Methotrexate Levels Research Articles

Clinical Risk Factors for High-Dose Methotrexate-Induced Oral Mucositis Following Individualized Dosing.

Oral mucositis affects about 20% of children undergoing high-dose methotrexate (HDMTX) for acute lymphoblastic leukemia (ALL), despite existing management strategies. Personalized HDMTX dosing, adjusted by pharmacokinetics and leukemia risk, has reduced mucositis incidence, but variations still occur with similar 24-h methotrexate levels. This retrospective study investigated risk factors for oral mucositis under individualized methotrexate protocols. Data from patients with ≥ Grade 2 oral mucositis (CTCAE v4.0) were analyzed from the St. Jude Children's Research Hospital total 16 trial. A 1:1 case-control matching method considered age, sex, risk classification, immunophenotype, and methotrexate course. McNemar's, Bowker's symmetry, and Wilcoxon signed-rank tests were used for statistical analyses. Risk factors for recurrent mucositis were identified in a case-only analysis. The study found significant associations between methotrexate-induced mucositis and new-onset skin rashes (p = 0.0027), fever (p = 0.0016), neutropenic fever (p = 0.0008), lower absolute neutrophil count (p < 0.0001), acute kidney injury (AKI) (p = 0.0164), delayed methotrexate clearance (p = 0.0133), and higher 42-h methotrexate levels (p = 0.0179). In the standard/high-risk group, mercaptopurine dose was also linked to mucositis (p = 0.0495). Multivariable analysis showed that skin rashes (OR 6.5, p = 0.0016), fever (OR 2.8, p = 0.009), and neutropenia (OR 2.3, p = 0.0106) were independent risk factors for mucositis. Female sex (OR 7.12, p = 0.015) and AKI (OR 3.819, p = 0.037) were associated with recurrent mucositis. Fever, skin rashes, AKI, delayed methotrexate clearance, and higher 42-h methotrexate levels were key risk factors for HDMTX-induced oral mucositis. Skin rashes, fever, and neutropenia were independent predictors, while female sex and AKI were linked to recurrent mucositis.

Serum Methotrexate Level Predicts Acute Kidney Injury After High-Dose Methotrexate: A Case Report and Single-Center Experience.

Acute kidney injury (AKI) is a serious complication following high-dose methotrexate (HD-MTX) treatment, despite established preventive measures. This study presents a case report and a retrospective review of patients treated with HD-MTX, aiming to identify risk factors for AKI and propose a modified treatment protocol. We report a case of a 43-year-old man with diffuse large B-cell lymphoma who developed severe AKI after HD-MTX therapy. Additionally, a retrospective review of 70 patients receiving HD-MTX at our institution was conducted to identify predictors of AKI. Serum methotrexate levels were analyzed to determine their significance in predicting AKI. The index patient developed methotrexate intoxication and severe AKI despite receiving standard prophylactic measures, requiring temporary hemodialysis. The retrospective review identified serum methotrexate levels as a significant predictor of AKI (OR 11.84, 95% CI: 2.62-53.53, p = 0.001). Higher initial serum methotrexate levels correlated with the development of AKI. Timely measurement of serum methotrexate levels is crucial in managing and preventing AKI in patients undergoing HD-MTX therapy. Based on our findings, we propose a modified HD-MTX treatment protocol to reduce the incidence of AKI. This includes earlier serum methotrexate level monitoring and adjustments in urine alkalization and leucovorin dosing strategies.

B-280 Nmr-based biomaker measurement determines high-dose methotrexate elimination rates in lymphoma and leukemia treatment

Abstract Background The cytotoxic methotrexate (MTX) is used in high doses in lymphoma and acute lymphocytic leukemia (ALL) treatment. A therapeutic plasma dose range is crucial to minimize MTX accumulation and prolonged exposure, which can lead to myelosuppression and other adverse effects. Determining MTX elimination prior to drug administration is difficult, given its non-linear clearance. Here we present a model to predict MTX elimination rates based on metabolite measurement in patients receiving high-dose methotrexate, using nuclear-magnetic resonance (NMR) spectroscopy. Methods A series of 26 residual sera from patients receiving MTX infusions as per treatment protocols for CNS lymphoma (MATRIX protocol, 3.5 g/m2 over four hours) and ALL (GMALL protocol, 1.5 g/m2 over 24 hours) were collected from patients at the University Hospital Regensburg at four timepoints (t0, t24, t42, and t48). Serum was NMR-measured in five replicates. NMR spectra for each sample were characterized and relevant parameters extracted. First order elimination constants were modeled for two MTX dosage regimes (&amp;lt;4 g, ≥4 g) using characterized NMR features from samples measured at t0. Results Metabolomic and lipidomic biomarkers measured at baseline (t0) were able to accurately predict MTX elimination rate constant k, determined by fitting MTX levels at a t24, t42, and t48, using first order c(t)∼c0*e(-kt) kinetic function (&amp;gt;4 g: R2 = 0.475, p-value &amp;lt; 0.05; ≤4 g: R2 = 0.918, p-value &amp;lt; 0.01) (Fig. A). Results were cross-validated with five-time repeated three-fold cross-validation. Performance values are given as means of the folds. Conclusions NMR-measured biomarkers can predict dose-dependent MTX elimination rates in patients receiving high-dose MTX, presenting a novel method to predict MTX clearance, allowing for personalized dose-monitoring and minimization of adverse events. Validation with a larger cohort is required.

Folylpolyglutamate synthetase inactivation in relapsed ALL induces a druggable folate metabolic vulnerability

AimsThe antifolate methotrexate (MTX) is an anchor drug used in acute lymphoblastic leukemia (ALL) with poorly understood chemoresistance mechanisms in relapse. Herein we find decreased folate polyglutamylation network activities and inactivating FPGS mutations, both of which could induce MTX resistance and folate metabolic vulnerability in relapsed ALL. MethodsWe utilized integrated systems biology analysis of transcriptomic and genomic data from relapse ALL cohorts to infer hidden ALL relapse drivers and related genetic alternations during clonal evolution. The drug sensitivity assay was used to determine the impact of relapse-specific FPGS mutations on sensitivity to different antifolates and chemotherapeutics in ALL cells. We used liquid chromatography-mass spectrometry (LC-MS) to quantify MTX and folate polyglutamate levels in folylpoly-γ-glutamate synthetase (FPGS) mutant ALL cells. Enzymatic activity and protein degradation assays were also conducted to characterize the catalytic properties and protein stabilities of FPGS mutants. An ALL cell line-derived mouse leukemia xenograft model was used to evaluate the in vivo impact of FPGS inactivation on leukemogenesis and sensitivity to the polyglutamatable antifolate MTX as well as non-polyglutamatble lipophilic antifolate trimetrexate (TMQ). ResultsWe found a significant decrease in folate polyglutamylation network activities during ALL relapse using RNA-seq data. Supported by functional evidence, we identified multifactorial mechanisms of FPGS inactivation in relapsed ALL, including its decreased network activity and gene expression, focal gene deletion, impaired catalytic activity, and increased protein degradation. These deleterious FPGS alterations induce MTX resistance and inevitably cause marked intracellular folate shrinkage, which could be efficiently targeted by a polyglutamylation-independent lipophilic antifolate TMQ in vitro and in vivo. ConclusionsMTX resistance in relapsed ALL relies on FPGS inactivation, which inevitably induces a folate metabolic vulnerability, allowing for an efficacious antifolate ALL treatment strategy that is based upon TMQ, thereby surmounting chemoresistance in relapsed ALL.

Involvement of the ABCB1 C3435T Variant but Not the MTHFR C677T or MTHFR A1298C Variant in High-Dose Methotrexate-Induced Toxicity in Pediatric Acute Lymphoblastic Leukemia Patients in China.

It remains unclear whether the MTHFR C677T, MTHFR A1298C and ABCB1 C3435T genetic variants are associated with methotrexate (MTX) elimination delay and high-dose MTX (HD-MTX) toxicities in the treatment of pediatric acute lymphoblastic leukemia (ALL). The aim of our study was to analyze the potential predictive role of MTHFR C677T, MTHFR A1298C and ABCB1 C3435T in toxicities and the relationship between these variants and MTX elimination delay during HD-MTX therapy in pediatric ALL patients. We conducted a retrospective study on ALL patients receiving HD-MTX treatment with available MTHFR C677T, MTHFR A1298C and ABCB1 C3435T genotype and 44-h plasma MTX levels. Logistic regression analyses and chi-square tests were used to assess the relationship between the variants and HD-MTX toxicities and MTX elimination delay. Genotype frequencies were in Hardy-Weinberg equilibrium. MTX elimination delay did not significantly differ between MTHFR C677T and MTHFR A1298C or ABCB1 C3435T. Leukopenia (P=0.028), neutropenia (P=0.034) and oral mucositis (P=0.023) were 6.444-fold, 4.978-fold and 9.643-fold increased, respectively, in ABCB1 C3435T homozygous genotype (TT) patients compared to wild-type (CC) patients. No significant association was found between the toxicities investigated and MTHFR C677T or MTHFR A1298C. This study showed that the ABCB1 C3435T homozygous allele genotype (TT) is associated with increased MTX-related toxicities (leukopenia, neutropenia and oral mucositis). These results may help to distinguish pediatric ALL patients with a relatively high risk of MTX-related toxicities before HD-MTX infusion and optimize MTX treatment.

Rechallenge With High-Dose Methotrexate After Treatment With Glucarpidase in Adult Patients With Lymphoma.

Limited evidence exists regarding methotrexate (MTX) resumption after patients with lymphoma receive glucarpidase for toxic MTX levels and acute kidney injury (AKI). This retrospective review included adults with lymphoma treated with glucarpidase after MTX at Mayo Clinic between January 31, 2020, and October 10, 2022. Descriptive statistics summarize patient characteristics and clinical outcomes. Of 11 patients treated with glucarpidase after MTX, seven (64%) were rechallenged with MTX. Indications for MTX rechallenge included confirmed CNS disease (n = 6, 86%) and intravascular lymphoma (n = 1, 14%). Compared with the nonrechallenged subgroup, before receiving MTX that required glucarpidase rescue, the rechallenged patients had lower median pretreatment serum creatinine (Scr; 0.7 v 1.2 mg/dL), and none had AKI with previous MTX doses, n = 0 (0%) versus n = 2 (50%). During the MTX dose requiring glucarpidase rescue, the rechallenged group had lower median peak Scr (1.26 v 3.32 mg/dL) and lower incidence of AKI stage III (n = 1 [14%] v n = 3 [75%]), and none of the rechallenged patients required renal replacement therapy (RRT; n = 0 [0%] v n = 1 [25%]). At the first rechallenge after glucarpidase administration, the median MTX dose reduction was 56% (range, 46%-75%), and the lowest used dose when prescribed according to each treatment protocol schedule was 1.5 g/m2. Two (29%) patients experienced AKI (n = 1 stage I, n = 1 stage II) after MTX rechallenge. Zero patients required RRT, and zero required another glucarpidase administration. Six (86%) patients completed all recommended MTX doses. In selected adults with lymphoma who required glucarpidase for toxic MTX levels after administration of high-dose MTX, resumption of MTX therapy at lower doses is safe. Patients selected for MTX resumption had experienced less severe AKI during the previous cycle compared with those not selected for MTX resumption.

Impact of Prophylactic Trimethoprim-Sulfamethoxazole on Clearance of High-Dose Methotrexate in Adult Patients.

High-dose methotrexate (HDMTX) is an antineoplastic dosing strategy used to treat various cancers including primary central nervous system lymphoma. Trimethoprim-sulfamethoxazole (TMP/SMX) is commonly used for antibiotic prophylaxis against Pneumocystis pneumonia infections in this patient population. Significant drug-drug interactions between TMP/SMX and methotrexate (MTX) leading to adverse outcomes have been documented, primarily in adult patients taking MTX for rheumatologic conditions. This study is a single-center, retrospective, cohort study comparing outcomes in patients where TMP/SMX was held during HDMTX and patients who received concurrent prophylactic TMP/SMX during treatment. The primary end point was mean MTX level at 24, 48, and 72 hours. Secondary end points included rate of nonhematologic toxicity, rate of hematologic toxicity, median days to MTX clearance, and frequency of glucarpidase utilization. In total, 248 cycles of HDMTX were analyzed from 221 individual patients. One hundred ninety-one cycles were administered without prophylactic TMP/SMX, and 57 were administered with TMP/SMX. The median MTX level at 24, 48, and 72 hours in those without versus with prophylactic TMP/SMX was 4.30 versus 4.30, 0.29 versus 0.30, and 0.14 versus 0.15, respectively. Similarly, rates of hematologic and nonhematologic toxicities did not differ significantly between groups with the exception of neutropenia; however, there was no corresponding increased rate of neutropenic fever. Only one patient received glucarpidase and had not received TMP/SMX. Prophylactic TMP/SMX had minimal interaction with HDMTX and does not lead to increased time to clearance or clinically relevant toxicities. Prophylactic TMP/SMX can be safely administered with HDMTX in adult patients.

Low serum level and chronic toxicity for methotrexate. Case Report.

Methotrexate (MTX) is a folic acid antagonist used to treat different immunological or proliferative illnesses because of its anti-proliferative and anti-inflammatory effects. MTX Toxicity is considered a severe problem. Although acute toxicity related to high-dose administration (doses ≥500 mg/m2) can be predicted based on the given dose, chronic toxicity still has no specific factors to predict it, so treatment depends on the history and symptoms of toxicity. MTX was initially used for oncology indications with high cyclic doses, then expanded to non-oncology indications with different low doses and frequencies. This significant change in doses resulted in dosing errors that contributed to MTX toxicity reports. Measures to prevent the toxicity of MTX should be implemented. A 66-year-old female patient ingested 10 mg of MTX daily for one month instead of the once-toxicity symptoms. The serum level of MTX was requested, and treatment with folinic acid was initiated until the patient improved with the discontinuation of MTX. There is limited literature about the lack the total cumulative dose, duration of intake, or serum level of MTX. All this information was provided in this case report, but drug-drug interactions were not reviewed, although aspirin and pantoprazole were identified as having interactions with methotrexate in this patient. Minimum total cumulative dose identification may help assess the toxicity risk in such patients. Low-dose MTX chronic toxicity still needs further information to guide the patient's risk of toxicity and when to initiate treatment. Safety-practical measures should be implemented to prevent such administration errors.

Influence of Hypoalbuminemia on Methotrexate Clearance in Children Receiving High Dose Methotrexate Therapy

Aim: Methotrexate, a structural counterpart of folic acid, is well recognized as a very efficacious pharmaceutical agent utilized in the treatment of several pediatric oncological conditions. A high-dose methotrexate is employed as a therapeutic intervention for a diverse range of malignant conditions including leukemia, lymphomas, and osteosarcoma. Hypoalbuminemia also decreases the methotrexate clearance leading to increased toxicity. Objective: To determine the frequency of side effects among patients receiving high dose methotrexate therapy based on baseline albumin levels. Study Design: A quasi-experimental trial was conducted at the Pakistan Institute of Medical Sciences, Islamabad, from January 1, 2023, to July 31, 2023. A total of thirty-four children receiving HDMTX were enrolled in the study. Albumin level, grade of liver toxicity, renal function tests, gastrointestinal and neurological toxicity, and mucositis were noted. Length of stay and methotrexate level at 24 hours were also noted. All the information was recorded in proforma.

An ultrahigh-performance liquid chromatography-tandem mass spectrometry method for quantification of methotrexate and 7-hydroxy-methotrexate and application for therapeutic drug monitoring in patients with central nervous system lymphoma.

A method using ultrahigh-performance liquid chromatography-tandem mass spectrometry was developed, validated, and applied to simultaneously determine plasma methotrexate (MTX) and 7-hydroxy-methotrexate (7-OH-MTX) in 117 patients with central nervous system (CNS) lymphoma. The ion transitions utilized were m/z 455.2 > 308.2 for MTX and m/z 471.2 > 324.1 for 7-OH-MTX. Samples were prepared through protein precipitation using methanol. Chromatographic separation was achieved within 3.0 min on a CMS9030 column (Ruixi, 2.1 × 50 mm, 3 μm) through a gradient elution of methanol and a 10% ammonium acetate solution at a flow rate of 0.4 mL/min. The method demonstrated linearity in the concentration range of 0.05-10 μM for MTX and 0.25-50 μM for 7-OH-MTX. The intra- and inter-day inaccuracy ranged from -7.38% to 7.83%, and the imprecision was less than 6.00% for both analytes. The recovery and matrix effect normalized by the internal standard (MTX-D3 ) remained consistent. Both analytes remained stable under nine different storage conditions. In patients with CNS lymphoma, MTX levels at 12 h and 7-OH-MTX levels at 12, 36, and 60 h after dosing in individuals with impaired renal function were significantly higher compared with those with normal renal function. 7-OH-MTX could potentially serve as a superior indicator for nephrotoxicity compared with MTX.

Early Post-Infusion Clearance of High-Dose Methotrexate Predicts Delayed Methotrexate Elimination and Acute Kidney Injury

Introduction High-dose methotrexate (HDMTX) is used for the treatment of hematologic malignancies but its use may be limited by delayed methotrexate elimination and acute kidney injury (AKI). Current guidelines for glucarpidase recommend administration between 48-60 hours following the start of a 24-hour HDMTX infusion based on methotrexate concentration at 36-, 42-, or 48-hours [Ramsey 2016]. However, this may be too late to prevent permanent toxicities in cases where significant AKI develops early during infusion. Recent evidence from pig models have shown that early methotrexate measurements can predict the development of AKI [Buddington 2023]. The aim of this study is to determine whether early elimination of methotrexate (soon after completion of the infusion) is a useful “early clearance biomarker” of delayed methotrexate elimination and AKI in humans. Methods Retrospective data from two sites in Spain that collected two or more early measurements of methotrexate levels from the end of HDMTX infusion were analyzed from a larger HDMTX registry. Methotrexate half-life was calculated employing exponential decay equations and the slope of a line connecting two out of three early time points; each combination was tested, and the best performing pair is reported. Early levels were tagged according to visual exploration and k-means cluster analysis (Figure 1). Delayed methotrexate elimination was defined as a concentration above 1 μmol/L after 42-hours from the start of infusion (micromolar criterion) or above 2 standard deviations (SD) from the population mean modeled using MTXPK.org at 42- or 48-hours from the start of infusion (SD criterion). Receiver operating characteristic (ROC) curves were fitted, with their respective area under the curve (AUC) and confidence intervals for delayed methotrexate elimination and AKI. Additionally, a logistic regression model was employed to predict AKI using a base model with covariates (age, sex, HDMTX dose, and first course) plus early methotrexate clearance. Results Data from 144 patients who received 453 HDMTX courses were included. Patient characteristics were as follows: 41% females, age ranged from 1.2 to 81.5 years at the time of HDMTX infusion; diagnoses included acute lymphoblastic leukemia, 79%; non-Hodgkin lymphoma, 21%. Median methotrexate dose was 4,856.9 mg/m 2 [IQR 2,871.7 - 4,985.0], with 88% of the courses infused over 24 hours and 12% over 4 hours. Delayed methotrexate elimination was found in 41% and 6.6% of the courses according to the micromolar criterion and SD criterion, respectively. AKI developed in 29.6% of courses. Median time for the first, second and third measurements were 0.25 [IQR 0.00 - 0.71], 6.32 [IQR 5.86 - 6.69] and 12 [IQR 11.68 - 12.50] hours, respectively. Half-life estimation between the second and third early time-points predicted delayed methotrexate elimination by the micromolar criterion with an ROC AUC of 0.81 [CI95 0.76 - 0.87] or SD criterion with an ROC AUC of 0.84 [CI95 0.68 - 1.00] (Fig. 2) and any grade AKI with an ROC AUC of 0.62 [CI95 0.55 - 0.69]. After adjusting for age, sex, HDMTX dose, and first course, half-life remained statistically significant in the logistic regression model, with an odds ratio of 1.4 [CI95 1.1 - 1.8] (p &amp;lt; 0.01). The addition of half-life to the base model improved the overall performance (ANOVA, p &amp;lt; 0.01). Conclusions The half-life of methotrexate elimination soon after completing the HDMTX infusion defines an early clearance biomarker that predicts delayed methotrexate elimination and AKI. It requires only the measurement of 2 early methotrexate levels soon after completing the infusion, and thus represents a practical and applicable method to identify patients at risk who may need pre-emptive interventions like increased hydration or glucarpidase.

Outcome of Newly Diagnosed Children and Adolescents with Localized Lymphoblastic Lymphoma Treated on JPLSG-LLB-NHL03 Trial: A Report from the Japan Children's Cancer Group

Background: Localized lymphoblastic lymphoma (LL) is rare in pediatric patients and have a favorable outcome. Although the Berlin-Frankfurt-Münster (BFM) 90/95 protocol has been considered standard therapy, this regimen is too intense for patients with localized disease and the total doses of anthracycline and alkylating agents are high. To resolve these problems, we planned a clinical trial to examine the efficacy and safety of a modified BFM regimen for patients with localized LL. We report the 3-year event-free survival (EFS) and overall survival (OS) rate for children and adolescents with localized LL treated on JPLSG-LLB-NHL03 trial. Procedure: From November 2004 to October 2019, 41 newly diagnosed patients up to 18 years of age enrolled on LLB-NHL03 with localized LL (Murphy stages I and II) and with either T- or B- cell immunophenotype. Staging procedures included: bilateral bone marrow aspirates and biopsies, lumbar puncture, CT or MRI of abdomen, chest X-ray, and bone scintigraphy or whole body bone X-ray. Central pathology review consisted of examination of morphology and immunophenotypic and any available genetic data from the original diagnostic biopsy to confirm the submission diagnosis and assign phenotypic lineage. Treatment consisted of 5 phases: induction (prednisolone 60mg/m 2, day 0-27; vincristine 1.5mg/m 2, day0,7,14,21; cyclophosphamide 1g/m 2, day 0; daunorubicin 30mg/m 2, day 0,7; E.coli L-asparaginase 6,000 units/m 2, 9 total doses, and intrathecal methotrexate and hydrocortisone, day 0,14), consolidation (mercaptopurine 60mg/m 2, day 0-27; cyclophosphamide 500mg/m 2, day0,14; cytarabine 75mg/m 2, 12 total doses and intrathecal therapy methotrexate and hydrocortisone, day 0,14) , CNS prophylaxis (high dose methotrexate 3g/m 2, day0,7,14; and intrathecal methotrexate and hydrocortison, day 0. Standard leucovorin dosing, 15mg/m 2 intravenous every 6h, is delivered 36h after the start of the methotrexate infusion and is continued until methotrexate levels are less than 0.1 μmol/L.), delayed intensification (prednisolone 60mg/m2, day 0-13; vincristine 1.5mg/m 2, day 0,7,14; cyclophosphamide 1g/m 2, day0; daunorubicin 30mg/m 2, day 0,7; L-asparaginase 6,000 units/m 2, 6 total doses and intrathecal methotrexate and hydrocortison, day 0,14), and maintenance (mercaptopurine 60mg/m 2 daily and methotrexate 20mg/m 2 weekly). Total duration of therapy was 24 months from diagnosis. Patients received 120mg/m 2 of anthracycline (daunorubicin) , 3 g/m 2 of cyclophosphamide and 7 doses of intrathecal therapy. Results: Of the 41 patients enrolled in the study, 35 were included in the primary efficacy and safety analysis, excluding 6 patients because they did not meet the eligibility criteria: three with diffuse large B-cell lymphoma, one with extranodal NK/T lymphoma, one with mixed phenotype LL, and one with stage III disease. Themedian [range] age at enrollment was 9.0 [2.1-16.1] years. Twenty-three (65.7%) were male. Twenty-nine had pre-B and 6 had pre-T immunophenotype. Fifteen (42.9%) were Stage I and 20 (57.1%) were Stage II. Thirty-four (97%) patients achieved complete remission at the end of induction. The median follow-up for EFS was 10.1 [0.0-15.2] years, and the primary endpoint of 3-year EFS rate [95% confidence interval: 95% CI] (n=35) was 97.1% [81.4-99.6%]. (Fig.1) The median follow-up for OS was 10.6 [2.9-16.1] years, and the 3-year OS rate (n=35) was 100%. One pre-T immunophenotype patient with stage II disease failed to achieve complete remission after induction therapy. Overall, patients tolerated therapy well and no treatment-related deaths were observed. There was only one unanticipated adverse event (retinal detachment), and a causal relationship to study treatment was ruled out. Conclusion: This is the largest trial ever conducted in the treatment of pediatric patients with newly diagnosed localized lymphoblastic lymphoma using an individual protocol. Outcomes of pediatric patients with localized LL treated with 2 years of less-intensive BFM-like therapy was excellent. The number of children diagnosed with localized LL limits the possibility of randomized trials focusing on therapeutic questions. Future studies should identify the biologic or clinical difference between LL and lymphoblastic leukemia, explore less intensive treatment for patients with localized disease, and explore novel targeted therapies.

Folate-functionalized CoFe2O4 nanozyme inspired by folate antagonist effect for methotrexate detection with high anti-interference ability and recyclability

Methotrexate (MTX) is a widely used chemotherapeutic drug to treat cancer and autoimmune diseases, which has a narrow therapeutic window and can cause systemic toxicity. The monitoring of MTX is therefore of crucial importance in clinical practice and precision medication. However, current detection techniques suffer from limitations in cost and time effectiveness, making them unsuitable for out-of-lab and point-of-care testing. Inspired by the antimetabolite mechanism of MTX, a signal-on colorimetric sensing platform based on CoFe2O4 nanozyme functionalized with folic acid (FA@CoFe2O4) was elaborately developed to monitor MTX. Due to the ability of the immobilized folate to be reduced by dihydrofolate reductase (DHFR) to tetrahydrofolate, it poses competitive effect on the chromogenic substrate by its strong reducibility when FA@CoFe2O4 exerts its peroxidase-like activity, thereby inhibiting the discoloration of the substrate. Once MTX is introduced, the active sites of DHFR will preferentially bind to it, leading to suppression in the generation of tetrahydrofolate and further restoring the substrate’s color rendering. Based on this principle, the developed sensing strategy exhibited a broad range from 0.1 μM to 40 μM towards MTX detection. In addition, the magnetic property endowed the FA@CoFe2O4 nanozyme excellent recyclability and allowed the MTX detection with high anti-interference ability from other drugs and coexisting antioxidants. The feasibility of the method was validated by monitoring MTX level in human serums. This work paves a new way towards therapeutic drug monitoring that serves personalized administration.

Primary CNS Lymphoma in Latin America: A Grupo De Estudio Latinoamericano De Linfoproliferativos (GELL) Survey in 80 Cancer Centers in Latin America

Introduction: Primary Central Nervous System Lymphoma (PCNSL) is a rare and aggressive subtype of lymphoma, representing 4-6% of extranodal lymphomas. Management of PCNSL involves a multidisciplinary approach which includes neurosurgery, ophthalmology, radiotherapy, autologous stem cell transplantation (ASCT) and the use of novel agents such as BTK-inhibitors, immunomodulatory agents and chimeric antigen receptor T-cell (CAR-T cell) therapy. These strategies can be costly and not readily available in developing countries. GELL, a multinational group for studying lymphoproliferative disorders, conducted a multicenter international survey to investigate the diagnosis, clinical staging and treatment patterns in Latin America (LATAM). Methods: We conducted an on-line survey consisting of 22 questions regarding diagnostic and treatment patterns of PCNSL in LATAM. All invited centers agreed to participate and contributed for the development of the survey. No identifiable patient information was abstracted at any time during the survey. Questions were structured based on 3 pillars: (1) diagnostic approach, including available biopsy procedures, immunohistochemistry markers, ophthalmology evaluation, flow cytometry and molecular testing; (2) first line therapy, including preferred regimens used for both transplant eligible and ineligible patients, and availability of novel agents, and (3) consolidation therapies including ASCT and whole brain radiation therapy (WBRT) Results: A total of 90 surveys were completed from 80 cancer centers in LATAM. Geographical distribution of the participating cancer centers are as followed: Argentina (n=11), Bolivia (n=2), Brazil (n=9), Chile (n=5), Colombia (n=17), Cuba (n=1), Ecuador (n=5), Mexico (n=10), Panama (n=1), Paraguay (n=4), Peru (n=5), Dominican Republic (n=1), Uruguay (n=5) and Venezuela (n=4). Most centers treat up to 5 new cases of PCNSL/year (84%), with only 3 centers treating more than 15 cases/year. Stereotaxic biopsy was the preferred diagnostic procedure in 78%, and complete resection was done in a minority of centers (3%). Cerebrospinal fluid studies were done in 82% and flow cytometry was available in 87% of the centers. Only 65% of centers had access to PET-CT at diagnosis. Interestingly, 80% of the physicians performed bone marrow biopsy at diagnosis, and ophthalmology evaluation was not available in 20% of the centers. Regarding IHC markers, EBV-LEMP was available in 55% and PDL-1 in only 38% of the centers. FISH for Myc/Bcl2 was performed in 32% and molecular testing in only 23% of the centers. The median time for treatment initiation was 0-10 days in 36%, 10-20 days in 37% and &amp;gt;30 days in 26% of the centers. Regarding first line therapy, MATRIX regimen was the preferred regimen for fit transplant eligible patients in 53% of the participants. However, thiotepa was not readily available as per 45% of the survey participants. Methotrexate (MTX) with Cytarabine was the preferred regimen for 36% of participants. Interestingly, serum monitoring of MTX was not available in 27% of the centers. ASCT was the preferred consolidation regimen for transplant eligible patients (78%), and the most used conditioning regimen was BEAM (49%), suggesting an impact of not availability of thiotepa in these centers. For transplant ineligible patients, MTR (27%), MATRIX (22%) and R-MVP (19%) were the preferred regimens, and 32% of survey participants considered WBRT as the preferred consolidation for these patients. Regarding relapsed/refractory (R/R) PCNSL, MTX-based salvage therapy was the preferred regimen in 38%, followed by cytarabine based in 23% and BTK-inhibitor based in 20% of the participants. CAR-T cell therapy was available in only 3 centers. Conclusion: Management of PCNSL is highly variable in LATAM. Access to targeted agents and novel therapies such as CAR-T cell therapy is limited. Moreover, most centers lack basic diagnostic approaches such as serum MTX level monitoring and ophthalmology evaluation. Lack of access to thiotepa was a common issue encountered by most cancer centers in LATAM. The use of thiotepa-containing regimens has demonstrated improved survivals both as consolidation and in the R/R setting. Thus, the absence of such agent in LATAM carries a profound impact on PCNSL outcomes in this area of the world. A retrospective evaluation on PNCSL outcomes is planned by the GELL group to better investigate our results.

A new pharmacokinetic model of urinary methotrexate to assess adherence in rheumatoid arthritis

Background and objectivesMethotrexate (MTX) is the first-line therapy for rheumatoid arthritis (RA). While therapeutic adherence is essential to successful management, no objective MTX assay is currently available. Using population pharmacokinetic modelling (PopPK), our objective was to describe the urinary MTX (MTXu) kinetics in treated patients and to evaluate its abilities to assess the MTX-adherence. MethodsThe association between urinary methotrexate level and methotrexate administration was assessed using a generalized linear model. Then, a population pharmacokinetic model was developed based on data from 59 patients using with Monolix 2021. R2. Simulations were run to establish a reference kinetic profile and evaluate the proportion of samples predicted as true positives. ResultsCompared to the control group, multivariate analysis showed that MTXu was independently associated with methotrexate administration (p < 0.0001) with a sensitivity and specificity greater than 99%. The final PopPK model selected was a two-compartment model with first-order absorption and elimination. Internal and external validation of the model met all predefined criteria. When using an analytical assay with a LOQ equal to 1 nM, the proportion of samples predicted as true positives is over 90%, as a function of MTX dose (7.5–25 mg/week) and post-administration sampling days (1–7 days). ConclusionWe developed a pharmacokinetic model able to describe expected patterns of urinary methotrexate. This allowed us to propose a new objective test of MTX adherence, which could help in routine practice to differentiate patients who are truly unresponsive to methotrexate from those who are unresponsive because of non-adherence.

Reagentless impedimetric immunosensor for monitoring of methotrexate in human blood serum using multiwalled carbon nanotube@polypyrrole/polytyramine film electrode

Ensuring effective monitoring of methotrexate (MTX) levels in the bloodstream of cancer patients undergoing high-dose methotrexate chemotherapy is crucial to prevent potentially harmful side effects. However, the absence of portable analytical devices suitable for point-of-care bedside monitoring has presented a significant obstacle to achieving real-time MTX monitoring. In this study, we developed an impedimetric immunosensor that doesn't require reagents for measuring MTX levels in undiluted human blood serum. This reagentless approach simplifies the assay process, enabling rapid and straightforward MTX quantification. The immunosensor transducer was fabricated by electrodepositing conductive network of porous multiwalled carbon nanotube@polypyrrole/polytyramine on screen-printed gold microchip electrode (SP–Au/MWCNT70@PPy-PTA). Polyclonal anti-MTX antibodies were immobilized on the film, acting as the immunorecognition element. Non-specific binding was prevented by blocking the transducer interface with denatured bovine serum albumin (dBSA) fibrils, resulting in SP-Au/MWCNT70@PPy-PTA/anti-MTXAb|dBSA film electrode. When MTX binds to the SP-Au/MWCNT70@PPy-PTA/anti-MTXAb|dBSA interface, the film conductance and electron transfer resistance changes. This conductivity attenuation allows for electrochemical impedimetric signal transduction without a redox-probe solution. The electrochemical impedance spectroscopy (EIS) results showed increased charge transfer resistance and phase angle as MTX concentrations increased. The SP-Au/MWCNT70@PPy-PTA/anti-MTXAb|dBSA demonstrated high sensitivity, with a linear response from 0.02 to 20.0 μM and a detection limit of 1.93 nM. The detection limit was 50 times lower than the intended safe level of MTX in human serum. The immunosensor exhibited minimal cross-reactivity with endogenous MTX analogs and serum proteins. The SP-Au/MWCNT70@PPy-PTA/anti-MTXAb|dBSA immunosensor presents a simple and rapid method for therapeutic drug monitoring compared to traditional immunoassay systems.

Access to Methotrexate Monitoring in Latin America: A Multicountry Survey of Supportive Care Capacity

High-dose methotrexate (HDMTX) is used to treat a broad spectrum of cancers. Methotrexate (MTX) monitoring and adequate supportive care are critical for safe drug administration; however, MTX level timing is not always possible in low- and middle-income countries. The aim of this study was to evaluate HDMTX supportive care capacity and MTX monitoring practices in Latin America (LATAM) to identify gaps and opportunities for improvement. A multicenter survey was conducted among LATAM pediatric oncologists. Twenty healthcare providers from 20 institutions answered the online questionnaire. HDMTX was used to treat acute lymphoblastic leukemia (ALL; 100%), non-Hodgkin lymphoma (84.2%), diffuse large B-cell lymphoma (47.4%), osteosarcoma (78.9%), and medulloblastoma (31.6%). Delays in starting HDMTX infusion were related to bed shortages (47.4%) and MTX shortages (21.1%). MTX monitoring was performed at an in-hospital laboratory in 52%, at an external/nearby laboratory in 31.6%, and was not available in 10.5%. Median interval between sampling and obtaining MTX levels was ≤ 2 h in 45% and ≥ 6 h in 30%, related to laboratory location. Sites without access to MTX monitoring reduced the MTX dose for patients with high-risk ALL or did not include MTX in the treatment of patients with osteosarcoma. Respondents reported that implementation of point-of-care testing of MTX levels is feasible. In LATAM, highly variable supportive care capacity may affect the safe administration of MTX doses. Improving accessibility of MTX monitoring and the speed of obtaining results should be prioritized to allow delivery of full doses of MTX required by the current protocols.

BSLD-01 DYNAMIC CONTROL OF CSF FOR PERSONALIZED CNS DRUG DELIVERY FOR LEPTOMINGEAL DISEASE

Abstract BACKGROUND Appropriate drug distribution is needed for effective treatment of brain metastases and leptomeningeal disease. Lack of proper drug bioavailability in the CNS contributes to the poor outcome in these patients. EnClear Therapies has developed a device delivering intrathecal therapeutics utilizing the dynamic control of CSF flow through an external pump. Herein, we look at intrathecal drug delivery of methotrexate with EnClear’s system and its impact of drug concentration in CNS tissue in sheep and non-human primates. METHODS EnClear’s system utilizes two implantable catheters (lateral ventricle and lumbar thecal sac) and an extracorporeal pump that controls the speed and direction of CSF flow. Software continuously monitors and reports the output from sensor arrays for pressure, respiration, and heart rate. Imaging studies were used to track the CSF flow characteristics during the use of EnClear’s system. Data from administration of methotrexate followed by the use of EnClear’s system was compared to the current standard of delivery, intracerebroventricular or lumbar intrathecal administration. Methotrexate levels were measured with LC/MS in CSF, blood, and multiple CNS and peripheral tissues. RESULTS There was a two-fold increase in methotrexate levels in multiple regions of the brain including deeper brain structures including the striatum using EnClear's system, compared to traditional lumbar intrathecal and intracerebroventricular delivery. Higher peak levels of methotrexate were reached in the CSF with the use of EnClear’s system, with simultaneous reduction in the peripheral nerves and systemic tissues – a potential source of toxicity. CONCLUSIONS In large animal studies, EnClear’s system has for the first time, demonstrated increased brain parenchymal drug levels via intrathecal delivery. Drug delivery with this novel device has shown improved distribution throughout the CNS and leptomeninges as compared to traditional intrathecal drug delivery and decreased peripheral distribution.

A pooled subgroup analysis of glucarpidase treatment in 86 pediatric, adolescent, and young adult patients receiving high-dose methotrexate therapy in open-label trials.

Delayed methotrexate elimination can occur in patients undergoing high-dose methotrexate cancer treatment. Effectiveness of glucarpidase for rapidly reducing methotrexate concentrations was shown in compassionate-use trials in patients aged 0-84years. We performed post hoc analyses of infants (≥28days to <2years), children (≥2 to <12years), adolescents (≥12 to <15years), and young adults (≥15 to <25years) from four multicenter, open-label, single-arm, glucarpidase compassionate-use trials. Patients had toxic methotrexate levels due to delayed methotrexate elimination and/or renal dysfunction, and received glucarpidase (50U/kg). The primary endpoint was clinically important reduction (CIR) in plasma methotrexate (methotrexate ≤1μmol/L at all post-glucarpidase measurements) based on high-performance liquid chromatography. Among 86 patients included in efficacy analyses, CIR was achieved by zero of one infant (0.0%), five of 16 children (31.3%), seven of 24 adolescents (29.2%), and 26/45 young adults (57.8%). Median methotrexate reduction was 98.7% or higher in each group 15minutes post-glucarpidase. Patients with pre-glucarpidase methotrexate less than 50μmol/L (35/42, 83.3%) were more likely to achieve CIR than those with methotrexate 50μmol/L or higher (1/37, 2.7%). The most common treatment-related adverse event was paresthesia, occurring in three adolescents (4.5%) and six young adults (5.2%). No other treatment-related adverse event occurred in 5% or higher of any age group. After accounting for pre-glucarpidase methotrexate levels, glucarpidase efficacy at inducing CIR in pediatric/young adult patients was consistent, with efficacy observed in the overall study population (i.e., patients aged 0-84), and no unexpected safety findings were observed. These findings demonstrate glucarpidase (50U/kg) is an effective and well-tolerated dose for pediatric, adolescent, and young adult patients.

High dose methotrexate (MTX) treatment: Value of post treatment MTX level monitoring and risk factors for acute kidney injury.

e19539 Background: High-dose methotrexate therapy is an effective treatment for primary central nervous system (CNS) lymphoma and other tumors. Methotrexate (MTX) is an antimetabolite which penetrates blood brain barrier when administered intravenously at high doses. Defective or delayed clearance of MTX may lead to toxicities, especially acute kidney injury (AKI) in 2-13% of patients. We developed a protocol to mitigate side effects by checking MTX levels every 24 hours, administering leucovorin or glucarpidase rescue, and hydration with urine alkalization. This study examines the risk factors for developing AKI after high dose MTX administration. Methods: Patients who began treatment with high dose methotrexate in 2020 and 2021 were eligible. We collected data on demographics, diagnosis, methotrexate levels, laboratory levels at baseline and after treatment, toxicities, interventions and outcomes. Results: 26 patients were included, who received 112 cycles of treatments (median 3.5, range 1-10). Median age was 62 years (range 25-83) and 57% were male. 20 patients experienced elevated MTX levels. Among the 112 cycles, MTX levels were elevated at 24h, 48h and 72h in 22 (19.6%), 16 (14.3%), and 45 (40.2%) of the cycles respectively, which trigged an increase in the rescue leucovorin dose and hydration dose in 26 (23.2%) and 16 (14.3%) cycles, respectively. AKI was recorded in 20 cycles (17.9%), with 17 (85%) being grade 1 (ULN – 1.5 x ULN), and 3 (2.7%) being grade 2 (1.5-3x ULN). In 20 cycles with AKI, 9 (45%) had grade 1 elevated creatinine level pretreatment; 8 (88.9%) of them remained grade1 without increase &gt; 0.2. The development of AKI occurred in all MTX dose levels, without a difference in frequency. 57% of cycles with elevated 24h MTX level, and 13% of cycles with delayed clearance &gt; 4 days, were associated with AKI. 75% of cycles with AKI had delayed clearance (avg 7 days). Two patients required glucarpidase rescue treatment, based on elevated 24-36 h MTX level and grade 1-2 AKI; one of these patients permanently discontinued MTX. Conclusions: Among 26 patients who received a total of 112 cycles of high dose MTX treatment, 11 developed AKI in 20 cycles, mostly grade 1 with spontaneous recovery. MTX level elevation at 24 hours predicted the development of AKI. Elevated baseline creatinine or MTX dose range is not a risk factor for AKI.