Serum Methotrexate Research Articles

Synthesis of Multi-Au-Nanoparticle-Embedded Mesoporous Silica Microspheres as Self-Filtering and Reusable Substrates for SERS Detection

Surface-enhanced Raman-scattering-based (SERS-based) biosensing in biological fluids is constrained by nonspecific macromolecule adsorptions and disposable property of the SERS substrate. Here, novel multi-Au-nanoparticle-embedded mesoporous silica microspheres (AuNPs/mSiO2) were prepared using a one-pot method, which served as reliable substrates for SERS enhancement associated with salient features of self-filtering ability and reusability. The fabrication and physical characterization of AuNPs/mSiO2 microspheres were discussed, and SERS activity of this novel substrate was investigated by using 4-mercaptobenzoic acid (4-MBA) as Raman probe. The responses of our substrates to Raman intensities exhibited a SERS enhancement factor of 2.01 × 107 and high reproducibility (relative standard deviation of 6.13%). Proof-of-concept experiments were designed to evaluate the self-filtering ability of the substrates in bovine serum albumin (BSA) and human serum solution, separately. The results clearly demonstrate that mesoporous SiO2 can serve as a molecular sieve via size exclusion and avoid Raman signal interference of biomacromolecules in biological fluids. Subsequently, feasibility of practical application of AuNPs/mSiO2 microspheres was assessed by quantitative detection of methotrexate (MTA) in serum. The method exhibited good linearity between 1 and 110 nM with the correlation coefficients of 0.996, which proved that the obtained AuNPs/mSiO2 microspheres were good SERS substrates for determination of small biomolecules directly in biological fluids without need of manipulating samples. In addition, the substrate maintained its SERS response during multiple cycles, which was evaluated by recording Raman signals for 4-MBA before and after thermal annealing, thereby demonstrating the high thermostability and satisfactory reusability. These results offered the AuNPs/mSiO2 microspheres attractive advantages in their SERS biosensing.

Association between MTHFR microRNA binding site polymorphisms and methotrexate concentrations in Chinese pediatric patients with acute lymphoblastic leukemia.

The pharmacokinetics and therapeutic response to methotrexate (MTX) display large variability in the treatment of acute lymphoblastic leukemia (ALL). The aim of the present study was to investigate the association of two microRNA (miRNA) binding site polymorphisms (rs3737966 G>A and rs35134728 DEL/TTC) in the 3'-untranslated region of MTHFR with serum MTX concentrations, in a Chinese pediatric population with ALL. Genotyping for MTHFR rs3737966 and rs35134728 in 144 children with ALL was performed using the Sequenom MassArray system (Sequenom, San Diego, CA, USA). Serum MTX concentrations were measured by a fluorescence polarization immunoassay 24h (C24h ) and 42h (C42h ) after administration. The effects of the polymorphisms on concentration-to-dose (C/D) ratios of MTX were assessed. Complete linkage disequilibrium between rs3737966 and rs35134728 polymorphisms (r2 =1) was found in the study population. The minor allele frequency observed in the present study (17.4%) was significantly lower than those in European and African samples reported in the 1000 Genomes Project (42.9% and 63.9%, respectively; p<0.01). The C/D ratios of MTX at 24 and 42h for the TTC/TTC-A/A haplotype carriers (11.74 and 0.07μmol/l per g/m2 , respectively) were significantly lower than those in DEL/DEL-G/G or DEL/TTC-G/G haplotype carriers (12.49 and 0.09μmol/l per g/m2 , respectively; p<0.05). Computational predictions suggested that the two polymorphisms overlapped with putative binding sites of several miRNAs. The rs3737966 and rs35134728 polymorphisms in MTHFR were associated with serum MTX concentrations. The findings of the present study indicate that miRNAs might be involved in the post-transcriptional regulation of MTHFR.

Determination of methotrexate in spiked human blood serum using multi-frequency electrochemical immittance spectroscopy and multivariate data analysis

This article describes an attempt to develop a sensor based on multi-frequency immittance spectroscopy for the determination of methotrexate (MTX) in blood serum using gold electrodes modified with antibodies. The attachment of antibodies was monitored with electrochemical immittance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS). The EIS measurements of MTX resulted in a data matrix of size 39 × 55. The data were analysed using multivariate data analysis and showed a concentration dependence and time dependence that could be separated. This allowed the calculation of a multivariate calibration model. The model showed good linear behavior on a logarithmic scale offering a detection limit of 5 × 10−12 mol L−1.

Biomarkers of brain injury and neurologic outcomes in children treated with chemotherapy for acute lymphoblastic leukemia (ALL).

10521 Background: Little is known about neurotoxic mechanisms associated with chemotherapy in children with ALL. Cerebrospinal fluid (CSF) biomarkers of brain injury may provide insight into this process. Methods: 235 patients (51% male; mean [SD] age diagnosed 6.8 [4.7] years) treated on a chemotherapy only protocol provided CSF samples following diagnosis and through consolidation. CSF was assayed for biomarkers of myelin degradation (myelin basic protein [MBP]), neuronal damage (nerve growth factor [NGF], total-Tau [T-Tau]) and astrogliosis (glial fibrillary acidic protein [GFAP]). Leukoencephalopathy was evaluated by brain MRI’s during therapy. At ≥5 years post-diagnosis, 138 (70%) of the 198 still eligible survivors (without relapse and unrelated neurologic injury) completed neurocognitive testing and brain diffusion tensor imaging of white matter integrity at age 13.6 [4.6] years. Log-binomial and general linear models were used to examine whether biomarker changes from baseline through consolidation were related to serum methotrexate exposure, acute leukoencephalopathy, and long-term brain outcomes. Results: NGF and T-Tau increased from baseline to consolidation ( P's &lt; 0.001), while MBP and GFAP were elevated at baseline and remained so through consolidation. The number of intrathecal injections (methotrexate, hydrocortisone, cytarabine) was positively correlated with NGF increase at consolidation ( P= 0.005). Increases in GFAP (RR 1.2; 95% CI [1.0 – 1.4]), MBP (RR 1.1 [1.0 – 1.1]) and T-Tau (RR 1.8 [1.1 – 2.8]) were related to higher risk for acute leukoencephalopathy, and higher diffusivity in frontal lobe white matter at ≥5 years post-diagnosis ( P’s &lt; 0.05). Increase in T-Tau at consolidation was associated with worse long-term sustained attention ( P= 0.03), and visual- ( P= 0.04) and visual-motor ( P= 0.02) processing speed. Conclusions: Glial injury, which is evident at diagnosis, may be related to leukemia and methotrexate exposure. Neuronal injury is associated with intrathecal chemotherapy and long-term neurocognitive and brain imaging outcomes. Monitoring CSF biomarkers may be useful in identifying individuals at risk for poor neurological outcomes.

Sensitive surface enhanced Raman spectroscopy (SERS) detection of methotrexate by core-shell-satellite magnetic microspheres

An effective approach for fabricating core-shell-satellite magnetic microspheres was developed to form a sub-10nm positively charged mesoporous silica (mSiO2) interlayer, which was applied to enrich samples and create sufficient hotspots between the inner Fe3O4@Ag core and outer assembled gold nanoparticles (AuNPs) satellites. Based on the prepared Fe3O4@Ag/mSiO2/AuNPs (FASA) as SERS substrates, a sensitive and label-free detection of methotrexate in serum was successfully realized. The positively charged mSiO2 interlayer exhibited strong capability in enriching methotrexate through the electrostatic interaction. The intraparticle plasmonic coupling between the inner Ag shell and the outside AuNPs satellites, and magnetism-induced aggregation greatly enhanced the SERS activity of methotrexate. The diluted serum containing different concentrations of methotrexate was employed as real samples and the minimum detected concentration of methotrexate was as low as 1nM with a linear response range from 1 to 200nM. This sensitive and label-free SERS detection of methotrexate based on FASA offers great potential for practical applications in medicine and biotechnology.

Neurotoxicity after high-dose methotrexate (MTX) is adequately explained by insufficient folinic acid rescue.

To challenge the view that the dose of folinic acid rescue after high-dose methotrexate (MTX) has no significance in the prevention of neurotoxicity and to present the minority view that neurotoxicity can be prevented by an adequate dose of folinic acid, without compromising treatment results. Several fallacies that led to the misunderstanding of post MTX neurotoxicity are presented. Data mining using search engines was used to find relevant publications, and an e-mail survey of more than 60 authors of articles in this field was performed. All relevant articles identified were read in their entirety. Examples of clinical studies with neurotoxicity following inadequate rescue are given. Some studies demonstrated no neurotoxicity when adequate doses of folinic acid rescue were started 24-36 h after the start of HDMTX rescue even after mega doses of MTX. Rescue started after 42 h was associated with neurotoxicity except in patients with low serum MTX levels after 24 and 36 h. ALL protocols with neurotoxicity, especially BFM-like protocols, are presented. Protocol is reported in which single protocol changes prevented neurotoxicity. From the published data, when folinic acid rescue is given in a sufficiently high enough dose and is started 24-36 h after the beginning of the methotrexate exposure, and virtually all forms of post MTX neurotoxicity can be prevented without compromising therapeutic results.

Methotrexate elimination and toxicity: MTHFR 677C>T polymorphism in patients with primary CNS lymphoma treated with high-dose methotrexate.

The genetic association of the methylenetetrahydrofolate reductase gene (MTHFR) 677C>T polymorphism with methotrexate (MTX)-associated toxicity has been evaluated and conflicting results have been reported. The substantial heterogeneity of the studied population was suggested to be a possible explanation because ethnicity, MTX dose, coadministered chemotherapeutic agents, and folinate rescue dosage regimen could alter the MTX toxicity profile. The patient population was homogenized by limiting the cancer type to primary central nervous system lymphoma and chemotherapy protocol to a high-dose MTX monotherapy regimen. A total of 111 patients with 402 chemotherapy courses were analyzed. MTHFR 677C>T polymorphism was identified as an independent predictive marker for MTX-associated hematologic toxicity (odds ratio, 2.60; 95% confidence interval, 1.32-5.09; P=.0055). Clinically significant nephrotoxicity occurred in patients without delayed elimination, suggesting roles for factors other than serum MTX levels. MTX-induced hepatotoxicity and oral mucositis occurred independently of plasma MTX levels.

Factors Affecting the Upper Limit of the Methotrexate (MTX) CSF Levels Achievable in Children With Brain Tumors Treated With High-dose Intravenous MTX.

Little has been published in the medical literature on serum and cerebrospinal fluid (CSF) methotrexate (MTX) levels in children with brain tumors. Matched 24-hour serum and CSF MTX levels were studied after 113 treatments in 35 brain tumors patients. A correlation between the 24-hour serum levels of MTX and MTX dosage was observed after 113 treatments in all 35 patients (r=0.39, P<0.001) but no statistical difference was found between CSF MTX levels in the irradiated and nonirradiated groups (P=0.12). Nonirradiated children received a lower dose of MTX (12.3±4.8 cf 14.8±3.7) (P=0.002). The 24-hour MTX CSF levels of these 2 groups were also found to be different (the nonirradiated group 7.6±9.8 cf 12.5±0.15.3). Using the Levene test for variances we found that these variances were not equal and therefore we used the Welch test which resulted in a P-value of 0.04. However, when an analysis of covariance was performed looking at evidence of CSF disease and MTX dose the radiation difference was no longer significant (P=0.15). The 24-hour CSF MTX levels in children without evidence of active CSF disease were consistently lower than those with active disease using a mixed-model analysis (P=0.002). Although a 24-hour CSF MTX level of at least 1 μM was observed after infusions of >5 g/m MTX in previously irradiated children and after infusion of ≥10 g/m in nonirradiated children this difference did not reach statistical significance. CSF MTX levels plateau at doses of MTX 15 g/m putting in doubt the value of administering even higher doses of MTX. The 24-hour MTX CSF levels are higher in patients with active CSF disease. Doses of <10 gm/m in children with brain tumors may not achieve a guaranteed 24-hour MTX CSF level of 1 μM. There may be little value in a given dose of >15 g/m MTX as CSF levels plateau at this dose.

Hepatoxicity in a Patient with Acute Lymphoblastic Leukemia Receiving High-Dose Methotrexate

Introduction: Methotrexate (MTX) is a folic acid analogue that inhibits purine and pyrimidine synthesis. The dosing regimen varies by indication; high, short term doses are used in the treatment of cancer and chronic, lower doses for autoimmune conditions. The pattern of liver toxicity is dose dependent. We present a case of high dose methotrexate induced acute liver injury. Case: A 31 year old female with a history of pre-B cell acute lymphocytic leukemia (ALL), and treated with one cycle of hyper-CVAD (Cyclophosphamide, Vincristine, Doxorubicin and Dexamethasone) was admitted for part B of her chemotherapy (MTX and cytarabine). Upon admission, she received 1000 mg/m2 of intravenous MTX. Leucovorin was administered and urine alkalinization performed per protocol. However, the patient's serum MTX levels remained elevated up to 96 hours after administration. Two days after MTX treatment her liver function tests (LFTs) became elevated from previously normal levels, with ALT 1608, AST 752, Alkaline Phosphatase 178, and Total Bilirubin 2.8. The LFTs peaked five days after the initial dose. The patient did not have encephalopathy, coagulopathy or jaundice. After other etiologies of acute liver injury were ruled out, the elevation of LFTs was determined to be related to MTX. LFTs began down trending 6 days after MTX dose. Approximately one month after administration of MTX the patient's LFTs normalized. Discussion: Methotrexate is an important part of ALL chemotherapy. With high dose intravenous MTX, serum ALT levels can rise to 10 to 20 times the upper limit of normal within 12 to 48 hours, but levels then rapidly return to normal with only rare instances of clinical liver injury. Gene polymorphisms play important role in MTX toxicity in ALL patients. It is important to assess for underlying liver disease in patients with excessive alcohol use, chronic viral hepatitis, elevations in serum enzymes, and in selected patients with diabetes and obesity before MTX therapy. Leucovorin is given in conjunction with high dose MTX to prevent death of non-malignant cells. However, this has not been proven to prevent hepatoxicity. Treatment of hepatotoxicity caused by MTX includes cessation of the drug and observation of LFTs. If LFTs do not normalize then liver biopsy is indicated to determine extent of damage. Liver injury can be minimized with MTX therapy by avoiding alcohol and hepatotoxic drugs, supplementing folic acid and closely observing LFTs.

Development of an Assay for Methotrexate and Its Metabolites 7-Hydroxy Methotrexate and DAMPA in Serum by LC-MS/MS.

Methotrexate (MTX) is a folic acid antagonist that is widely used as an immunosuppressant and chemotherapeutic agent. After high-dose administration of MTX serum levels must be monitored to determine when to administer leucovorin, a folic acid analog that bypasses the enzyme inhibition caused by MTX and reverses its toxicity. We describe a rapid and simple turbulent flow liquid chromatography (TFLC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of MTX, 7-hydroxymethotrexate (7-OH MTX), and 4-amino-4-deoxy-N(10)-methylpteroic acid (DAMPA) concentrations in serum. MTX is isolated from serum samples (100 μL) after protein precipitation with a methanolic solution containing internal standard (MTX-D3) followed by centrifugation. The supernatant is injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFLC-ESI-MS/MS) and quantified using a six-point calibration curve. For MTX, 7-OH MTX, and DAMPA the assays were linear from 20 to 1000 nmol/L. Dilutions of 10-, 100-, and 1000-fold were validated giving a clinically reportable range of 20 to 1.0 × 10(6) nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10 % for all three analytes.

Monitoring and Treatment of Acute Kidney Injury in Children with Acute Lymphoblastic Leukemia After High Dose Methotrexate Chemotherapy.

To investigate acute kidney injury (AKI) in children with acute lymphoblastic leukemia (ALL) who received high dose methotrexate (MTX) chemotherapy and explore the corresponding treatment. Methods 180 children who received high dose MTX chemotherapy were observed with serum MTX concentration and serum creatinine. Patients with AKI of stage 3 or poor response to conventional treatment were performed on hemodialysis and assessed the treatment outcome. Results 9 patients (5%) have appeared AKI, including 7 cases of AKI of stage 3. However, there were not any significant correlation between age, gender, serum MTX concentration and AKI, respectively. Compared with normal serum MTX concentration, the patients with high serum MTX concentration easily were developed to AKI, the MTX and serum creatinine concentration had been significantly decreased in 9 patients after hemodialysis. Conclusion AKI has appeared in some children with ALL who receive high dose MTX chemotherapy, and this may due to increase of serum MTX concentration. The monitoring of serum MTX concentration and AKI index could help to find out AKI, and even to prevent the occurrence of it. Furthermore, once AKI is present, those patients with AKI stage 3 or poor response to conventional treatment should be performed on hemodialysis treatment.

PTCT-05CHEMOTHERAPY ADMINISTRATION DIRECTLY INTO THE FOURTH VENTRICLE IN CHILDREN WITH MALIGNANT FOURTH VENTRICULAR BRAIN TUMORS: A PILOT CLINICAL TRIAL

BACKGROUND: We hypothesize that chemotherapy can be safely and repeatedly administered directly into the fourth ventricle to treat recurrent malignant brain tumors in children. METHODS: For the first time in humans, methotrexate was infused into the fourth ventricle in children with recurrent, malignant brain tumors. A catheter was surgically placed into the fourth ventricle under direct visualization and attached to a ventricular access device. Cerebrospinal fluid (CSF) flow was confirmed by CINE MRI postoperatively. Each cycle consisted of 4 consecutive daily methotrexate infusions (2 milligrams). Disease response was monitored with serial MRI scans and CSF cytologic analysis. Trough CSF methotrexate levels were sampled. RESULTS: Five patients (3 with medulloblastoma and 2 with ependymoma) received 18, 18, 12, 9, and 3 cycles, respectively. There were no serious adverse events or new neurological deficits attributed to methotrexate. Two additional enrolled patients were withdrawn prior to planned infusions due to rapid disease progression. Median serum methotrexate level four hours after infusion was 0.04 micromoles per liter (µmol/L). Range was 0.02–0.13 µmol/L. Median trough CSF methotrexate level 24 hours after infusion was 3.18 µmol/L (range 0.53 - 212.36 µmol/L). All three patients with medulloblastoma had partial response or stable disease until one patient had progressive disease after cycle 18. Both patients with ependymoma had progressive disease after 9 and 3 cycles, respectively. CONCLUSIONS: These data establish the safety of neurosurgical implantation of a catheter into the fourth ventricle and direct infusion of low-dose methotrexate into the fourth ventricle. Some patients with recurrent medulloblastoma experience a beneficial anti-tumor effect both within the fourth ventricle and at distant sites. These results support future clinical trials of direct infusion of chemotherapy and novel agents into the fourth ventricle for children with malignant posterior fossa tumors.

Development and validation of a turbulent flow chromatography and tandem mass spectrometry method for the quantitation of methotrexate and its metabolites 7-hydroxy methotrexate and DAMPA in serum.

A rapid and simple turbulent flow liquid chromatography (TFC-LC) method implementing positive heated electrospray ionization (HESI) for the accurate and precise determination of methotrexate (MTX), 7-hydroxy methotrexate (7-OH MTX), and 4-amino-4-deoxy-N(10)-methylpteroic acid (DAMPA) concentrations in serum was developed. MTX was isolated from serum samples (100μL) after protein precipitation with methanol containing formic acid and internal standard (MTX-D3) followed by centrifugation. The supernatant was injected into the turbulent flow liquid chromatography which is followed by electrospray positive ionization tandem mass spectrometry (TFC-LC-MS/MS) and quantified using a six-point calibration curve. For MTX and DAMPA the assays were linear from 10 to 1000nmol/L and for 7-OH MTX from 20 to 2000nmol/L. Dilutions of 10, 100 and 1000-fold were validated giving a clinically reportable range of 10nmol/L to 5×10(5)nmol/L. Within-day and between-day precisions at concentrations spanning the analytical measurement ranges were less than 10% for all three analytes. MTX, DAMPA and 7-OH MTX were sufficiently stable under all relevant analytical conditions. No significant matrix effect was observed during the method validation. The TFC-LC-MS/MS MTX method was also compared with three other clinically validated MTX assays: a dihydrofolate reductase (DHFR) inhibition assay, an immunoassay based on fluorescence polarization and a previously developed LC-MS/MS assay.

A reversed-phase high performance liquid chromatography method for quantification of methotrexate in cancer patients serum.

A simple, rapid and sensitive reversed-phase high performance liquid chromatography (HPLC) method has been developed for the determination of methotrexate in human serum. After deproteinization of the serum with 40% silver nitrate solution, methotrexate and internal standard (IS) were separated on a reversed-phase column with a mobile phase consisting of 10mM sodium phosphate buffer (pH6.40)-methanol (78:22%, v/v) and ultraviolet detection at 310nm. The linearity is evaluated by a calibration curve in the concentration range of 0.05-10.0μg/mL and presented a correlation coefficient of 0.9995. The absolute recoveries were 97.52±3.9% and 96.87±3.7% for methotrexate and ferulic acid (internal standard), respectively. The intra- and inter-day precision were less 6.19 and 5.89%, respectively (n=6). The limit of quantitation was 0.02μg/mL and the limit of detection was 0.006μg/mL. The complete analysis was achieved less than 10min with no interference from endogenous components or 22 examined drugs. This method was validated by using serum samples from high-dose methotrexate treated patients with osteosarcoma, breast cancer, acute leukemia and lymphoma. The method was demonstrated to be a simple, rapid and reliable approach in quantification of methotrexate in serum samples from patients with high-dose methotrexate therapy.

Methotrexate administration directly into the fourth ventricle in children with malignant fourth ventricular brain tumors: a pilot clinical trial.

We hypothesize that chemotherapy can be safely administered directly into the fourth ventricle to treat recurrent malignant brain tumors in children. For the first time in humans, methotrexate was infused into the fourth ventricle in children with recurrent, malignant brain tumors. A catheter was surgically placed into the fourth ventricle and attached to a ventricular access device. Cerebrospinal fluid (CSF) flow was confirmed by CINE MRI postoperatively. Each cycle consisted of 4 consecutive daily methotrexate infusions (2 milligrams). Disease response was monitored with serial MRI scans and CSF cytologic analysis. Trough CSF methotrexate levels were sampled. Five patients (3 with medulloblastoma and 2 with ependymoma) received 18, 18, 12, 9, and 3 cycles, respectively. There were no serious adverse events or new neurological deficits attributed to methotrexate. Two additional enrolled patients were withdrawn prior to planned infusions due to rapid disease progression. Median serum methotrexate level 4 h after infusion was 0.04 µmol/L. Range was 0.02–0.13 µmol/L. Median trough CSF methotrexate level 24 h after infusion was 3.18 µmol/L (range 0.53–212.36 µmol/L). All three patients with medulloblastoma had partial response or stable disease until one patient had progressive disease after cycle 18. Both patients with ependymoma had progressive disease after 9 and 3 cycles, respectively. Low-dose methotrexate can be infused into the fourth ventricle without causing neurological toxicity. Some patients with recurrent medulloblastoma experience a beneficial anti-tumor effect both within the fourth ventricle and at distant sites.

Methotrexate Associated Renal Impairment Is Related to Delayed Elimination of High-Dose Methotrexate.

Although Methotrexate (MTX) is an effective drug for the treatment of acute lymphoblastic leukemia (ALL), the toxicity remains a significant problem. In this prospective study, fifty-four patients with ALL were enrolled. 3 g or 5 g MTX/m2 was administered over 24 hours. Serum MTX concentrations were determined in 24, 48, and 96 hours after MTX infusion. Serum creatinine concentrations and creatinine clearance rate (CCR) were determined before and 24 and 48 hours after MTX infusion. A total of 173 courses of MTX infusion were administered. The serum creatinine concentrations did not change much after MTX infusion while the CCR was gradually decreased. MTX clearance status was independently related to CCR decrease, with the risk of 8.07 to develop renal impairment in patients with delayed MTX elimination. Serum creatinine concentration, serum creatinine ratio, CCR, and CCR ratio at 24 hours were all related to MTX elimination delay. Patients with serum creatinine level >35.0 μmol/L, creatinine ratio >1.129, or CCR <100.0 mL/min were more likely to undergo MTX elimination delay. In conclusion, MTX could induce transient renal impairment and compromised renal function will delay MTX clearance. The serum creatinine concentration and the ratio and CCR are useful tools for evaluating MTX elimination status.

Correlation of serum methotrexate levels with drug induced side effects

Often employed in the treatment of malignancies with high cellular proliferation rates, including acute lymphoblastic leukemia, choriocarcinoma, trophoblastic tumours, and carcinomas of the breast, tongue, pharynx, and testis; and certain nonmalignant conditions like rheumatoid arthritis, psoriasis, asthma, sarcoidosis, and transplantation therapy, methotrexate is an antifolate agent. It works by inhibiting the enzyme dihydrofolate reductase. The molecule inhibits DNA synthesis by depletion of nucleotide precursors and is associated with a number of diverse side effects. The present series comprises of 4 patients: Each demonstrated significant drug-induced side effects even though their serum methotrexate levels were found to be normal.

Influence of the folate pathway and transporter polymorphisms on methotrexate treatment outcome in osteosarcoma.

Osteosarcoma is the most common primary bone malignancy that occurs mostly in adolescents. Treatment protocols usually include multiagent preoperative and postoperative chemotherapy based on methotrexate, cisplatin, doxorubicin and ifosfamide. Despite a favourable prognosis, there are considerable interindividual differences in treatment outcome. Genetic variability of enzymes involved in the metabolism and transport of methotrexate could contribute towards observed differences in response to chemotherapy. Our aim was to evaluate how polymorphisms in the folate pathway and transporter genes influence treatment outcome in osteosarcoma patients. In total, 44 osteosarcoma patients treated with methotrexate were genotyped for eleven polymorphisms in four folate pathway and five folate transporter genes. Cox regression was used in survival analysis. Logistic regression was used to assess the influence of polymorphisms on treatment efficacy and toxicity and nonparametric tests were used to determine the influence on serum methotrexate levels. Polymorphic SLCO1B1 rs4149056 and rs11045879 alleles were associated with significantly higher serum methotrexate area under the curve (P=0.001 and 0.011, respectively). Carriers of at least one polymorphic SLCO1B1 rs4149056 and rs11045879 allele tended to have longer event-free survival compared with patients with two wild-type alleles [P=0.040, hazard ratio (HR)=0.26, 95% confidence interval (CI)=0.07-0.94; and P=0.034, HR=0.20, 95% CI=0.05-0.89, respectively]. Compared with the most common haplotype, carriers of both polymorphic alleles had significantly longer event-free survival (P=0.009, HR=0.27, 95% CI=0.10-0.72). We have shown that SLCO1B1 polymorphisms influence methotrexate disposition and survival in methotrexate-treated osteosarcoma patients and therefore might serve as pharmacogenetic markers of treatment outcome.

Abstract 3678: An immunoassay for measurement of methotrexate on ARCHITECT i system

Abstract Methotrexate (MTX) is a cancer therapeutic drug for leukemia, osteosarcoma, non-Hodgkin's lymphoma and others. MTX levels in blood are monitored in patients to avoid toxic effects caused by the excessive dosage of this drug, and to determine when to intervene with the counter-acting ‘rescue’ therapy. An assay for serum and plasma MTX measurement on ARCHITECT i System (ARCHITECT Methotrexate) is being developed and the analytical feasibility of this assay is presented. METHODS: The ARCHITECT Methotrexate is a competitive immunoassay. First, the instrument mixes and incubates a sample (calibrators, controls, sera or plasma) with anti-methotrexate antibody-coated magnetic microparticles, biotinylated anti-methotrexate antibody and acridinium-conjugated MTX. Then, the instrument washes the microparticles and adds pre-trigger and trigger solutions to initiate the chemiluminescence reaction. Signals obtained as relative luminescent units are inversely proportional to the amount of MTX in the sample. RESULTS: The limit of quantitation was ≤ 0.040 µmol/L MTX. The 20-day imprecision study showed a total CV ≤ 7.1% within the range of 0.056 to 1705.433 µmol/L MTX with 6 controls and 5 panels on 4 instruments using 3 lots of reagents (n = 80). Dilution linearity showed the deviations from linearity were -4.1% to 7.2% within the range of 1.881 to 0.012 µmol/L MTX. Spike recovery revealed a mean recovery of 92% - 94% for samples at 0.045, 0.909 and 9.090 µmol/L of MTX. In the interference studies, the average levels of MTX in the individual interfering substance-spiked samples (10 endogenous substances including human anti-mouse antibody and rheumatoid factor, and 21 therapeutic drugs) were within 90% - 110% of that in the unspiked control samples. Method comparison of ARCHITECT Methotrexate to TDx/TDxFLx Methotrexate II (TDx) generated a Passing Bablok correlation as [ARCHITECT] = 0.00 + 1.01 [TDx] for samples within the range of 0.040 to 1.415 µmol/L (n = 92) and [ARCHITECT] = 0.00 + 1.04 [TDx] for samples within the range of 0.040 to 1624.760 µmol/L MTX , n = 142). Specimen storage study denoted that specimens could be stored at room temperature for 24 hours or at 2-8oC for 48 hours since the MTX values of the stored specimens described above were within 10% deviation from that of the baseline control specimens. Specimen Tube type study demonstrated that specimens collected in K2-EDTA, sodium heparin, and lithium heparin tubes was within ± 10% deviation from that collected in serum tubes across the range of 0.040 to 10.00 μmol/L MTX. Reagent on-board stability showed that the ARCHITECT Methotrexate reagents could remain on the analyzer for a minimum of 30 days with no more than 10% shift from baseline. CONCLUSION: The ARCHITECT Methotrexate under development was demonstrated to be an accurate, precise, sensitive and robust assay for the measurement of Methotrexate in human serum and plasma. Citation Format: Robert J. Smalley, Raymond E. Picard, Beth A. Burkhart, Robert L. Frescatore, Curtis L. Glover, Lisa C. Zhu, Elizabeth A. Roessner, Karin MajnesjÖ, Anders Öhrvik, Kuanglin He, Zhong Qian Li, Timothy R. Kettlety. An immunoassay for measurement of methotrexate on ARCHITECT i system. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3678. doi:10.1158/1538-7445.AM2014-3678

Facile synthesis of N, S-codoped fluorescent carbon nanodots for fluorescent resonance energy transfer recognition of methotrexate with high sensitivity and selectivity

In this report, N, S-codoped fluorescent carbon nanodots (NSCDs) were prepared by a facile, simple, low-cost, and green thermal treatment of ammonium persulfate, glucose, and ethylenediamine. The as-prepared NSCDs displayed bright blue emission with a relatively high fluorescent quantum yield of 21.6%, good water solubility, uniform morphology, and excellent chemical stability, compared to pure CDs. The fluorescence of NSCDs can be significantly quenched by methotrexate (MTX) via fluorescence resonance energy transfer (FRET) between NSCDs and MTX, which was used for highly selective and sensitive detection of MTX with a wide linear range up to 50.0μM and a low detection limit of 0.33nM (S/N=3). Moreover, this method was explored for practical detection of MTX in human serum with satisfied results.