disturb the physiological environment. The validation parameters for the methodological results were excellent. The procedure was successfully applied to monitoring the concentrations of four reduced aminothiols (Hcy, Cys, CG, and GSH) in 96 clinical blood samples. There were no significant differences in Hcy, Cys, CG, or GSH for the different genders, ages, or combinations with methotrexate or vancomycin (P > 0.05). However, there was a significant increase in Hcy concentration in patients treated with valproic acid who were diagnosed with epilepsy (p=0.0007). It is advisable to measure reduced Hcy level in patients taking valproic acid. The developed HFCF-UF method was simple and accurate. It can be easily applied in clinical research to evaluate oxidative stress in further study.

Comparison of two dose escalation strategies of methotrexate in active rheumatoid arthritis: a multicentre, parallel group, randomised controlled trial.

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.

Delayed methotrexate elimination in a patient with primary central nervous system lymphoma: A case report.

WHAT IS KNOWN AND OBJECTIVE: Methotrexate (MTX) is an important agent for the treatment of primary central nervous system lymphomas (PCNSL) but needs to be given in big doses by intravenous infusions to achieve therapeutic concentrations in the cerebrospinal fluid. However, co-administration with many drugs may delay the excretion of MTX which may cause serious adverse effects if the serum concentration exceeds 0.1 µmol/L 72 h after an intravenous infusion. CASE SUMMARY: A 67-year-old Japanese female with PCNSL was treated with high-dose MTX-based chemotherapy. The serum MTX concentration 72 h post-infusion was 0.153 µmol/L when she was taking levofloxacin (LVFX) but <0.1 µmol/L 72 h after 4 subsequent infusions when she was not taking LVFX. She was given many other drugs but the timing of the short course of LVFX and the fact that ciprofloxacin also delays MTX excretion suggests that LVFX was the cause. WHAT IS NEW AND CONCLUSION: Co-administration of LVFX may delay the excretion of MTX. Therefore, serum concentrations of MTX should be monitored to help prevent and improve the management of potentially serious MTX drug-drug interaction.

A single assessment of methotrexate levels at 42 hours permits safe administration and early discharge in children with lymphoblastic lymphoma and leukemia receiving high-dose methotrexate.

High-dose methotrexate (HDMTX) is an important component of treatment in pediatric acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LL). Optimal rescue therapy is essential for the safe administration of HDMTX. A cost-effective strategy that does not compromise safety is necessary for low- and middle-income countries. Consecutive admissions for HDMTX in children with ALL and LL over 12 months were analyzed. The dose of HDMTX was 3 g/m2 in B-ALL and B-LL and 5 g/m2 in T-ALL and T-LL. A methotrexate level was measured at 42 hours of starting HDMTX infusion (T42-MTX). Three doses of folinic acid at T42, T48, and T54 and alkalinized hydration till T54 were administered if T42-MTX <1 µM. A total of 282 cycles of HDMTX that were administered in 71 patients were analyzed. T42-MTX was <1 µM in 266 (94.3%) cycles. T42-MTX was ≥1 µM in 12% and 3% of cycles of HDMTX administered at a dose of 5 g/m2 and 3 g/m2, respectively (p = .074). The median duration of hospitalization for HDM was three days and did not differ with the dose of HDMTX administered (p = .427). Mucositis, delayed recovery of blood counts, and hospitalization for reversible toxicity occurred after 21 (7.4%), 28 (9.9%), and 19 (6.7%) cycles of HDMTX, respectively. Mucositis was greater following the administration of 5 g/m2 of HDMTX. A single T42-MTX measurement permits the safe administration of HDMTX and an expedited discharge from the hospital within three days in more than 90% of children with ALL/LL.

Study on Relationships of Tumor Status and Gene Polymorphism With Blood Concentration of MTX and Toxicities in 63 Pediatric Mature B Cell Lymphoma in Chinese Population.

OBJECTIVE: This study investigated the relationships of tumor status (stage, renal involvement, bone marrow status, bulky disease, liver function), tumor gene polymorphism, and methotrexate (MTX) dosage (stratified by treatment group) with blood MTX levels and adverse reactions (ADR). METHODS: We retrospectively reviewed 63 mature B cell lymphoma patients who were treated in our center. Genotyping of the MTHFR 677 and SLCO1B1 genes was carried out, and the relationships between tumor status, polymorphism of the genes, MTX level, and ADR were analyzed. RESULTS: Altogether, 63 children were included. The mean blood MTX concentration was 0.25 ± 0.2 umol/L at 45 h. Liver dysfunction and bulky disease were both correlated with MTX level (both P < 0.05). ADRs were higher among patients with blood MTX > 0.5 mmol/l at 45 h than for the groups with lower blood MTX. The MTHFR 677 CT genotype was correlated with liver function damage (P = 0.04); the rs11045879 locus CC genotype of SLCO1B1, stage IV, and bulky disease at the time of diagnosis were correlated with 4° neutropenia (P < 0.05). Stage IV, bulky disease, leukemia stage at the time of diagnosis, and C2 treatment group were correlated with severe anemia (P < 0.05). Stage IV, bulky disease, leukemia stage, renal invasion at the time of diagnosis, and C2 treatment group were associated with severe thrombocytopenia (P < 0.05). Bulky disease and renal invasion at the time of diagnosis were associated with severe mucositis and severe infection (P < 0.05). CONCLUSION: Taken together, our data demonstrate that gene polymorphism, MTX levels, tumor status, and treatment group might be useful to optimize MTX therapy and estimate toxicity.

Sensitive determination of methotrexate in plasma of children with acute leukemia using double-solvent supramolecular systemas a novel extractant for dispersive liquid-liquid microextraction.

Methotrexate, as a folate antagonist, is one of the first anti-neoplasm drugs offered and is still used as an effective drug in the treatment of various malignancies. Methotrexate has a narrow treatment index and is associated with numerous side effects.In thisresearch, for the first time a double-solvent supramolecular system (DSS) was developed as an extractant without disperser solvent for dispersive liquid-liquid microextraction (DLLME). DSS - DLLME was applied to the extraction of methotrexate in plasma of children with acute leukemiaprior to itsdetermination by high-performance liquid chromatography-ultraviolet detection (HPLC - UV). In the present method, two long normal chain alcohols are mixed in a particular ratio, and then it is injected into the sample solution, which is on the magnetic stirrer. In this case, the mixture of the two alcohol changes to new supramolecular aggregate. This new supermolecule is used as an extractant, which has a higher extraction power than any of its components alone. Under the optimum conditions, the calibration graph was linear in the rage of 0.1-150 µg L-1 with detection limit of 0.03 µg L-1. Relative standard deviations (RSDs) including intra-day and inter-day of method based on7 replicate determinations of 100.0 µg L-1of methotrexate were 2.6% and 4.8%,respectively. The results proved that DSS - DLLME is a sensitive, very simple, inexpensive, environmental friendly, rapid and efficient method for the preconcentration of trace amount of drugs in biological samples.

Methotrexate Triglutamate as a Determinant of Clinical Response in Mexican Patients With Rheumatoid Arthritis: Pharmacokinetics and Dose Recommendation.

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/m2 ) * (red blood cells/4.6 × 106 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.

Studies on the intracellular accumulation process of methotrexate and its correlation with the key protein using an LC-MS/MS method: a novel way to realize prospective individualized medication.

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 (MTXPG1) and its metabolites MTXPG2-7 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).

Risk factors for delayed elimination of high-dose methotrexate in childhood acute lymphoblastic leukemia and lymphoma.

High-dose methotrexate (HD-MTX) therapy is widely used in patients with acute lymphoblastic leukemia (ALL) and lymphoma. However, some patients experience delayed MTX elimination, which requires treatment suspension or dose reduction to avoid organ damage. This single-center retrospective analysis reviewed the clinical data of 88 children with ALL or non-Hodgkin lymphoma who received a total of 269 courses of HD-MTX therapy between April 2008 and April 2019. HD-MTX was defined as MTX administration at 2.0, 3.0, or 5.0 g/m2 over a 24-h period, and delayed MTX elimination was defined as a serum MTX concentration ≥ 1.0 µmol/L at 48 h after the start of HD-MTX. Clinical factors were compared between courses with and without delayed MTX elimination. MTX elimination was delayed in 21 of the 269 courses (7.8%). Multivariate analysis showed that first HD-MTX course (OR 4.04), lower urine volume per BSA on the first day of HD-MTX administration (< 2,675 mL/m2, OR 5.10), higher total bilirubin (> 0.5 mg/dL, OR 5.11), lower eGFR (< 136 mL/min/1.73 m2, OR 3.90), higher dose of MTX(> 3.0 g/m2, OR 10.8), and lower urine volume per BSA on the next day of starting HD-MTX (< 2,107 mL/m2, OR 3.43) were independent risk factors for delayed MTX elimination.