Polyglutamate Metabolites Research Articles

Up-regulation of ABCG1 is associated with methotrexate resistance in acute lymphoblastic leukemia cells.

Acute lymphoblastic leukemia (ALL) is a prevalent hematologic malignancy in children, and methotrexate (MTX) is a widely employed curative treatment. Despite its common use, clinical resistance to MTX is frequently encountered. In this study, an MTX-resistant cell line (Reh-MTXR) was established through a stepwise selection process from the ALL cell line Reh. Comparative analysis revealed that Reh-MTXR cells exhibited resistance to MTX in contrast to the parental Reh cells. RNA-seq analysis identified an upregulation of ATP-binding cassette transporter G1 (ABCG1) in Reh-MTXR cells. Knockdown of ABCG1 in Reh-MTXR cells reversed the MTX-resistant phenotype, while overexpression of ABCG1 in Reh cells conferred resistance to MTX. Mechanistically, the heightened expression of ABCG1 accelerated MTX efflux, leading to a reduced accumulation of MTX polyglutamated metabolites. Notably, the ABCG1 inhibitor benzamil effectively sensitized Reh-MTXR cells to MTX treatment. Moreover, the observed upregulation of ABCG1 in Reh-MTXR cells was not induced by alterations in DNA methylation or histone acetylation. This study provides insight into the mechanistic basis of MTX resistance in ALL and also suggests a potential therapeutic approach for MTX-resistant ALL in the future.

Clinical and Laboratory Associations with Methotrexate Metabolism Gene Polymorphisms in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that causes loss of joint function and significantly reduces quality of life. Plasma metabolite concentrations of disease-modifying anti-rheumatic drugs (DMARDs) can influence treatment efficacy and toxicity. This study explored the relationship between DMARD-metabolising gene variants and plasma metabolite levels in RA patients. DMARD metabolite concentrations were determined by tandem mass-spectrometry in plasma samples from 100 RA patients with actively flaring disease collected at two intervals. Taqman probes were used to discriminate single-nucleotide polymorphism (SNP) genotypes in cohort genomic DNA: rs246240 (ABCC1), rs1476413 (MTHFR), rs2231142 (ABCG2), rs3740065 (ABCC2), rs4149081 (SLCO1B1), rs4846051 (MTHFR), rs10280623 (ABCB1), rs16853826 (ATIC), rs17421511 (MTHFR) and rs717620 (ABCC2). Mean plasma concentrations of methotrexate (MTX) and MTX-7-OH metabolites were higher (p < 0.05) at baseline in rs4149081 GA genotype patients. Patients with rs1476413 SNP TT or CT alleles have significantly higher (p < 0.001) plasma poly-glutamate metabolites at both study time points and correspondingly elevated disease activity scores. Patients with the rs17421511 SNP AA allele reported significantly lower pain scores (p < 0.05) at both study intervals. Genotyping strategies could help prioritise treatments to RA patients most likely to gain clinical benefit whilst minimizing toxicity.

Optimising low-dose methotrexate for rheumatoid arthritis-A review.

Methotrexate at low doses (5-25mg/week) is first-line therapy for rheumatoid arthritis. However, there is inter- and intrapatient variability in response, with contribution of variability in concentrations of active polyglutamate metabolites, associated with clinical efficacy and toxicity. Prescribing remains heterogeneous across population groups, disease states and regimens. This review examines current knowledge of dose-response of oral methotrexate in the setting of rheumatoid arthritis, and how this could help inform dosage regimens.

Methotrexate disposition, anti-folate activity and efficacy in the collagen-induced arthritis mouse model

Methotrexate (MTX) efficacy in autoimmune arthritis is variable and unpredictable resulting in the need for the identification of biomarkers to guide drug therapy. This study utilizes the collagen-induced arthritis mouse model to investigate erythrocyte MTX disposition and anti-folate activity as biochemical markers of efficacy in autoimmune arthritis. Following induction of arthritis, DBA/1J mice were treated with once-weekly subcutaneous MTX at varying doses over a period of 40 days. At the completion of the study tissue samples were analyzed for MTX and folate content and assessed for their relationship with MTX efficacy. MTX treatment resulted in a reduction in disease activity that was variable and dose-dependent. Erythrocyte accumulation of MTX and its polyglutamate metabolites were dose proportionate, however, polyglutamate metabolites represented a mean ± S.E.M. of 8.9 ± 0.4% of total erythrocyte MTX, which is markedly lower than previously observed in humans and failed to display any significant association with MTX efficacy. MTX treatment resulted in reductions in erythrocyte 5-methyl-tetrahydrofolate (5mTHF) levels that were similar to those previously observed in human studies. Disease induction was associated with a decrease in liver 5mTHF and increased formyl-tetrahydrofolate (fTHF) that was normalized in MTX treated mice. MTX efficacy was associated with reductions in erythrocyte 5mTHF (P = 0.04) and increases in liver 5mTHF (P = 0.0001). Together, these findings demonstrate a relationship between alterations in tissue folate levels and MTX efficacy, and supports erythrocyte levels of 5mTHF as a marker of MTX efficacy in autoimmune arthritis.

Constitutive Activation of Stat Signaling in Fibroblast‐like Synoviocytes Derived from Patients with Juvenile Idiopathic Arthritis Is Inhibited by Methotrexate

BACKGROUNDMethotrexate (MTX) is the cornerstone of treatment in the management of autoimmune arthritis, including juvenile idiopathic arthritis (JIA). Despite being in use for over 35 years the mechanism through which MTX inhibits inflammation remains unclear. In this study we investigate activation of JAK/STAT signaling in fibroblast‐like synoviocytes from patients with JIA and the effect of MTX.METHODSSynoviocytes were isolated from the synovial fluid of juvenile idiopathic arthritis patients following routine arthrocentesis and maintained in culture. Synoviocytes were treated with MTX and assessed for cellular levels of MTX and its polyglutamated metabolites, as well as intermediates of nucleotide biosynthesis, including dUMP and ZMP. Analytes were detected by UPLC‐MS/MS. JAK/STAT signaling was assessed in synoviocytes and A549 human lung carcinoma cells by Western blotting and immunofluorescence. Experiments were performed in triplicate and statistical testing was done by unpaired t test analysis.RESULTSMTX was actively taken up and metabolized in synoviocytes to form polyglutamated metabolites of MTX, however, MTX failed to cause measurable inhibition of de novo nucleotide synthesis by dUMP and ZMP analysis. Synoviocytes displayed a high level of STAT phosphorylation with 58.8±4.8% of total measureable STAT3 signal and 69.9±7.3% of total measureable STAT5 signal. In contrast, phosphorylation of STAT3 and STAT5 was undetectable in A549 cells and treatment with 100 ng/mL of recombinant IL‐6 resulted in a marked increase in STAT phosphorylation with 64.4±6.0% of measureable STAT3 signal and 76.6±8.5% of measureable STAT5 signal. Treatment with MTX resulted in inhibition of STAT3 and STAT5 phosphorylation by 38.1±3.4% (p&lt;0.05) and 61.2±7.8% (p&lt;0.05) in primary synoviocytes and 65.3±2.8 (p&lt;0.05) and 92.8±8.1% (p&lt;0.01) in IL‐6 stimulated A549 cells, respectively. In comparison, phosphorylation of STAT3 and STAT5 in response to IL‐6 stimulation in A549 cells was decreased by 91.6±11.3% (p&lt;0.001) and 94.5±12.3% (p&lt;0.001) by 50 μM of JAK Inhibitor I (CAS 457081‐03‐7). In A549 cells inhibition of STAT phosphorylation by MTX was completely reversed by co‐treatment with 100 μM folinic acid.CONCLUSIONThese findings support constitutive activation of JAK/STAT signaling in fibroblast‐like synoviocytes isolated from patients with JIA that is inhibited by treatment with MTX. Although the underlying biochemical mechanism through which MTX inhibits JAK/STAT signaling is not known, it appears to be dependent on the anti‐folate activity of MTX. Together, this data supports inhibition of JAK/STAT signaling as a possible mechanism of anti‐inflammatory activity of MTX.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High and individually variable enzymatic activity precludes accurate determination of pemetrexed, methotrexate and their polyglutamate metabolite concentrations in plasma

Most drugs are metabolized in the human body. Therefore, it is essential for therapeutic drug monitoring studies to also take into account the concentrations of drug metabolites. One of the possible metabolic activities on drugs such as pemetrexed or methotrexate is (poly)glutamation. Here, we report on a series of experiments that we performed to investigate the stability of polyglutamate metabolites in plasma.Removal of glutamate residues from pemetrexed polyglutamate by most likely proteases in human plasma is influenced by temperature as it is observed at 25°C and even more strongly at 37°C, but not at 4°C. The observed protease activity is highly variable among patients; in approximately 15–20% of the patients tested it is not observed, whereas in other individuals the activity is so extensive that after 10min, more than 50% of spiked polyglutamated pemetrexed is degraded at room temperature (5–10% of the tested individuals). Similar observations also pertain to methotrexate polyglutamates. These observations do not extend to pemetrexed and methotrexate themselves which are unaffected by this activity.Due to the considerable and, among individuals, variable protease activities on polyglutamated drug metabolites in plasma, these metabolites are virtually impossible to quantify if no precautions are taken.

Review of the bioanalytical methods for the determination of methotrexate and its metabolites in in vitro, preclinical and clinical studies: Case studies and perspectives

Methotrexate is an old drug that has found use in several therapeutic areas, such as cancer to treat various malignancies, rheumatoid arthtritis and inflammatory bowel disease. Owing to its structural properties of possessing two carboxylic acid groups and having low native fluorescence, it has provided technical challenges for development of bioanalytical methods. Also, in vivo metabolism leading to circulatory metabolites such as 7-hydroxymethotrexate and 2,4-diamino N10 -methylpteroic acid, as well as the formation of polyglutamate metabolites intracellularly have added further complexity for the assays in terms of the analytes that need to be quantified in addition to methotrexate. The present review is aimed at providing a concise tabular summary of chromatographic assays with respect to method nuances including assay/chromatographic conditions, key validation parameters and applicable remarks. Several case studies are reviewed under various subheadings to provide the challenges involved in the method development for methotrexate and metabolites. Finally, a discussion section is devoted to overall perspectives obtained from this review.

A new quantification method for assessing plasma concentrations of pemetrexed and its polyglutamate metabolites

Currently no quantification method exists for potentially therapeutically relevant polyglutamate metabolites of the drug pemetrexed which is used for the treatment of lung carcinoma patients.We developed and tested an LC–MS/MS-based analytical assay that uses isotope-labeled internal standards to quantify pemetrexed and its (poly)glutamate metabolites in clinical human plasma samples of lung carcinoma patients.UHPLC chromatography and triple quadrupole mass spectrometry showed an LLOQ of 0.2nmol/L for pemetrexed and an LLOQ of 0.5nmol/L for the two metabolites (one glutamate and two glutamate moieties covalently bound to the pemetrexed molecule, for which no other quantification methods have previously been published). The recoveries for PMTX and its metabolites ranged between 30% and 67%. Precision and accuracy at a concentration of 20nmol/L for all four analytes was well below 15% CV. The precision (RSD) in the biological replicates of the separate days (within-run precision) as well as the reproducibility over several days (between-run precision), tested in the range of 5–250nmol/L, were all below 15%. Autosampler, benchtop and freeze-thaw cycle stability of the analytes was also demonstrated. To illustrate the new assay in a relevant biological context, concentrations of pemetrexed and the two metabolites were quantified in plasma samples of lung carcinoma patients treated with pemetrexed.The assay is straightforward, relatively easy to perform, and has potential for use in therapeutic drug monitoring in non-small cell lung carcinoma patients.

Simplification of a pharmacokinetic model for red blood cell methotrexate disposition.

A pharmacokinetic (PK) model is available for describing the time course of the concentrations of methotrexate (MTX or MTXGlu1) and its active polyglutamated metabolites (MTXGlu2-5) in red blood cells (RBCs). In this study, we aimed to simplify the MTX PK model and to optimise the blood sampling schedules for use in future studies. A proper lumping technique was used to simplify the original MTX RBC PK model. The sum of predicted RBC MTXGlu3-5 concentrations in both the simplified and original models was compared. The sampling schedules for MTXGlu3-5 or all MTX polyglutamates in RBCs were optimised using the Population OPTimal design (POPT) software. The MTX RBC PK model was simplified into a three-state model. The maximum of the absolute value of relative difference in the sum of predicted RBC MTXGlu3-5 concentrations over time was 6.3%. A five blood sample design was identified for estimating parameters of the simplified model. This study illustrates the application of model simplification processes to an existing model for MTX RBC PK. The same techniques illustrated in our study may be adopted by other studies with similar interest.

Abstract 4482: A new quantification method for assessment of plasma concentrations of pemetrexed and its polyglutamate metabolites

Abstract Pemetrexed (PMTX) is a multi-targeted antifolate drug applied in the treatment of diseases such as malignant pleural mesothelioma and non-small cell lung cancer. Assessment of therapeutic plasma drug and drug metabolite concentrations offers a useful tool for monitoring drug dosage, avoiding drug toxicity and obtaining therapeutic efficacy. Stock solutions of synthesized PMTX and its (poly)-glutamyl metabolites (PMTXPG1-3), as well as their corresponding heavy labeled analogues (13C5, 15N (6 Da mass difference)) were prepared and spiked into plasma from healthy donors, and 2 M trichloroacetic acid (TCA) was added for protein precipitation. The samples were centrifuged at 5223 g and subsequently the supernatant was taken for measurement on a UHPLC-QqQ mass spectrometry set-up. After assessment of analytical factors such as linear response, stability, reproducibility and recovery, plasma of 4 non-small cell cancer patients undergoing PMTX treatment were measured to establish the methods ability for measuring clinically relevant concentrations. The stability of patient samples stored at -80°C was monitored during a six month period, in which no statistically significant change in recovery between them (all recoveries were between 97-101%, no trend of decrease in time) was observed. The recovery of PMTX and its metabolites after the sample pretreatment procedure ranged between 45% and 79%. The relative standard deviations (RSD) in the biological replicates of the reproducibility analysis were 7.9% for PMTX, 5.9% for PMTXPG1, 7.2% for PMTXPG2 and 37.9% for PMTXPG3. For all analytes the linear response was determined over three orders of magnitude in concentration (0.2 to 500 nmol/L). Additionally, the limit of quantification (lowest quantifiable concentration (RSD &amp;lt; 20%)) was determined for all analytes. For PMTX, PMTXPG1 and PMTXPG2 subnanomolar limits of quantification were determined. The linear response for the compounds was determined by calculation of the correlation coefficient, which was better than 0.99 for all four analytes. In all four PMTX-treated patients the plasma concentrations of PMTX, PMTXPG1 and PMTXPG2 could be detected and quantified. PMTXPG3 was not detected in any of the tested clinical samples of the four patients. In conclusion, a LC-MS/MS-based method to measure PMTX and its polyglutamate metabolites with minimal sample pretreatment is described. To our knowledge this is the first time measurement method for pemetrexed metabolites in human plasma is reported. The method also has relatively short measurement times and good performance in being able to quantify the drug and two of its metabolites at clinically relevant concentrations in plasma of patients with lung carcinoma. Citation Format: Marcel Stoop, Sabine Visser, Evert van Dijk, Bruno Stricker, Theo Luider, Joachim Aerts. A new quantification method for assessment of plasma concentrations of pemetrexed and its polyglutamate metabolites. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4482. doi:10.1158/1538-7445.AM2015-4482

Folate depletion and increased glutamation in juvenile idiopathic arthritis patients treated with methotrexate.

Folates exist as a fluctuating pool of polyglutamated metabolites that may serve as a clinical marker of methotrexate (MTX) activity. This study was undertaken to evaluate circulating folate content and folate polyglutamate distribution in juvenile idiopathic arthritis (JIA) patients and in a cell culture model based on MTX exposure and folate supply. Blood, plasma, and red blood cell (RBC) measurements of MTX and folates were obtained from previously published data sets and an additional analysis of JIA patients receiving MTX (n = 98) and those not receiving MTX (n = 78). Erythroblastoid cells maintained in culture were exposed to MTX and grown under varying levels of folic acid supplementation. Samples were analyzed for cellular folate and MTX content. Circulating folate levels were lower in JIA patients receiving MTX, with reduced levels of blood, plasma, and RBC 5-methyl-tetrahydrofolate (5mTHF) (P < 0.0001). Average polyglutamate chain length (Gluavg ) of RBC 5mTHF was elevated in JIA patients receiving MTX (median ± interquartile range 5.63 ± 0.15 versus 5.54 ± 0.11 in those not receiving MTX; P < 0.001) and correlated with both RBC MTX accumulation (P = 0.02) and reduced plasma 5mTHF levels (P = 0.008). MTX exposure and folate deprivation in erythroblastoid cells resulted in a depletion of bioactive folate species that was associated with a shift to higher Gluavg values for several species, most notably tetrahydrofolate (THF) and 5,10-methylene-tetrahydrofolate (CH2 THF). Increased Gluavg resulted from the depletion of short-chain and the accumulation of long-chain glutamate species. Our findings indicate that folate content and polyglutamate distribution are responsive markers of MTX activity and folate supply in vivo and in vitro, and may provide novel clinical markers of pharmacologic activity of MTX.

A Population Pharmacokinetic Model for Low-Dose Methotrexate and its Polyglutamated Metabolites in Red Blood Cells

Measurement of intracellular concentrations of methotrexate (MTX) and its polyglutamated metabolites (MTXGlu(2-5)) in red blood cells (RBCs) has been suggested as a potential means of monitoring low-dose MTX treatment of rheumatoid arthritis (RA). However, a possible correlation between RBC MTX and MTXGlu2-5 concentrations and clinical outcomes of MTX treatment in RA is debated. A better understanding of the dose-concentration-time relationship of MTX and MTXGlu(2-5) in RBCs by population pharmacokinetic modelling is desirable and will facilitate assessing a potential RBC concentration-effect relationship in the future. The purpose of this analysis was to describe the pharmacokinetics of MTX and MTXGlu(2-5) in RBCs. Secondary objectives included investigation of deglutamation reactions and the loss of MTX and MTXGlu(2-5) from the RBC. A model was developed using NONMEM(®) version 7.2 based on RBC data obtained from 48 patients with RA receiving once-weekly low-dose MTX treatment. This model was linked to a fixed two-compartment model that was used to describe the pharmacokinetics of MTX in the plasma. A series of five compartments were used to describe the intracellular pharmacokinetics of MTX and MTXGlu(2-5) in RBCs. Biologically plausible covariates were tested for a significant effect on MTX plasma clearance and the intracellular volume of distribution of all MTX species in RBCs ([Formula: see text]). The developed model was used to test hypotheses related to the enzymatic deglutamation of MTXGlu(2-5) and potential loss of MTXGlu(2-5) from RBCs. The final RBC pharmacokinetic model required the intracellular volumes of distribution for the parent and metabolites to be set to the value estimated for the parent drug MTX alone, and the rate constants describing the polyglutamation steps were fixed at literature values. Significant covariates included effect of body surface area-adjusted estimated glomerular filtration rate on renal plasma clearance and effect of allometrically scaled total body weight with a fixed exponent of 0.75 on non-renal plasma clearance of MTX. The only significant covariate with an effect on [Formula: see text] was mean corpuscular volume (MCV). The model supported single deglutamation steps and a single mechanism of MTX and MTXGlu(2-5) loss from RBCs. The developed model enabled acceptable description of the intracellular kinetics of MTX and MTXGlu(2-5) in RBCs. In the future it can form the basis of a full pharmacokinetic-pharmacodynamic model to assess the time-RBC concentration-effect relationship of low-dose MTX treatment in RA.

Therapeutic targeting malignant mesothelioma with a novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate via its selective uptake by the proton-coupled folate transporter

We examined whether the novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate, compound 2, might be an effective treatment for malignant pleural mesothelioma (MPM), reflecting its selective membrane transport by the proton-coupled folate transport (PCFT) over the reduced folate carrier (RFC). HeLa sublines expressing exclusively PCFT (R1-11-PCFT4) or RFC (R1-11-RFC6) and H2452 MPM cells were assayed for transport with [(3)H]compound 2. [(3)H]Polyglutamate metabolites of compound 2 were measured in R1-11-PCFT4 and H2452 cells. In vitro cell proliferation assays and colony formation assays were performed. Inhibition of glycinamide ribonucleotide formyltransferase (GARFTase) was assayed by nucleoside protection assays and in situ GARFTase assays with [(14)C]glycine. In vivo efficacy was established with early- and advanced-stage H2452 xenografts in severe-combined immunodeficient (SCID) mice administered intravenous compound 2. [(3)H]Compound 2 was selectively transported by PCFT and was metabolized to polyglutamates. Compound 2 selectively inhibited proliferation of R1-11-PCFT4 cells over R1-11-RFC6 cells. H2452 human MPM cells were sensitive to the antiproliferative effects of compound 2. By colony-forming assays with H2452 cells, compound 2 was cytotoxic. Compound 2 inhibited GARFTase in de novo purine biosynthesis. In vivo efficacy was confirmed toward early- and advanced-stage H2452 xenografts in SCID mice administered compound 2. Our results demonstrate potent antitumor efficacy of compound 2 toward H2452 MPM cells in vitro and in vivo, reflecting its efficient membrane transport by PCFT, synthesis of polyglutamates, and inhibition of GARFTase. Selectivity for non-RFC cellular uptake processes by tumor-targeted antifolates such as compound 2 presents an exciting new opportunity for treating solid tumors.

Assessment of intracellular methotrexate and methotrexate‐polyglutamate metabolite concentrations in erythrocytes by ultrafast matrix‐assisted laser desorption/ionization triple quadrupole tandem mass spectrometry

A new ultrafast quantitative and high-throughput mass spectrometric method using matrix-assisted laser desorption/ionization triple quadrupole tandem mass spectrometry has been developed and validated for determination of intracellular erythrocyte concentrations of the antifolate drug methotrexate (MTX) and its polyglutamate metabolites. The method consists of a solid-phase extraction of MTX and MTX-polyglutamate metabolites from deproteinized erythrocyte lysates spiked with aminopterin as internal standard. The newly developed method was validated according to the most recent FDA guidelines on linearity, recovery, within-run and between-run accuracy and precision and stability of the analytes. The low limit of quantification (LLOQ) was 10 nmol/L for all analytes while the limit of detection (LOD) determined at a signal-to-noise (S/N) ratio = 3:1 in drug- free erythrocyte lysate was on average 0.3 nmol/L. After validation, the new method was used in the measurement of intracellular erythrocyte concentrations of MTX and MTX-polyglutamate metabolites (MTXPG2 to MTXPG7) in packed human erythrocyte samples collected from patients with rheumatoid arthritis receiving low-dose oral methotrexate therapy. Mean (SD) intracellular erythrocyte concentrations observed in patient samples were 12.8 (12.6), 12.4 (9.4), 44.4 (30.0), 33.6 (35.9) and 9.4 (8.2) nmol/L for MTX to MTXPG5, respectively, in 10(6) erythrocytes. The highest observed glutamylation degree of MTX was MTXPG5, the very long chain MTX-polyglutamate metabolites MTXPG6 and MTXPG7 were not detected in the packed erythrocyte pellets collected from rheumatoid arthritis patients.

Expedient methodology for total methotrexate polyglutamation pool determination in human erythrocytes

The measurement of methotrexate polyglutamate metabolites in red blood cells has potential to aid in individualization of methotrexate therapy in rheumatoid arthritis and juvenile idiopathic arthritis. In this report a method is presented for rapid analysis of these metabolites in human red blood cells. The analytical procedure is a simple “one pot” pre-column reaction involving the addition of sodium dithionite as a reducing agent followed by 15 minutes of boiling. After centrifugation the supernatant is introduced into a conventional HPLC system equipped with a fluorescence detector, without the need for further workup. By performing the derivatization reaction pre-column, a time consuming (6–14 h) commonly used deglutamation procedure utilizing blank human plasma, becomes obsolete. Using the described procedure the total sample preparation time for a 50 sample run should not exceed 1–1.5 hours. The chromatographic run time per sample is 7 minutes using isocratic elution conditions. The method was found to be linear over the clinical relevant concentration range of 10–500 nM of intra-cellular methotrexate polyglutamates. The intra-run mean accuracy of the target value was between 98.1% and 106.0%. The intra-run precision was between 1.2% and 8.8%.

Simultaneous determination of methotrexate and its polyglutamate metabolites in Caco-2 cells by liquid chromatography–tandem mass spectrometry

In normal and malignant human cells, the folate antagonist methotrexate (MTX) is converted to a series of polyglutamates (MTXGlu n , n = 2–5) which play a role in its therapeutic efficacy. Here we report an assay to determine MTX and MTXGlu n in Caco-2 cells exposed to MTX. After a simple protein precipitation step, cell homogenates (2 × 10 6 cells) were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) using aminopterin as internal standard. Separation was by reversed phase HPLC on a C8 column using gradient elution with 0.1% formic acid and acetonitrile. Detection was by electrospray ionization in the positive ion mode followed by multiple reaction monitoring of the transitions of the [M+H] + ions of MTX and MTXGlu n to their common product ion at m/ z 308.2 and of aminopterin at m/ z 441.3 → 294.2. Calibration curves for all analytes were linear in the range 2–250 nM ( r 2 > 0.996). Intra- and inter-day precisions (as coefficient of variation) were 3.4–15.1% and 4.3–18.4%, respectively with corresponding accuracies (as relative error) of −3.6 to +6.6% and −5.5 to +7.5%, respectively. Recoveries were in the range 60 ± 4 to 108 ± 13%. It was found that MTX undergoes only limited polyglutamation in Caco-2 cells exposed to MTX over 24 h.

Phase I Trial of Edatrexate in Advanced Breast and Other Cancers

Edatrexate (EDX) (10-ethyl-10-deazaaminopterin or 10-EDAM) is an analogue of methotrexate with improved pre-clinical antitumor activity, more selective cellular uptake, and with more extensive formation of intracellular polyglutamate metabolites. A phase I trial in advanced cancer using EDX was initiated to determine the toxicities associated with the use of a biweekly schedule of intravenous EDX, and to carry out a dose escalation to define the maximum tolerated dose employing this schedule.Thirty-four patients were enrolled in this phase I trial. Thirty-three patients were treated in cohorts of at least three patients (except at one dose level, 210 mg m−2, where there were only two patients). Dose escalations of EDX were administered starting with 100 mg m−2, and progressing through 120, 140, 160, 180, 210, 240, and 270 mg m−2. Edatrexate was administered by intravenous infusion over 20 min, and cryotherapy using ice chips was given prophylactically for 5 min before, during, and 15 min after each EDX treatment.The dose-limiting toxicity could not be reached in this study because it had to be closed on account of competing protocols using EDX in combination regimens. Of note though, was that the delivered dose intensity at the 160 mg m−2 week−1, was higher than the previously used or recommended phase II doses. Anemia was mild and white blood corpuscle toxicity was mostly of grade 1 or 2. One patient had grade 4 neutropenia and one had grade 3 thrombocytopenia. Of the non-hematological toxicities, nausea, vomiting, and diarrhea were mild and tolerable. Mucositis, which was the dose-limiting toxicity in previous studies, was seen in 30% of the patients, but was predominantly a grade 1 toxicity. This could have been due to either the different schedule of EDX used in this study or the use of cryotherapy. Substantial antitumor effects were noted, with two near-complete complete responses at the 120 and 160 mg m−2 levels. Additionally, six partial responses and one minor response were observed, and stable disease was observed in seven patients.Despite achieving antitumor activity at different dose levels, a clear-cut dose response was not evident at the levels tested. We feel that the biweekly EDX schedule is a tolerable regimen, which allows a higher dose intensity than weekly administration, and that EDX is an active agent for the treatment of patients with metastatic cancer.

Variable expression of the folylpolyglutamate synthetase gene at the level of mRNA transcription in human leukemia cell lines sensitive, or made resistant, to various antifolate drugs.

We investigated the expression of the folylpolyglutamate synthetase (FPGS) gene at the mRNA level in MOLT-3 and K562 human leukemia cell lines sensitive, or made resistant, to methotrexate (MTX) and/or trimetrexate (TMQ), or raltitrexed (ZD1694). Northern blot analysis demonstrated approximately 3-fold higher FPGS mRNA expression in K562 cells than that in MOLT-3 cells, being consistent with graded polyglutamation capacities of these cell lines. A slight increase in the expression of the FPGS gene was observed in the TMQ-resistant MOLT-3 cells (MOLT-3/TMQ800); moreover, sequential development of MTX resistance in the TMQ-resistant cells (MOLT-3/TMQ800-MTX10,000) resulted in a further enhancement of FPGS mRNA expression despite of decreased polyglutamation capacity in this subline. Another MTX-resistant subline with impaired reduced folate carrier (MOLT-3/MTX10,000) also showed overexpression of FPGS mRNA. Conversely, both raltitrexed-resistant sublines (MOLT-3/ZD1694 x C and K562/ZD1694 x C) displayed a moderately decreased expression of FPGS mRNA. These findings did not correspond to the virtual absence of ZD1694 polyglutamates inside the former cells nor to possibly intact polyglutamation capacity in the latter cells. These results indicate that FPGS mRNA expression may predict cellular ability to produce polyglutamate metabolites of antifolate drugs in the sensitive cells, but does not necessarily reflect FPGS function at the enzyme level in the antifolate-resistant tumor cells.

'Tomudex' (raltitrexed) development: preclinical, phase I and II studies.

Raltitrexed ('Tomudex', formerly ZD1694) is a new active drug for advanced colorectal cancer, an area that has been without new drugs for over 40 years. It has a convenient dosing schedule and a potential for lower toxicity which represent important advantages over existing treatments. Advanced colorectal cancer is currently treated with 5-fluorouracil, generally in combination with other agents such as leucovorin. This leads to complex dosing schedules with increased activity but potentially serious toxicity. Raltitrexed is a novel cytotoxic agent, rationally designed to inhibit a specific molecular target, thymidylate synthase. In contrast to other current agents, raltitrexed inhibits thymidylate synthase directly, specifically and non-competitively, which may lead to an improved toxicity profile. It is retained within cells as polyglutamate metabolites, allowing a more convenient dosing schedule than for 5-fluorouracil. Phase I and pharmacokinetic studies established the optimum dosing schedule as 3 mg/m2, administered in a single 15-min intravenous infusion once every 3 weeks. In a phase II study in 177 patients with advanced colorectal cancer, this dose produced a response rate of 26% and median survival of 11.2 months. The safety profile was acceptable, the main adverse events being myelosuppression, gastrointestinal toxicity, asthenia and transient asymptomatic increases in liver transaminases without evidence of any other liver dysfunction. Activity of raltitrexed has also been observed in a range of other solid tumours, including breast, pancreatic, non-small-cell lung and refractory ovarian cancer.

The measurement of polyglutamate metabolites of the thymidylate synthase inhibitoR, ICI D1694, in mouse and human cultured cells

A method is described for the measurement of the polyglutamates of the quinazoline thymidylate synthase inhibitor, N-(5-[N-(3,4- dihydro-2- methyl-4- oxoquinazolin-6- ylmethyl)-N- methylamino]-2- theonyl)- l - glutamic acid (ICI D1694). This involved incubation of cells with [5- 3H]ICI D1694, extraction of the polyglutamates and their analysis by HPLC using an ion-pairing method. Co-chromatography with ICI D1694 and its synthetic di-hexaglutamate standards (UV detection) aided identification of the [ 3H]polyglutamates in the fractions recovered from the HPLC. Recovery of the polyglutamates at each stage of extraction and analysis was very good (77–84% overall recovery). Polyglutamates readily accumulated as the tri-, tetra and penta forms and occasionally a small amount of hexaglutamate was found. After mouse L1210 leukemia or human W1L2 lymphoblastoid cells were incubated for 30 min with 0.1 μM [ 3H]ICI D1694 there was a ∼6-fold concentration effect intracellularly with most of the 3H associated with polyglutamate forms (∼75% and 96% for the L1210 and W1L2, respectively). Even some of the higher chain length tetra- and pentaglutamates could be detected at this time. After 4 hr incubation the total level of intracellular 3H had risen to 2–3 μM, greater than 96% of which was associated with polyglutamates (mainly tetra- and pentaglutamates). Four other human cell lines, two ovarian (CH1 and 41M), the MCF-7 breast and the HT-29 colon, were examined for their ability to form intracellular polyglutamates. A 4 hr incubation with 0.1 μM [ 3H]ICI D1694 resulted in a substantial intracellular accumulation of the drug (20–100-fold) in its polyglutamate forms with only 2–20% remaining as the parent monoglutamate, depending on the cell line. The major polyglutamate was again cell line dependent, ranging from the tri to the penta form. Prolonging the incubation time to 24 hr allowed a further accumulation of drug with a larger percentage appearing as tri- to hexaglutamates. Although cell lines differed in the total level of polyglutamates formed and the pattern of chain length observed, rapid and extensive polyglutamation of ICI D1694 occurred in all the cell types examined.