Monitoring Of Methotrexate Research Articles

Simultaneous Determination of Methotrexate Concentrations in Human Plasma and Cerebrospinal Fluid Using Two-Dimensional Liquid Chromatography: Applications in Primary Central Nervous System Lymphoma.

In this study, we aimed to develop a method for the simultaneous quantification of methotrexate (MTX) samples extracted from human plasma and cerebrospinal fluid (CSF), using two-dimensional liquid chromatography (2D-LC). Furthermore, we intended to verify whether intravenous mannitol could increase MTX concentration in the CSF of patients. The mobile phase of PUMP1 consisted of 10.0 mmol/L ammonium acetate and acetonitrile. PUMP2 solution consisted of an aqueous solution of 10.0 mmol/L ammonium acetate. The mobile phase of PUMP3 comprised 50.0 mmol/L ammonium acetate and acetonitrile, with a flow rate of 1.0 mL/min. The developed method was successfully employed to simultaneously determine drug levels in plasma and CSF from the patients treated with MTX. CSF samples were obtained by lumbar puncture 0.5 - 2 h after starting the high-dose methotrexate (HD-MTX) infusion (over 4 h) and immediately before the intrathecal (IT) administration of MTX. Venous blood samples were drawn 4 h after the start of infusion. The calibration curve was linear, with a range of 0.07 - 2.38 µmol/L for CSF samples and a range of 0.11 - 5.51 µmol/L for plasma samples. Precision (> 95%) and accuracy (> 97%) were within the acceptance criteria for each quality control (QC) level. Inter- and intra-day accuracy and precision values met the acceptance criteria for each QC level. The correlation between MTX concentrations in the plasma and CSF was moderate (r = 0.502). No significant difference was observed in MTX concentration in CSF between patients using intravenous mannitol and those not using intravenous mannitol (P = 0.682). The developed method was useful for therapeutic drug monitoring of MTX and suitable for assessing the risks and benefits of chemotherapy in patients with primary central nervous system lymphoma. Intravenous mannitol did not increase MTX concentration in the CSF of patients.

Early therapeutic drug monitoring of methotrexate and its association with acute kidney injury: A retrospective cohort study.

High-dose methotrexate (HDMTX) use can be limited by the development of acute kidney injury (AKI). Early AKI detection is paramount to prevent further renal injury and irreversible toxicities. This study sought to determine whether early elimination patterns of MTX would be useful as a biomarker of AKI in HDMTX treatment. This retrospective cohort study included two sites that collected ≥2 MTX levels within 16 h from completion of MTX infusion. Early levels were tagged and MTX elimination half-life (t½) were calculated from combinations of two of three different early time periods. Receiver operating characteristic (ROC) curves were synthesized for each elimination t½ (biomarker) with respect to AKI and delayed methotrexate elimination (DME); the biomarker with the highest area under the ROC curve (AUC) was tested in a multiple variable logistic regression model. Data from 169 patients who received a total of 556 courses of HDMTX were analyzed. ROC analysis revealed MTX elimination t½ calculated from the second and third time periods had the highest AUC for AKI at 0.62 (interquartile range [IQR] 0.56-0.69) and DME at 0.86 (IQR 0.73-1.00). After adjusting for age, sex, dose (mg/m2), infusion duration, HDMTX course, and baseline estimated glomerular filtration rate, it remained significant for AKI with an OR of 1.29 and 95% confidence interval of 1.03-1.65. Early MTX elimination t½ measured within 16 h of infusion completion was significantly associated with the development of AKI and serves as an early clearance biomarker that may identify patients who benefit from increased hydration, augmented leucovorin rescue, and glucarpidase administration.

Clinical feasibility of a label-free SERS assay for therapeutic drug monitoring of methotrexate

We present a therapeutic drug monitoring (TDM) feasibility study using a label-free surface-enhanced Raman spectroscopy (SERS)-based assay for quantifying methotrexate (MTX) from samples collected from children with acute lymphoblastic leukemia undergoing high-dose MTX therapy. We show that, when combined with an appropriate sample preparation (solid phase extraction) and multivariate data analysis (partial least squares regression), the SERS assay demonstrated a good correlation with a reference chromatographic method (r = 0.889, p < 0.005). We also found that the SERS-based approach underestimated MTX concentration, with a 10% bias calculated, but both methods showed similar variability (RSD of 8.4% for HPLC and 11.6% for SERS). The presented results are strong evidence that SERS can be used for TDM of MTX, and this work brings us one step closer to implementing a SERS-based assay in clinics.

OpenSAFELY: The impact of COVID-19 on azathioprine, leflunomide and methotrexate monitoring, and factors associated with change in monitoring rate.

The COVID-19 pandemic created unprecedented pressure on healthcare services. This study investigates whether disease-modifying antirheumatic drug (DMARD) safety monitoring was affected during the COVID-19 pandemic. A population-based cohort study was conducted using the OpenSAFELY platform to access electronic health record data from 24.2 million patients registered at general practices using TPP's SystmOne software. Patients were included for further analysis if prescribed azathioprine, leflunomide or methotrexate between November 2019 and July 2022. Outcomes were assessed as monthly trends and variation between various sociodemographic and clinical groups for adherence with standard safety monitoring recommendations. An acute increase in the rate of missed monitoring occurred across the study population (+12.4 percentage points) when lockdown measures were implemented in March 2020. This increase was more pronounced for some patient groups (70-79 year-olds: +13.7 percentage points; females: +12.8 percentage points), regions (North West: +17.0 percentage points), medications (leflunomide: +20.7 percentage points) and monitoring tests (blood pressure: +24.5 percentage points). Missed monitoring rates decreased substantially for all groups by July 2022. Consistent differences were observed in overall missed monitoring rates between several groups throughout the study. DMARD monitoring rates temporarily deteriorated during the COVID-19 pandemic. Deterioration coincided with the onset of lockdown measures, with monitoring rates recovering rapidly as lockdown measures were eased. Differences observed in monitoring rates between medications, tests, regions and patient groups highlight opportunities to tackle potential inequalities in the provision or uptake of monitoring services. Further research should evaluate the causes of the differences identified between groups.

Functional Group-Assisted Fluorescence Sensing Platform for Nanomolar-Level Detection of an Antineoplastic Drug and a Neurotransmitter from Environmental Water and Human Biofluids.

A fast, sensitive, selective, and biocompatible dual sensor of an antineoplastic medication (methotrexate) and a neurotransmitter (adrenaline) is still being searched by present-day scientists. To overcome this issue, we have designed a functionalized, robust, bio-friendly luminescent MOF for the sensitive, selective, and rapid monitoring of methotrexate and adrenaline. This probe is the first ever reported MOF-based fluorescence sensor of methotrexate and second only for adrenaline. This fluorescence probe has a very low limit of detection (LOD) of 0.34 and 11.2 nM for adrenaline and methotrexate, respectively. The sensor can detect both the targeted analytes rapidly within 5 s. It can also detect adrenaline and methotrexate from human blood serum and urine accurately and precisely. This reusable sensor is equally efficient in detecting methotrexate from environmental water specimens. Biocompatible, user-friendly, and inexpensive chitosan@MOF@cotton composites were fabricated for the detection of adrenaline and methotrexate from the nanomolar to the micromolar range by the naked eye under a fluorescence lamp. This probe displayed high reproducibility, precision, and accuracy in sensing methotrexate and adrenaline. Fluorescence resonance energy transfer (FRET) and the inner filter effect (IFE) are the possible mechanisms for adrenaline and methotrexate sensing, respectively. The possible mechanism was supported by using required instrumental techniques and theoretical simulations.

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.

Methotrexate toxicity-an alarming bell!

Since its introduction in 1951, methotrexate (MTX) has been widely used as the primary anti-psoriatic agent. However, unsupervised dosing by patients or concurrent use of excessive nonsteroidal anti-inflammatory drugs (NSAIDs) can lead to toxicity. Gastrointestinal symptoms are the most commonly observed manifestations, followed by cutaneous toxicity. In this retrospective study conducted at a tertiary care center in Gujarat, data from patients admitted for MTX toxicity between April 2017 and December 2019 were analyzed. A detailed history, including dosage, duration, signs and symptoms, investigations, treatment, and outcomes, was collected and evaluated. The study included 12 hospitalized patients (aged 35-70 years) with MTX toxicity. Among them, 10 patients had psoriasis, 1 had psoriatic arthritis, and 1 had rheumatoid arthritis with discoid lupus erythematosus. Eleven patients had taken daily oral MTX for one week along with painkillers, while one patient had taken an unknown amount without undergoing any investigations. Mucosal ulceration was observed in all cases, and skin necrosis was seen in 2 patients. Hematological investigations revealed myelosuppression in all patients, with altered renal function in 1 patient. Leucovorin (15 mg/ml) was administered to all patients, resulting in improvement for 10 patients, but 2 patients experienced persistent myelosuppression and succumbed to the toxicity. This study emphasizes the importance of pre-treatment investigations, proper monitoring, and strict avoidance of self-administration when administering MTX. Additionally, the co-administration of drugs such as NSAIDs should be judiciously managed. Folinic acid has shown usefulness in cases of MTX toxicity and overdose. In summary, this study underscores the significance of careful administration and monitoring of MTX, along with the avoidance of self-dosing. It highlights the need for judicious use of co-administered medications, and emphasizes the utility of folinic acid in managing MTX toxicity and overdose.

Wearable Microneedle-Based Array Patches for Continuous Electrochemical Monitoring and Drug Delivery: Toward a Closed-Loop System for Methotrexate Treatment.

Wearable devices based on microneedle (MN) technology have recently emerged as tools for in situ transdermal sensing or delivery in interstitial fluid (ISF). Particularly, MN-based electrochemical sensors allow the continuous monitoring of analytes in a minimally invasive manner through ISF. Exogenous small molecules found in ISF such as therapeutic drugs are ideal candidates for MN sensors due to their correlation with blood levels and their relevance for the optimal management of personalized therapies. Herein, a hollow MN array patch is modified with conductive pastes and functionalized with cross-linked chitosan to develop an MN-based voltammetric sensor for continuous monitoring of methotrexate (MTX). Interestingly, the chitosan coating avoids biofouling while enabling the adsorption of MTX at the electrode's surface for sensitive analysis. The MN sensor exhibits excellent analytical performance in vitro with protein-enriched artificial ISF and ex vivo under a Franz diffusion cell configuration. The MN sensor shows a linear range from 25 to 400 μM, which fits within the therapeutic range of high-dose MTX treatment for cancer patients and an excellent continuous operation for more than two days. Moreover, an iontophoretic hollow MN array patch is developed with the integration of both the anode and cathode in the single MN array patch. The ex vivo characterization demonstrates the transdermal on-demand drug delivery of MTX. Overall, the combination of both MN patches represents impactful progress in closed-loop systems for therapeutic drug management in disorders such as cancer, rheumatoid arthritis, or psoriasis.

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.

Therapeutic drug monitoring of methotrexate in patients with Crohn's disease.

Therapeutic drug monitoring (TDM) has the potential to improve efficacy and diminish side effects. Measuring methotrexate-polyglutamate (MTX-PG) in erythrocytes might enable TDM for methotrexate in patients with Crohn's disease (CD). To investigate the relationship between MTX-PGs and methotrexate drug survival, efficacy and toxicity METHODS: In a multicentre prospective cohort study, patients with CD starting subcutaneous methotrexate without biologics were included and followed for 12 months. Primary outcome was subcutaneous methotrexate discontinuation or requirement for step-up therapy. Secondary outcomes included faecal calprotectin (FCP), Harvey Bradshaw Index (HBI), hepatotoxicity and gastrointestinal intolerance. Erythrocyte MTX-PGs were analysed at weeks 8, 12, 24 and 52 or upon treatment discontinuation. We included 80 patients with CD (mean age 55 ± 13y, 35% male) with a median FCP of 268 μg/g (IQR 73-480). After the 12-month visit, 21 patients (26%) were still on subcutaneous methotrexate monotherapy. Twenty-one patients stopped because of disease activity, 29 because of toxicity, and four for both reasons. Five patients ended study participation or stopped methotrexate for another reason. A higher MTX-PG3 concentration was associated with a higher rate of methotrexate drug survival (HR 0.86, 95% CI 0.75-0.99), lower FCP (β -3.7, SE 1.3, p < 0.01) and with biochemical response (FCP ≤250 if baseline >250 μg/g; OR 1.1, 95% CI 1.0-1.3). Higher MTX-PGs were associated with less gastrointestinal intolerance. There was no robust association between MTX-PGs and HBI or hepatotoxicity. Higher MTX-PG3 concentrations are related to better methotrexate drug survival and decreased FCP levels. Therefore, MTX-PG3 could be used for TDM if a target concentration can be established.

Fluorescent immunochromatographic test strip for therapeutic drug monitoring of methotrexate with high sensitivity and wide dynamic range.

As a front-line chemotherapeutic drug for maintenance and consolidation therapy, methotrexate (MTX) has widely been applied to treat various tumors and some inflammatory diseases. However, because of its severe toxicity ascribed to low selectivity, it is necessary to monitor therapeutic drugs in high-dose MTX therapeutic regimens to ensure treatment safety. In this work, we developed a fluorescent immunochromatographic test strip (FITS) for monitoring MTX by employing time-resolved fluorescent microspheres as signal probes. With a competitive immunoassay mode, the FITS for MTX shows a super-wide dynamic range of 10pM-10μM, covering the entire clinical therapeutic concentration range of MTX. Therapeutic drug monitoring of MTX can be achieved within 7min with high specificity, facilitating the timely rescue of drug poisoning led by high-dose MTX treatment. The methodwasemployedfor monitoring MTX in the spiked human serum, urine, and milk, showing acceptable recoveriesranging from 94.0 to 110.0%. The established FITS has been applied toMTX detection in serum obtained from high-dose MTX treatment. The results from FITS and enzyme multiplied immunoassay technique showed no significant difference, suggesting its reliability for usage in real biological samples. The device shows promise in point-of-caretherapeutic drug monitoring for resource-limited countries and institutes, which significantly facilitates overcoming the lag time between sampling and results.

E007 Is there an urgent need to change the methotrexate monitoring guidelines to incorporate fibroscan?

Abstract Background/Aims Methotrexate (MTX) is a widely used DMARD for which routine blood monitoring is required to identify hepatotoxicity and bone marrow suppression. Despite routine monitoring and normal liver function tests some patients develop hepatic fibrosis. Transient elastography (fibroscan) offers an excellent non-invasive approach to measure the stiffness of the liver as a form of assessing hepatic fibrosis and would prove extremely useful to monitor the effects of MTX in our patient groups. Methods A retrospective study was conducted between January 2021 and August 2022. Inflammatory arthritis patients who have been on MTX for more than five years were selected for the study. Their fibroscan results were obtained and their median value (kilopascals), if they had an ultrasound in the past showing cirrhosis, metabolic risk factors such as obesity (BMI&amp;gt;30), presence of type 2 diabetes and alcohol consumption (&amp;gt;14 units/week) were recorded from their clinic letters. Results Of the 56 patients, there were 18 males and 38 females. 29% of the patients had a median fibroscan value of more than 7 kpa (n = 16). Of these 16 patients, 50% of the patients had a metavir score of F2 (n = 8), 25% of the patients had a metavir score of F3 (n = 4) and 25% of the patients had a metavir score of F4 (n = 4). Of the 16 patients (11 RA and 5 PSA) with high median value, 46% had a BMI &amp;gt; 30 kg/m2 (n = 7), 13% had a diagnosis of type 2 diabetes mellitus (n = 2) and none of the patients had consumed more than 14 units/week of alcohol. Of the 16 patients with high median value, 12% (n = 2) had a diagnosis of cirrhosis on ultrasound. One patient had extremely high median value of 26 had underlying ILD, right heart failure and possible cardiac cirrhosis. 1 patient had developed frank cirrhosis and the high fibroscan results were noted subsequently. For all the patients with high metavir score of F4 and above, MTX therapy was discontinued. Conclusion A significant percentage of the cohort (29%) had a high median value above 7 of which 25% of the patients had F4 fibrosis despite having normal liver function tests. The study illustrates the need for a different modality in addition to the routine blood monitoring for the patients on MTX with metabolic risk factors. Therefore, it is vital to obtain a baseline fibroscan in high-risk patient group with preexisting metabolic risk factors prior to initiating MTX therapy and monitor every 5 years thereafter. This will help the clinician to safely continue MTX in this group of patients without the fear of worsening liver fibrosis. Disclosure S. Ramalingam: None. M. Sayed: None. S. Alam: None. D. Makkuni: None.

Aptamer/proximity hybridization-based label-free and highly sensitive colorimetric detection of methotrexate via polymerization/nicking recycling amplifications

Methotrexate (MTX) is one of the commonly used therapeutic drugs for treating various tumors and autoimmune diseases. However, high dose usage of MTX may cause severe side effects and the monitoring of MTX is therefore critical. By coupling a new MTX aptamer-based proximity hybridization with polymerization/nicking reaction (PNR) recycling amplifications, we develop here a sensitive and label-free colorimetric approach for MTX detection in diluted human serums. The MTX molecules can bind and switch the conformation of aptamers in the DNA duplex probes to initiate subsequent proximity hybridization-induced PNR recycling processes for the yield of a great deal of G-quadruplexes with the assistance of two single-stranded assistant DNA sequences. Hemin subsequently combines with these G-quadruplexes to produce lots of G-quadruplex/hemin horseradish peroxidase (HRP) mimicking DNAzymes, which then catalyze intensified color transition of the substrate solution to exhibit highly magnified UV–Vis absorption for label-free and ultrasensitive detection of MTX at concentration as low as 5.66 nM in the range of 10 nM to 1 μM. High selectivity of the developed method also enables it to monitor low levels of MTX in diluted serum samples, which offers such a method enormous potentials for convenient and highly sensitive detection of other small molecule drugs for various clinical applications.

A Rapid Method for Determination of Serum Methotrexate Using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry and Its Application in Therapeutic Drug Monitoring.

Objectives Methotrexate (MTX) has anticancer therapeutic potential with multiple doses-related adverse effects and toxicities. Immunoassays for therapeutic monitoring of serum MTX have their own limitations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered as the reference method; however, commercially availability of them is limited. We aimed to adapt/develop an in-house LC-MS/MS method for therapeutic monitoring of serum MTX. Materials and Methods Serum protein precipitation was performed using acetonitrile-water containing 250 μM solution of aminoacetophenone as internal standard (IS). Chromatographic separation was achieved on a C18 column with mobile phase of 0.1% solution of formic acid (solvent A) and acetonitrile (solvent B) at a flow rate of 0.4 mL/min. MS was performed under positive ion mode with mass transition for MTX and IS as m/z 455.1→308.1 and 136.2→94.1, respectively. The method was validated by following Bioanalytical Method Validation Guidance for Industry, 2018 and applied on leukemia patients' samples on MTX therapy. Results The correlation coefficient of eight serially diluted calibration standards of 0.09 to 12.5 μM was >0.99 and had linearity with > 95% precision and accuracy at analytical quality control levels. The lower limit of MTX quantification achieved was 0.09 μM with good intensity and sharp peak as compared with blank sample. The total run time of the assay was 5 minutes. The serum MTX levels obtained by this method in leukemia patients exhibited clinical correlation and an excellent agreement with commercial immunoassay used in parallel. Conclusion We were able to develop a rapid, sensitive, and cost-effective LC-MS/MS method suitable for therapeutic drug monitoring of MTX in routine clinical diagnostic laboratories.

Ultrasensitive therapeutic drug monitoring of methotrexate by a structure-switching aptamer with cascade primer exchange reaction.

As a folate antagonist, methotrexate (MTX) has been widely used in clinics with good effects on various tumors and inflammatory diseases. While the optimum dosage and total body clearance of MTX usually varies between individuals and even low-dose MTX has side effects, high-dose MTX may cause life-threatening side effects. Therefore, a convenient and simple method toward MTX sensing is highly demanded. Herein, we report a highly sensitive and selective method for therapeutic drug monitoring (TMD) of MTX by integrating a highly specific MTX-dependent structure-switching aptamer with a primer exchange reaction-based signal amplification technique. The detection limit is down to 1.7 nM with a linear range from 0.01 to 1 μM in buffer. More importantly, the sensing strategy can effectively detect MTX in a complex bio-environment with a linear response range from 0.05 to 2 μM and a LOD of 12.4 nM in 10% FBS and a range of 0.2 to 5 μM with a LOD of 63.73 nM in 10% whole blood. Considering the high sensitivity and selectivity and good performance in blood, the method reported herein paves a new avenue for the effective determination of MTX in clinics.

Pharmacokinetics of oral and subcutaneous methotrexate in red and white blood cells in patients with early rheumatoid arthritis: the methotrexate monitoring trial

ObjectiveTo investigate the pharmacokinetics of methotrexate polyglutamate (MTX-PG) accumulation in red blood cells (RBCs) and peripheral blood mononuclear cells (PBMCs) in patients with early rheumatoid arthritis (RA) after oral and...

Large-scale assembly and pattern transfer of SERS-active nanoparticles for application in drug monitoring of methotrexate in blood serum

Self-assembly of plasmonic gold nanoparticles (AuNPs) in periodic arrays with nanometric gaps results in the optimization of near- and far-field effects, which provides excellent surface-enhanced Raman spectroscopy (SERS) sensitivity. Herein, we report the assembly of ∼30 nm AuNPs on electron beam lithography (EBL) substrates using an inexpensive and simple method of stamping. This method yields a highly regular arrangement of AuNPs in the nanochannels of EBL substrates. An experimental approach reveals that, when using the stamping method, the groove width of the gratings plays an important role in linearly arranging the AuNPs on the EBL substrates and is a result of unidirectional flow of AuNPs due to the capillary effect. For assessment of the substrate’s SERS efficiency, rhodamine 6 G and brilliant blue were tested with a limit of detection (LOD) of 10.2 nM. To practically test the clinical-based translation of SERS substrates, detection of the chemotherapy drug methotrexate (MTX) was performed with an LOD of 13.7 nM in human blood serum to illustrate its potential in therapeutic drug monitoring (TDM) and its translation into clinics.

Safety analysis of high-dose methotrexate in pediatric non-Hodgkin lymphomas.

High-dose methotrexate (HD-MTX) with rigorous supportive care is essential to the treatment of pediatric non-Hodgkin lymphomas (NHL). We describe the safety and tolerability of HD-MTX in patients with NHL treated at our center. In our cohort of 46 patients, the majority had at least one course of delayed clearance and/or creatinine elevation. Additionally, more than one-third of patients experienced an episode of grade ≥3 mucositis. Creatinine elevations and delayed clearance were independently associated with subsequent grade ≥3 mucositis. We advocate for greater availability of methotrexate monitoring to allow dose escalation of this essential modality around the world.