Intracellular Methotrexate Research Articles

Methotrexate accumulation in target intestinal mucosa and white blood cells differs from non-target red blood cells of patients with Crohn's disease.

Intracellular methotrexate polyglutamates (MTX-PGs) concentrations are measurable in red blood cells (RBCs) during MTX treatment. MTX-PG3 concentrations correlate with efficacy in patients with Crohn's disease (CD). Since RBCs are not involved in pathogenesis of CD and lack extended MTX metabolism, we determined MTX-PGs accumulation in peripheral blood mononuclear cells (PBMCs: effector cells) and intestinal mucosa (target cells) and compared those with RBCs as a potential more precise biomarker. In a multicentre prospective cohort study, blood samples of patients with CD were collected during the first year of MTX therapy. Mucosal biopsies were obtained from non-inflamed rectum and/or inflamed intestine. MTX-PGs concentrations in mucosa, PBMCs and RBCs were measured by liquid chromatography-tandem mass spectrometry. From 80 patients with CD, a total of 27 mucosal biopsies, 9 PBMC and 212 RBC samples were collected. From 12 weeks of MTX therapy onwards, MTX-PG3 was the most predominant species (33%) in RBCs. In PBMCs, the distribution was skewed towards MTX-PG1 (48%), which accounted for an 18 times higher concentration than in RBCs. Long-chain MTX-PGs were highly present in mucosa: 21% of MTX-PGtotal was MTX-PG5. MTX-PG6 was measurable in all biopsies. MTX-PG patterns differ between mucosa, PBMCs and RBCs of patients with CD.

Contribution of aldehyde oxidase to methotrexate-induced hepatotoxicity: in vitro and pharmacoepidemiological approaches

ABSTRACT Background Methotrexate (MTX) is partially metabolized by aldehyde oxidase (AOX) in the liver and its clinical impact remains unclear. In this study, we aimed to demonstrate how AOX contributes to MTX-induced hepatotoxicity in vitro and clarify the relationship between concomitant AOX inhibitor use and MTX-associated liver injury development using the U.S. Food and Drug Administration Adverse Event Reporting System (FAERS). Methods We assessed intracellular MTX accumulation and cytotoxicity using HepG2 cells. We used the FAERS database to detect reporting odds ratio (ROR)-based MTX-related hepatotoxicity event signals. Results AOX inhibition by AOX inhibitor raloxifene and siRNA increased the MTX accumulation in HepG2 cells and enhanced the MTX-induced cell viability reduction. In the FAERS analysis, the ROR for MTX-related hepatotoxicity increased with non-overlap of 95% confidence interval when co-administered with drugs with higher Imax, u (maximum unbound plasma concentration)/IC50 (half-maximal inhibitory concentration for inhibition of AOX) calculated based on reported pharmacokinetic data. Conclusion AOX inhibition contributed to MTX accumulation in the liver, resulting in increased hepatotoxicity. Our study raises concerns regarding MTX-related hepatotoxicity when co-administered with drugs that possibly inhibit AOX activity at clinical concentrations.

MicroRNA-411-3p motivates methotrexate's cellular uptake and cytotoxicity via targeting Yin-yang 1 in leukemia cells.

This study aimed to figure out how microRNA (miR)-411-3p's impacts on methotrexate (MTX)'s cellular uptake and cytotoxicity in acute lymphoblastic leukaemia (ALL) CEM-C1 cells by targeting Yin-yang 1 (YY1). miR-411-3p and YY1 were detected by RT-qPCR or Western blot. Intracellular MTX concentration was measured by enzyme-linked immunosorbent assay. Cell viability and apoptosis were evaluated by CCK-8, clonal formation assay, and flow cytometry. Verification of miR-411-3p and YY1's targeting link was manifested. It came out that miR-411-3p mimic or si-YY1 elevated intracellular MTX, MTX-induced cytotoxicity and apoptosis rate in CEM-C1. However, the inverse results were noticed in cells introduced with miR-411-3p inhibitor or oe-YY1. Meanwhile, it was found that cell relative luciferase activity was reduced after co-transfection of miR-411-3p mimic with YY1-WT, indicating that miR-411-3p targeted YY1. Elevation of YY1 could turn around elevating miR-411-3p's impacts on MTX's cellular uptake and cytotoxicity in CEM-C1 cells. These findings convey that miR-411-3p motivated MTX's cellular uptake and cytotoxic impacts via targeting YY1 in leukemia cells. This study is helpful for learning about the mechanisms underlying MTX responses in ALL patients.

Quantitation of methotrexate polyglutamates in human whole blood, erythrocytes and leukocytes collected via venepuncture and volumetric absorptive micro-sampling: a green LC–MS/MS-based method

Low-dose methotrexate (MTX) plays a key role in treatment of rheumatoid arthritis. However, not all patients respond satisfactorily, and no therapeutic drug monitoring has been implemented in clinical practice, despite the fact that MTX therapy has now been available for decades. Analysis of individual intracellular MTX metabolites among rheumatoid arthritis (RA) patients is hampered by the low intracellular concentrations of MTX-PGs which require a highly sensitive method to quantify. Here, we present a rapid and highly sensitive LC (HILIC) MS/MS method with LLOQ 0.1 nM, 0.8 nmol/L for each metabolite of MTX-PG1-5 and MTX-PG6-7 respectively. Over a linear range of 0.1–100 nM, 0.8–100 nmol/L for each metabolite of MTX-PG1-5 and MTX-PG6-7, respectively, the inter- and intra- accuracy and precision were within 15% of the nominal value for all MTX metabolites. The presented assay was used to assess and compare MTX metabolite concentrations extracted from four different matrices: red blood cells, plasma, peripheral blood mononuclear cells, and whole blood that have been collected either using traditional venepuncture or volumetric absorptive micro-sampling (VAMS) sampling techniques. The presented method not only improves analyte coverage and sensitivity as compared to other published methods; it also improves the greenness.Graphical abstract

OA26 Erythrocyte mean corpuscular volume as a surrogate marker for methotrexate polyglutamation during early treatment in rheumatoid arthritis

Abstract Background/Aims The long-term outcomes for patients with rheumatoid arthritis (RA) depend on early and effective disease control. Methotrexate remains the key first-line disease-modifying therapy for the majority of patients. The gradual onset of action of methotrexate has been proposed to be linked to the build-up of intracellular stores of the drug, achieved through the process of polyglutamation. This is a complex and expensive assay. Recent work has shown an association between the increase in mean corpuscular volume (MCV) of erythrocytes and improvements in disease activity. This study seeks to relate intracellular methotrexate and 5-methyl-tetrahydrofolate polyglutamate concentrations with changes in MCV and their association with early ‘steroid free’ remission in RA patients. Methods 68 drug-naïve, newly diagnosed RA patients were recruited form the Newcastle Early Arthritis Clinic (NEAC). These patients were followed up for 6 months with serial blood sampling and quantification of the erythrocyte methotrexate and 5-methyl-tetrahydrofolate polyglutamate levels by liquid chromatography-mass spectrometry (LC-MS). This was then related to the contemporaneous erythrocyte MCV values from the clinical monitoring bloods on those occasions and the treatment outcome at 6 months. The outcome measure used was 4 component DAS28CRP remission (<2.4) with only the baseline intramuscular steroid permitted for the definition of remission. Results The majority of patients displayed a gradual increase in MCV over the first 6 months of treatment (MCV change from patient baseline used to normalise the data). When grouped by the ‘steroid free’ remission outcome, there was a divergence in MCV change with greater MCV increases observed in those achieving remission from 4 months of treatment onward (p < 0.05). In the same patient group, the accumulation of longer chain methotrexate polyglutamates (chain length 3 to 5) was observed to follow a similar trend to MCV with higher polyglutamate concentrations in those achieving the remission outcome from 2 months of treatment (P < 0.05). When the contemporaneous MCV and MTX polyglutamate samples were compared, an association was observed between the longer chain MTX polyglutamates and the change in MCV values (R2 = 0.39, p < 0.001), but not between intracellular 5-methyl-tetrahydrofolate and MCV (R2 = 0.01, p = NS). Conclusion Change in MCV shows an association with early remission induction with MTX therapy in treatment-naïve RA patients. This change in MCV appears to be associated with the accumulation of intracellular MTX metabolites, rather than via depletion of intracellular folates. MCV may therefore represent a tractable proxy measure of methotrexate polyglutamation for application in larger studies or for the purpose of drug monitoring. Disclosure P.M. Brown: None. A.G. Pratt: None. A.E. Anderson: None. A.W. Morgan: None. J.D. Isaacs: None.

In Vitro and In Vivo Efficacy of a Novel Glucose-Methotrexate Conjugate in Targeted Cancer Treatment.

Methotrexate (MTX) is a commonly used antimetabolite, which inhibits folate and DNA synthesis to be effective in the treatment of various malignancies. However, MTX therapy is hindered by the lack of target tumor selectivity. We have designed, synthesized and evaluated a novel glucose–methotrexate conjugate (GLU–MTX) both in vitro and in vivo, in which a cleavable linkage allows intracellular MTX release after selective uptake through glucose transporter−1 (GLUT1). GLU–MTX inhibited the growth of colorectal (DLD-1), breast (MCF-7) and lung (A427) adenocarcinomas, squamous cell carcinoma (SCC-25), osteosarcoma (MG63) cell lines, but not in WI-38 healthy fibroblasts. In tumor cells, GLU–MTX uptake increased 17-fold compared to unconjugated MTX. 4,6-O-ethylidene-α-D-glucose (EDG), a GLUT1 inhibitor, significantly interfered with GLU–MTX induced growth inhibition, suggesting a glucose-mediated drug uptake. Glu-MTX also caused significant tumor growth delay in vivo in breast cancer-bearing mice. These results show that our GLUT-MTX conjugate can be selectively uptake by a range of tumor cells to cause their significant growth inhibition in vitro, which was also confirmed in a breast cancer model in vivo. GLUT1 inhibitor EDG interfered with these effects verifying the selective drug uptake. Accordingly, GLU–MTX offers a considerable tumor selectivity and may offer cancer growth inhibition at reduced toxicity.

Actively Targeted and Redox Responsive Delivery of Anticancer Drug by Chitosan Nanoparticles.

The clinical efficacy of methotrexate (MTX) is limited by its poor water solubility, its low bioavailability, and the development of resistance in cancer cells. Herein, we developed novel folate redox-responsive chitosan (FTC) nanoparticles for intracellular MTX delivery. l-Cysteine and folic acid molecules were selected to be covalently linked to chitosan in order to confer it redox responsiveness and active targeting of folate receptors (FRs). NPs based on these novel polymers could possess tumor specificity and a controlled drug release due to the overexpression of FRs and high concentration of reductive agents in the microenvironment of cancer cells. Nanoparticles (NPs) were prepared using an ionotropic gelation technique and characterized in terms of size, morphology, and loading capacity. In vitro drug release profiles exhibited a glutathione (GSH) dependence. In the normal physiological environment, NPs maintained good stability, whereas, in a reducing environment similar to tumor cells, the encapsulated MTX was promptly released. The anticancer activity of MTX-loaded FTC-NPs was also studied by incubating HeLa cells with formulations for various time and concentration intervals. A significant reduction in viability was observed in a dose- and time-dependent manner. In particular, FTC-NPs showed a better inhibition effect on HeLa cancer cell proliferation compared to non-target chitosan-based NPs used as control. The selective cellular uptake of FTC-NPs via FRs was evaluated and confirmed by fluorescence microscopy. Overall, the designed NPs provide an attractive strategy and potential platform for efficient intracellular anticancer drug delivery.

CD44-Mediated Methotrexate Delivery by Hyaluronan-Coated Nanoparticles Composed of a Branched Cell-Penetrating Peptide

Branched polymers as drug delivery carriers have been widely attempted due to their outstanding drug loading capability and complex stability like branched polyethyleneimine (B-PEI). However, branched polymers without biodegradability may cause toxicity as they can accumulate in the body. Herein, we report branched modified nona-arginine (B-mR9) composed of redox-cleavable disulfide bonds to form stable complexes with methotrexate (MTX) as an anticancer agent, which is further coated with hyaluronic acid (HA). The HA-coated nanoparticles provide targetability for the CD44 cell surface receptor. The B-mR9-MTX/HA can effectively aid in intracellular MTX delivery to CD44 overexpressing cancer cells being degradable by the reducing environments of the cancer cells. The B-mR9-MTX/HA exhibits not only a glutathione-triggered degradability but also an outstanding CD44-mediated MTX delivery efficacy. In addition, its superior tumor inhibition capability was confirmed through an in vivo study. The results suggest that the HA-coated B-mR9 nanoparticle can be used as a drug delivery platform.

Evaluation of Methotrexate Clearance in Adult and Pediatric Patients with Hypoalbuminemia

Introduction: Methotrexate (MTX) is a weak acid that is approximately 50% bound to serum albumin. Although data are limited, recent publications have shown higher MTX concentrations in patients with low albumin levels. It has been proposed that hypoalbuminemia alters MTX clearance through two distinct mechanisms: 1) increased formation of intracellular MTX polyglutamates, and 2) development of third-space fluid collections in the absence of appropriate colloidal osmotic pressure. The purpose of our study was to evaluate the effect of albumin on MTX clearance in cancer patients. Methods: This single center, retrospective analysis included hospitalized adult and pediatric patients receiving high-dose MTX therapy (≥ 1 g/m2). The study was approved by the local IRB. Adult patients' records were reviewed from January 2015 to December 2018, and pediatric patients' records were reviewed from August 2016 to December 2018. Patients were included in the study more than once if they had a repeat but separate hospital encounter. The primary outcome measure was the average time to MTX clearance in patients with normal (3.5 g/dL to 5 g/dL) versus low (< 3.5 g/dL) serum albumin levels. MTX clearance was defined as the first recorded time that the level was ≤ 0.1 μM/L. Secondary outcomes included MTX levels at 24, 48, and 72 hours; rate of delayed clearance; rate of toxicities; hospital length of stay; and other factors that contribute to delayed clearance. Group comparisons for continuous data were made using Student's t-test or Mann-Whitney U test. Chi-square or Fisher's exact test was used for categorical data. To evaluate the relationship between time to MTX clearance and toxicities, Cox proportional hazards models were used. Analyses were conducted separately for pediatric and adult patients. P values < 0.05 were considered statistically significant. Results: Of 510 encounters initially screened, 204 adult patient encounters and 106 pediatric patient encounters were included. Among the adults and pediatric patients, 52% and 23% had hypoalbuminemia, respectively. In both cohorts, baseline characteristics were largely similar between the groups (Table 1). The most common cancer diagnosis among adults was lymphoma (60%). High-dose MTX ± rituximab (58%) and HyperCVAD (39%) were the most common protocols used in the adult population. The majority of pediatric patients had leukemia (55%). Results are shown in Table 2. Adult outcomes: MTX clearance was prolonged in patients with hypoalbuminemia when compared to those with normal albumin levels (108 hours vs 78.7 hours; p=0.0012) with a significantly higher percentage of the former group experiencing delayed clearance (42.8% vs 24.2%; p=0.00077). Mean length of stay was longer among hypoalbuminemic patients (17.6 days vs 8.7 days; p=0.0001). There were no statistically significant differences in rate of toxicities (Table 3). However, there was a trend towards an increased rate of grades 3-4 hepatitis among patients with low albumin levels (28.6% vs 15.8%; p=0.073). Further analysis utilizing a Cox proportional hazards model revealed that a shorter infusion time (HR 0.4, 95% CI 0.22-0.75) had a protective effect on MTX clearance. Conversely, MTX doses of 1-2.9 g/m2 (HR 3.26, 95% CI 1.52-6.97) were associated with delayed excretion of MTX. Age (HR 0.94, 95% CI 0.83-1.06), hypoalbuminemia (HR 0.94, 95% CI 0.49-1.79) and a MTX dose of 3-5.9 g/m2 (HR 1.57, 95% CI 0.64-3.88) were not significant predictors of delayed clearance. Pediatric outcomes: There was no statistically significant difference between the two groups with regard to mean time to MTX clearance. Overall mean length of stay was 7.6 days in the low albumin group and 4.4 days in the normal albumin group (p<0.00001). There were no differences in mean MTX levels at 24, 48, or 72 hours. As seen in Table 3, hypoalbuminemic patients had an increased rate of hepatitis (58.3% vs 47.9%; p= 0.017). Using the Cox analysis, the most significant predictors of delayed MTX clearance were serum creatinine and age. Conclusion: Adult patients with hypoalbuminemia had prolonged MTX excretion but this observation did not translate into an increased rate of toxicities. No difference in clearance time was detected between the two pediatric groups. To our knowledge, this is the largest study to demonstrate that high-dose MTX in combination with aggressive supportive care can be safely administered to patients with low albumin levels. Disclosures No relevant conflicts of interest to declare.

Ex vivo resistance in childhood acute lymphoblastic leukemia: Correlations between BCRP, MRP1, MRP4 and MRP5 ABC transporter expression and intracellular methotrexate polyglutamate accumulation

Chemoresistance is an important factor in the treatment failure of childhood acute lymphoblastic leukemia (ALL). One underlying mechanism of chemoresistance involves (over)expression of ATP-dependent drug efflux transporters such as multidrug resistance protein 1–5 (MRP1–5) and breast cancer resistance protein (BCRP), which can extrude the important antileukemia drug methotrexate (MTX). Survival of childhood ALL critically depends on the leukemic blasts' capacity for intracellular retention of MTX and MTX-polyglutamates. This pilot study assessed whether expression of MRP1, MRP4, MRP5 and BCRP (real-time PCR) in primary childhood ALL blasts (n = 23) correlated with ex vivo resistance to MTX (assayed by in situ thymidylate synthase inhibition assay (TSIA)), ex vivo accumulation of (radioactive) MTX polyglutamates, and patient survival. Results show that high MRP4 expression is correlated with ex vivo MTX resistance assayed by TSIA (P = 0.01). Moreover, elevated MRP4 and BCRP expression correlated with lower accumulation of MTX-PGs (P = 0.004 and P = 0.03, respectively). Combined high expression of BCRP and MRP4 even further impacted reduced MTX-PG accumulation (P = 0.02). Overall survival was lower (P logrank = 0.04) in children with ALL cells which featured a relatively high expression of both BCRP and MRP4 transporters. These results underscore the impact of high drug efflux transporter expression, notably MRP4 and BCRP, in diminished MTX response in childhood ALL.

P050 METHOTREXATE POLYGLUTAMATES AS BIOMARKERS OF TREATMENT RESPONSE IN PEDIATRIC INFLAMMATORY BOWEL DISEASE

Therapeutic drug monitoring methods are lacking for methotrexate (MTX), an immunomodulator used in Inflammatory Bowel Disease (IBD) treatment, alone or in conjunction with infliximab (IFX). Although studies demonstrate a lack of relationship between MTX plasma concentrations and efficacy/toxicity, recent studies in rheumatoid and juvenile idiopathic arthritis suggest a positive correlation between therapeutic response and accumulation of intracellular MTX metabolites, MTX polyglutamates (MTX-Glu). The aim of this cross-sectional investigation was to test the hypothesis that intracellular MTX-Glu are associated with therapeutic response in pediatric IBD. 21 children (5-21 years; 90% Crohn’s disease), receiving combination therapy with IFX and MTX for maintenance treatment of IBD, had MTX-Glu1-6 measured in erythrocytes, using HPLC-MS/MS methodologies, and serum IFX troughs and anti-IFX antibodies (Anti-IFX) measured, using a NF-kB luciferase gene-reporter assay (ARUP Laboratories). Medical records were reviewed and disease activity determined (active vs quiescent) via consensus by 2 pediatric gastroenterologists on Physician Global Assessment. Only children on stable dosing were included (i.e., no changes to dose or interval of either drug for at least 2 IFX cycles). Nonparametric tests (e.g., Mann-Whitney U) were used to compare MTX-Glu, IFX, anti-IFX, laboratory and demographic data in children with active vs quiescent disease, and associations between MTX-Glu and parameters of interest explored via Pearson’s correlation (SPSS24; α=0.05). To account for variability in standard-of-care IFX and MTX dosing, data were normalized for mg/kg and interval, as appropriate. MTX dose ranged 5-25mg weekly (86% oral) and, adjusted for weight (mg/kg), correlated with total MTX-Glu accumulation (ρ=0.6; α=0.01), especially long-chain MTX-Glu3-5(ρ=0.7; p=0.01). Children with quiescent disease (n=11) had significantly higher dose-adjusted total MTX-Glu than children with active disease (n=10), 224 ± 94.6 vs 133 ± 85.6 (p=0.04), despite receiving comparable MTX doses (0.22 ± 0.12 vs 0.31 ± 0.18 mg/kg; p=0.39). IFX troughs (μg/ml) unadjusted (24.3 ± 16.1 vs 16.7 ± 13.0) or adjusted (adj) for IFX dose and interval (86.9 ± 59.7 vs 57.4 ± 46.8) were comparable between study groups, which were also comparable for age and disease duration (p>0.25). Neither MTX dose nor MTX-Glu correlated significantly with IFX trough or adjtrough (ρ -0.09 to 0.39; p>0.1). Only 1 child, with undetectable IFX trough, had anti-IFX. The observed positive correlation between MTX dose and MTX-Glu suggests that MTX-Glu reflect systemic MTX exposure in IBD. Despite comparable IFX troughs, higher total MTX-Glu, particularly long-chain MTX-Glu, were associated with quiescent vs active disease, suggesting that MTX-Glu may be important markers of disease response in IBD.

Genetic polymorphism of the methotrexate transporter ABCG2, blood pressure and markers of arterial function in patients with rheumatoid arthritis: repeated cross-sectional study.

PurposeMethotrexate (MTX) treatment is associated with lower blood pressure (BP) and arterial stiffness in rheumatoid arthritis (RA). We investigated associations between single-nucleotide polymorphism (SNP) of the ATP-binding cassette efflux transporter gene ABCG2 (rs2231142), BP, and arterial stiffness in RA patients treated with MTX.Patients and methodsClinical and 24-hour peripheral and central BP, arterial wave reflection (Augmentation Index, AIx), arterial stiffness (Pulse Wave Velocity, PWV), and intracellular MTX polyglutamate (MTXPGs) concentrations were assessed in 56 RA patients on stable treatment with MTX using a repeated cross-sectional study design with measurements at baseline and after 8 months.ResultsMajority of the RA patients were homozygotes for the normal allele (CC, n=46) whereas 10 were rs2231142 heterozygotes (AC, n=10). MTXPGs concentrations were non-significantly higher in AC when compared to CC (144.3 vs 116.3 nmol/L packed RBCs, P=0.10). At baseline, the AC group had significantly lower age-adjusted clinical systolic BP (SBP) (P=0.01), 24-hour peripheral SBP (P=0.003), and central SBP (P=0.02) when compared to the CC group. However, AIx and PWV values were not significantly different between the two groups. When data from both visits were combined in a single analysis, and additionally adjusted for visit, gender, body mass index, and Disease Activity Score 28, the trend in SBP differences between-groups persisted but was no longer significant.ConclusionFuture studies are required to test the hypothesis that this genetic polymorphism is associated with lower BP, arterial stiffness, and possibly, cardiovascular risk, in RA patients treated with MTX.

Reduced folate carrier-mediated methotrexate transport in human distal lung epithelial NCl-H441 cells.

We had previously found that reduced folate carrier (RFC; SLC19A1) is mainly involved in an influx of transport of methotrexate (MTX), a folate analogue, using alveolar epithelial A549 cells. Therefore, we examined MTX uptake in NCl-H441 (H441) cells, another invitro alveolar epithelial model, focusing on the localization of RFC in the present study. Transport function of RFC in H441 cells was studied using [3 H]MTX. The uptake of MTX was increased remarkably after pretreatment of the cell monolayer with ethylenediaminetetraacetic acid (EDTA) in H441 cells but not in A549 cells, indicating the contribution of the basolaterally located transporter. In addition, folic acid and thiamine monophosphate, RFC inhibitors, inhibited the uptake of MTX from the basolateral side of the H441 cells. In order to compare the function of RFC on the apical and basolateral sides of the cells, the uptake of MTX from each side was examined using a Transwell chamber. Intracellular MTX amounts from the basolateral side were found to be significantly higher than those from the apical side. These findings suggest that the distribution of MTX in the lung alveolar epithelial cells may be mediated by basolaterally located RFC in alveolar epithelial cells.

Do SNPs in folate pharmacokinetic pathway alter levels of intracellular methotrexate polyglutamates and affect response? A prospective study in Indian patients.

This study investigated the impact of seven polymorphisms in genes of folate transport and (de)glutamation pathway on methotrexate polyglutamate levels and response in patients with rheumatoid arthritis. This prospective study included patients with rheumatoid arthritis. They were treated with methotrexate (up to 25mg per week) for 24weeks and categorized by EULAR response criteria into responders (good and moderate) and non-responders. Using real-time Taqman discrimination assay, SNPs were genotyped-rs1045642 (ABCB1 3435C>T), rs1128503 (ABCB1 1236C>T), rs10106 (FPGS 1994A>G), rs1544105 (FPGS G>A), rs11545078 (GGH 452C>T), rs3758149 (GGH -401C>T), and rs1051266 (RFC1 80G>A). RBC methotrexate polyglutamate1-5(MTX-glu1-5) levels were determined at 4, 8, 16, and 24weeks using by reverse phase HPLC using C-18 column followed by post column photo-oxidation. This study included 117 patients with rheumatoid arthritis (M:F = 14:103). The mean dose of methotrexate at 24weeks was 22.0 ± 4.0mg, with data on DAS28(3) at 24weeks available in 96 patients-61 responders and 35 non-responders. Minor allele of GGH 452C>T had an association with non-response (odds ratio 2.9, 95% CI 1.4-5.6) and assuming the dominance of C, the recessive genetic model found GGH 452C>T CC genotype (odds ratio 9.5, 95% CI 1.2 to 76.0) was significantly associated with response. However, there was no difference in MTX-glu1-5 levels among the various genotypes of this SNP (p = 0.9). Other SNPs were neither associated with response nor with alteration in methotrexate polyglutamate levels. On logistic regression, GGH 452C>T CC genotype and DAS28(3) at baseline were independent predictors of response. GGH 452C>T CC genotype was associated with response to methotrexate. None of the SNPs affected MTX-glu1-5levels.

Predicting methotrexate resistance in rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is an incurable, systemic autoimmune disease that decreases quality of life and can lead to severe disability. While there are many medications available to treat RA, the first-line of therapy is low-dose methotrexate (MTX), a small molecule disease-modifying anti-rheumatic drug (DMARD). MTX is the recommended therapy due to its affordability and efficacy in reducing symptoms in most RA patients. Unfortunately, there is great person-to-person variability in the physiological response to MTX, with up to 50% of patients showing little response to the medication. Thus, many RA patients initially placed on MTX do not experience an adequate reduction of symptoms, and could have benefited more in both the short and long terms if initially prescribed a different drug that was more effective for them. To combat this problem and better guide treatment decisions, many research groups have attempted to develop predictive tools for MTX response. Currently, there is no reliable, clinical-grade method to predict an individual's response to MTX treatment. In this review, we describe progress made in the area of MTX non-response/resistance in RA patients. We specifically focus on application of the following elements as predictive markers: proteins related to MTX transport and function, intracellular MTX concentration, immune cell frequencies, cytokines, and clinical factors.

MiR-595 Suppresses the Cellular Uptake and Cytotoxic Effects of Methotrexate by Targeting SLC19A1 in CEM/C1 Cells.

The human solute carrier family 19 member 1 (SLC19A1) is the gene coding for reduced folate carrier 1 (RFC1). In our previous work, we showed that the miR-595-related polymorphism, rs1051296G>T, which was located in the 3'-untranslated region (3'-UTR) of SLC19A1, was associated with high methotrexate (MTX) plasma concentrations in patients with paediatric acute lymphoblastic leukaemia (ALL). This study aimed to investigate the role of miR-595 in the regulation of SLC19A1 expression and its effects on the cellular uptake and cytotoxicity of MTX in ALL CEM/C1 cells. Luciferase reporter assay was performed to validate SLC19A1 as a miR-595 target. RFC1 protein expression was determined via Western blotting. Intracellular MTX concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Cell viability and apoptosis were assessed using Cell Counting Kit-8 (CCK-8) assay and flow cytometer, respectively. Compared to the negative control, miR-595 mimics induced a significant decrease in the relative luciferase activity by binding to the 3'-UTR of SLC19A1 harbouring the rs1051296 T allele (p<0.01). Treatment of CEM/C1 cells with miR-595 mimics substantially reduced RFC1 protein expression, intracellular MTX levels, MTX-induced cytotoxicity and apoptosis rates compared to those of negative control. However, opposite results were observed in cells transfected with a miR-595 inhibitor. These findings suggested that miR-595 acts as a phenotypic regulator of MTX sensitivity in CEM/C1 cells by targeting SLC19A1. This study helped us to understand the mechanisms underlying the variable MTX responses observed in patients with ALL.

Folylpolyglutamate synthase is a major determinant of intracellular methotrexate polyglutamates in patients with rheumatoid arthritis.

We investigated major determinants of the intracellular concentrations of methotrexate polyglutamates (MTXPGs) in patients with rheumatoid arthritis (RA). In 271 RA patients on stable oral low dose weekly pulse MTX therapy, the concentrations of MTXPGs in red blood cells (RBCs) were measured by liquid chromatography-electrospray ionization-tandem mass spectrometry. Polymerase chain reaction-restriction fragment length polymorphism analysis was performed to determine the genotypes of solute carrier family 19 member 1 (SLC19A1), folylpolyglutamate synthase (FPGS), and gamma-glutamyl hydrolase (GGH). The mean total MTXPG concentration and the concentrations of individual MTXPGs increased dose-dependently, but reached a plateau at MTX doses >10 mg weekly. The MTXPG3-5/1-2 ratio was lower in patients with adverse events related to MTX than in patients without adverse events. Three polymorphisms of FPGS significantly influenced the MTXPG3-5/1-2 ratio in RBCs, while polymorphisms of SLC19A1 and GGH had no impact. The minor allele frequencies of 2 FPGS genotypes were significantly increased in our patients compared with a Caucasian population. FPGS may have a major role in regulating intracellular polyglutamation of MTX in RA patients receiving low-dose weekly MTX therapy.

Abstract 1784: The impact of the natural (6S) isomer of 5-formyltetrahydrofolate on the activities of pralatrexate and methotrexate in vitro

Abstract Introduction: Pralatrexate, the 10-propargyl-10-deaza analog of aminopterin, is a much superior substrate for the reduced folate carrier and folylpolyglutamate synthetase than methotrexate (MTX) resulting in the rapid build-up and retention of high levels of the polyglutamate derivatives of this agent within cells. In HeLa cells in vitro, the growth inhibitory effect of a 6h exposure to pralatrexate is two orders of magnitude greater than that of MTX. Yet the drug can be administered weekly at similar doses than MTX, consistent with a high level of selectivity. Nonetheless, mucosites can result in dosage reductions. This has led to the application of “rescue” regimens with the natural isomer of 5-formyltetrahydrofolate, (6S)5-formylTHF. Purpose: To investigate the impact of (6S)5-formylTHF on the antitumor activity of pralatrexate in HeLa cells in vitro, simulating the exposures to this folate as occurs in the clinical setting. These properties were compared to those of MTX. Experimental design: HeLa cells were grown with 25 nM (6S)5-formylTHF as the folate growth source. Experiments evaluated the impact of a 24h exposure to different concentration of (6S)5-formylTHF varying intervals prior to, or after, a 6h exposure to antifolate. The impact on the accumulation of [3H](6S)5-formylTHF and [3H]antifolate was also assessed. Results: A 24h interval between antifolate, followed by (6S)5-formylTHF, was sufficient to sustain the full activities of the antifolates up to a (6S)5-formylTHF level of 4 µM. A 72h interval was required between (6S)5-formylTHF, followed by the antifolate, to sustain full antifolate activities. HeLa cells were then exposed to 4 µM (6S)5-formylTHF for 24h weekly X 4; a concentration of the folate in excess of what is achieved in “rescue” regimens. This had no significant effect on the pralatrexate IC50; however, the MTX IC50 increased 2.5-fold. The four cycles of exposure to 4 µM [3H](6S)5-formylTHF resulted in a 16% increase in the tritiated intracellular folate level (P=0.02) as compared to cells growing continuously in 25 nM [3H](6S)5-formylTHF.There was no significant decrease in total intracellular pralatrexate; there was a 15% decrease in total intracellular MTX (P=0.004). Conclusions: Exposure of HeLa cells to (6S)5-formylTHF either before or after pralatrexate or MTX, simulating the sequence of administration in “rescue” regimens, and at concentrations in excess of what is achieved clinically, had no effect on pralatrexate activity but decreased the activity of MTX. This cumulative effect was associated with a small increase in the intracellular (6S)5-formylTHF level and a small decrease in total intracellular MTX. The data suggests that standard “rescue” regimens should have little or no impact on the antitumor activity of pralatrexate. Citation Format: Michele Visentin, Ersin Selcuk Unal, I David Goldman. The impact of the natural (6S) isomer of 5-formyltetrahydrofolate on the activities of pralatrexate and methotrexate in vitro. [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 1784. doi:10.1158/1538-7445.AM2014-1784

THU0122 Effect of Two MTX Dosing Regimens on Intracellular MTX Accumulation in Rheumatoid Arthritis Patients

BackgroundLow-dose methotrexate (MTX) is gold standard in the treatment of rheumatoid arthritis (RA). Response to MTX is very variable and is related to the intracellular MTX-polyglutamate (MTX-PG) levels.1,2 Similar to...

The impact of 5-formyltetrahydrofolate on the anti-tumor activity of pralatrexate, as compared to methotrexate, in HeLa cells in vitro

To investigate the impact of 5-formyltetrahydrofolate on the activities of pralatrexate, as compared to methotrexate (MTX), in vitro. Cells were exposed to (6S)5-formyltetrahydrofolate (5-formylTHF) for 24 h, before or after a 6-h exposure to antifolates following which the cellular accumulation and activities of the drugs were evaluated in HeLa cells. A 24-h delay between a 6-h exposure to antifolates and a subsequent 24-h exposure to 4 μM 5-formylTHF sustained the full activities of both antifolates. A 72-h interval was required between a single exposure of up to 4 μM 5-formylTHF and subsequent exposure to drugs to sustain activities of the antifolates. When cells were incubated with 4 μM 5-formylTHF for 24 h weekly, for 4 weeks, there was no significant increase in the IC50 for pralatrexate, but the MTX IC50 increased 2.5-fold as compared to cells growing continuously in 25 nM 5-formylTHF. This cyclical exposure to 5-formylTHF increased the cell folate pool by 16 %, had no significant effect on the intracellular pralatrexate level, but decreased intracellular MTX by 15 %. An extracellular concentration of MTX 50-fold higher than that of pralatrexate was required to achieve an intracellular level, and growth inhibition, comparable to that of pralatrexate. Cyclical exposures to 5-formylTHF at levels in excess of what is achieved in most clinical "rescue" regimens do not affect pralatrexate accumulation nor antitumor activity in HeLa cells, in contrast to MTX. An important element in preserving pralatrexate activity is achieving a sufficient interval between exposure to 5-formylTHF and the next dose of antifolate.