Methotrexate Concentrations Research Articles

MiR-29 Family Inhibits Resistance to Methotrexate and Promotes Cell Apoptosis by Targeting COL3A1 and MCL1 in Osteosarcoma.

BackgroundMicroRNAs (miRNAs) play a crucial role in regulating diverse biological processes, including drug resistance. We investigated the potential roles of the miR-29 family in methotrexate (MTX) resistance in osteosarcoma.Material/MethodsTwo MTX-resistant osteosarcoma cell lines, MG-63/MTX and U2OS/MTX, were generated by continuous exposure to stepwise increasing concentrations of MTX. miR-29abc, COL3A1, and MCL1 mRNA expression levels were determined using quantitative real-time PCR (qRT-PCR). Protein expression levels of COL3A1 and MCL1 were detected by Western blot. Cell viability, IC50 value, and cell apoptosis were assessed by CCK-8 assay and flow cytometry, respectively. The target relationship between the miR-29 family and COL3A1 or MCL1 was confirmed by luciferase reporter assay.ResultsmiR-29a, miR-29b, and miR-29c were significantly downregulated in MG-63/MTX and U2OS/MTX cells and in chemotherapy poor-response osteosarcoma tissues. Overexpression of the miR-29 family sensitized MG-63/MTX and U2OS/MTX cells to MTX and obviously promoted cell apoptosis compared with negative control. COL3A1 and MCL1 were identified to be target genes of the miR-29 family, and transfection with miR-29abc mimics in MG-63/MTX and U2OS/MTX cells decreased COL3A1 and MCL1 mRNA and protein expression. Meanwhile, overexpression of COL3A1 and MCL1 partly neutralized the effects of the miR-29 family on MTX resistance and cell apoptosis.ConclusionsTaken together, our findings suggested a tumor-suppressor role of the miR-29 family in control of MTX resistance and cell apoptosis through regulating COL3A1 or MCL1. Targeting the miR-29 family might provide new strategies to overcome the high-dosage MTX-induced cytotoxicity in osteosarcoma treatment.

Methotrexate-induced apoptosis in human ovarian adenocarcinoma SKOV-3 cells via ROS-mediated bax/bcl-2-cyt-c release cascading.

IntroductionThe communication between a substance and a cell may depend on whether the cell is normal or pathological. The disease cells and drug interaction may occasionally overcome beneficial action of the drug; subsequently, it is important to investigate the effect of the drug in both the normal and target cells. This study aimed to evaluate the methotrexate (MTX) antiproliferative effect and explore the mechanistic approach to investigate the cell death index in SKOV-3 ovarian cells during treatment with MTX.MethodsIn vitro studies of SKOV-3 cells were examined by tetrazolium assay after exposure to various concentrations of MTX. Moreover, reactive oxygen species (ROS) generation, mitochondrial membrane potential, DNA damage, and AO/EtBr staining morphological analysis of necrotic/apoptotic cells were detected; cellular impairment in mitochondria and DNA was confirmed by JC-1 mitotracker/DAPI, respectively, and cell death pathway markers; bax/bcl-2 were analyzed.ResultsA dose-dependent antiproliferative effect of MTX treatment was observed in SKOV-3 cells; the prominent inhibitory concentration was 40 µM of MTX (P<0.01). The growth inhibition rates of the cancer cells reached 24.07% in MTX. The MTX showed increase in ROS generation and mitochondrial depolarization, and DNA integrity cells collectively advocated the apoptotic cell death at higher concentration. In addition, the results of reverse transcription polymerase chain reaction also supported the apoptosis by upregulating the bax and downregulating the bcl-2 (P<0.01). Thus the MTX moderately provokes apoptosis.ConclusionOur findings suggest that MTX acts on SKOV-3 cancer cells by increasing intracellular ROS levels, leading to DNA damage and altering the MMP along with apoptotic gene upregulation. This mechanism may provide new therapeutic targets to improve tumor treatment.

Methotrexate-Loaded Biodegradable Polymeric Micelles for Lymphoma Therapy in Mouse Model

Purpose: Methotrexate (MTX), a folate antimetabolite, competitively inhibits dihydrofolate reductase, the intracellular enzyme responsible for converting folic acid to folate cofactor, thus blocking the synthesis of thymine and purine, causing impairment of tumor growth and induction of cell death. High dose MTX (HD-MTX) is infused in doses of 3-5g/m2 and over a period of 6-24h to maintain the serum MTX at high levels, which shows benefit in the treatment of childhood leukemia and certain adult NHL, particularly the Burkitt type. However, the clinical implication of MTX is hampered for some reasons. On the one hand, the adverse effects of HD- MTX are severe, including bone marrow suppression, mucositis, hepatotoxicity, nephrotoxicity and neurological symptoms. On the other hand, leucovorin rescue, adequate hydration, urinary alkalization and monitoring of MTX concentration is required to avoid life-threating side effects of HD-MTX, which makes the administration rather complicated. In addition, the poor water solubility also impedes the application of MTX. To overcome these obstacles, a new drug delivery system is required to reduce the side effects of HD-MTX as well as to increase the solubility of the drug. So we designed a self-assembled micellar system to load MTX to overcome water solubility and to enhance therapeutic efficacy while reduce toxicity.Materials and Methods: Monomethyl poly (ethylene glycol)-poly (ε-caprolactone) (MPEG-PCL) our group previously synthesized was employed to load MTX through a one-step solid dispersion method. The preparation was done without any surfactants, organic solvents, or vigorous stirring.Results: MTX loaded micelles had a small particle size of 25.64±0.99nm and polydisperse index (PDI) of 0.176±0.05. Drug loading and encapsulation efficiency of MTX loaded micelle were 5.57±0.14% and 92.46±2.38%, respectively. Compared with free MTX, MTX loaded micelles demonstrated a much slower and sustained release behavior in vitro. MTT assay and cell apoptosis study suggested that MTX loaded micelles were more effective in inhibiting proliferation (IC50 value 137.8ug/ml vs 86.5ug/ml, p<0.05) and inducing apoptosis (p<0.01) on Raji lymphoma cells than MTX treatment, which was especially distinct in high dose groups (38.78% vs 22.17%). Cellular uptake study indicated that MPEG-PCL micelle had a 1.5 times higher uptake rate in Raji cells. As for in vivo studies, MTX loaded micelles were more competent to suppress tumor growth (p<0.01) and prolong survival time than MTX injection (p<0.05) in the subcutaneous Raji lymphoma mouse model. Notably, the high dose group of micelle formulation exhibited the strongest anti-tumor effect without additional toxicity. As for body weight, which represented systematic toxicity, no difference between control group and two kinds of MTX formulation group was observed (p >0.05). Furthermore, immunofluorescent and immunohistochemical studies showed that tumors of MPEG-PCL-MTX treated mice had more apoptotic cells (p<0.05) and fewer proliferative cells (p<0.01).Conclusions: In this work, MPEG-PCL-MTX micelle was fabricated with a thin-film hydration method to improve the water solubility of MTX and to meet the requirements of intravenous administration, which was stable, safe, effective, easy to produce and scale up, and showed potential clinical application. The enhanced therapeutic efficiency of MPEG-PCL-MTX micelle might be attributed to enhanced permeability and retention (EPR) effect, which increased drug accumulation in the tumor site. Besides, compared with MTX injection with highest concentration in our experiment, MPEG-PCL-MTX micelle with lowest concentration achieved similar or better therapeutic outcome, which indicated the amount of drug could be reduced to achieve the same therapeutic efficiency and alleviate adverse effects towards normal tissues and organs. Moreover, MPEG-PCL-MTX micelle of high dose (7mg/ml) achieved great therapeutic advantage without obvious toxicity, which makes micelle an effective and safe choice when HD-MTX is clinically employed. DisclosuresNo relevant conflicts of interest to declare.

Improving recombinant bone morphogenetic protein-4 (BMP-4) production by autoregulatory feedback loop removal using BMP receptor-knockout CHO cell lines

A Chinese hamster ovary (CHO) cell line producing recombinant human bone morphogenetic protein-4 (rhBMP-4) (CHO-BMP-4), which expresses essential components of BMP signal transduction, underwent autocrine BMP-4 signaling. RNA seq analysis on CHO host cells (DG44) treated with rhBMP-4 (20 µg/mL) suggested that rhBMP-4 induced signaling in CHO cells could be a critical factor in limiting rhBMP-4 production and should be removed to improve rhBMP-4 production in recombinant CHO (rCHO) cells. The inhibition of autocrine BMP signaling in CHO-BMP-4 cells by the addition of LDN-193189, a chemical inhibitor of BMP receptor type I, significantly increased the mRNA expression levels of rhBMP-4. To establish BMP signaling-free host cells, a BMP receptor, the BMPRIA or BMPRII gene in DG44 cells, was knocked out using CRISPR/Cas9 gene-editing technology. Using three different knockout (KO) host cell lines as well as a DG44 wild-type (wt) cell line, rCHO cell clones producing rhBMP-4 were generated by a stepwise selection with increasing methotrexate concentrations. KO-derived clones showed a significantly higher maximum rhBMP-4 concentration than wt-derived clones in both batch and fed-batch cultures. Unlike wt-derived clones, KO-derived cell clones were able to produce higher amounts of hBMP-4 transcripts and proteins in the stationary phase of growth and did not experience growth inhibition induced by rhBMP-4. The mean maximum rhBMP-4 concentration of KO host-derived clones was approximately 2.4-fold higher than that of wt-derived clones (P < 0.05). Taken together, the disruption of BMP signaling in CHO cells by knocking out the BMP receptor significantly improved rhBMP-4 production.

Effects of Red Ginseng Extract on the Pharmacokinetics and Elimination of Methotrexate via Mrp2 Regulation.

We aimed to investigate the effects of red ginseng extract (RGE) on the expression of efflux transporters and to study the pharmacokinetics of representative substrate. For this, rats received single or repeated administration of RGE (1.5 g/kg/day) for 1 and 2 weeks via oral gavage. mRNA and protein levels of multidrug resistance-associated protein2 (Mrp2), bile salt export pump (Bsep), and P-glycoprotein (P-gp) in the rat liver were measured via real-time polymerase chain reaction and Western blot analysis. Ginsenosides concentrations from the rat plasma were also monitored using a liquid chromatography–tandem mass spectrometry (LC–MS/MS) system. Plasma concentrations of ginsenoside Rb1, Rb2, Rc, and Rd following repeated administration of RGE for 1 and 2 weeks were comparable but significantly higher than those after single administration of RGE. These dosing regimens did not induce significant biochemical abnormalities in the liver, kidneys, and lipid homeostasis. In the RGE repeated oral administration groups, the mRNA and protein levels of Mrp2 significantly decreased. Accordingly, we investigated the changes in the pharmacokinetics of methotrexate, a probe substrate for Mrp2, following intravenous administration of 3 mg/kg methotrexate to rats in the RGE 1-week repeated oral administration group, compared to that in the control group. Biliary excretion, but not urinary excretion, of methotrexate decreased in the RGE repeated administration group, compared to that in the control group. Consequently, the plasma concentrations of methotrexate slightly increased in the RGE repeated administration group. In conclusion, repeated administration of RGE for 1 week resulted in a decrease in Mrp2 expression without inducing significant liver or kidney damage. Pharmacokinetic herb–drug interaction between RGE and methotrexate might occur owing to the decrease in the mRNA and protein levels of Mrp2.

A novel mixed hemimicelles dispersive micro-solid phase extraction using ionic liquid functionalized magnetic graphene oxide/polypyrrole for extraction and pre-concentration of methotrexate from urine samples followed by the spectrophotometric method

Methotrexate (MTX) is an anticancer drug that is widely used in a variety of cancers including primary central nervous system lymphoma. It is also administrated in the treatment of some autoimmune diseases. A simple, accurate, sensitive, and precise mixed hemimicelles dispersive micro-solid phase extraction was proposed for MTX quantification in human urine samples. MTX was quantified by spectrophotometer after dispersive micro-solid phase extraction using ionic liquid functionalized magnetic graphene oxide/polypyrrole. Interactions of adsorbent and MTX were modeled by molecular docking and the interaction energy was predicted to be −8.35 kcal/mol. A larger absolute value of binding energy represents larger adsorption strength, indicating that graphene oxide nanosheets could perform higher adsorption strength toward MTX. The concentrations of MTX were proportional to analytical response in amounts ranging from 10 to 1000 ng/mL with a good correlation (R2 = 0.99). Inter- and intra-day precisions and accuracies were within the acceptable limit according to FDA guideline (15% for biological determination). The recoveries were ranging from 89 to 93% and the method was specific for routine analysis of MTX. This protocol was applied to the urine of two patients under MTX therapy received an intravenous administration of 1 mg/kg/dose of MTX with acute lymphoblastic leukemia. The accuracy of the method was confirmed by HPLC measurements.

Renal Function and Plasma Methotrexate Concentrations Predict Toxicities in Adults Receiving High-Dose Methotrexate.

BackgroundMethotrexate (MTX) is an effective drug for the treatment of adult malignancies, but toxicity remains a significant problem. Toxic reactions may occur when patients use high-dose MTX (HD-MTX), but the correlation between its toxicity and concentration in adults is controversial. The purpose of this study was to examine the relationship between MTX concentration and renal function, as well as to assess toxic reactions to MTX in Chinese adults with acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL).Material/MethodsThis retrospective study enrolled 97 patients who had been diagnosed with ALL or NHL, and who were treated at the Hemopathology Department of Shanghai Changhai Hospital from January 2015 to June 2016.ResultsForty-one (27.5%) episodes of elimination delay were observed. We found negative correlations between creatinine clearance rate before MTX infusion and the plasma concentrations of MTX at 36 h after MTX infusion (P=0.005). The serum creatinine at 48 h and plasma concentrations of MTX at 48 h and72 h were significantly and positively correlated (both p=0.000). High blood concentration of MTX was positively associated with nephrotoxicity > grade 1 (P<0.01). Infection > grade 1 was more likely to occur if a patient had high MTX levels at 36 h, 48 h, and 72 h (P<0.01).ConclusionsOur results show that renal function is associated with MTX concentration, and high MTX concentration can predict the occurrence of renal toxicity and infection related to MTX.

Core-shell-satellite microspheres-modified glass capillary for microsampling and ultrasensitive SERS spectroscopic detection of methotrexate in serum

An ultrasensitive and highly reproducible novel surface-enhanced Raman scattering (SERS)-enabled capillary platform was developed for on-site detection of methotrexate (MTA) in serum. By employing a facile mirror reaction and a self-assembly procedure, glass capillaries were compactly coated with core-shell-satellite microspheres (CSSM) via a batch-fabrication process without using any pre-surface modifications or specialized equipment. The quality of the prepared capillaries was facilely evaluated and controlled by detecting SERS signals of 4-mercaptobenzoic acid (4-MBA) that were specifically anchored in the middle gap of CSSM. The CSSM-modified glass capillaries exhibit strong enrichment of MTA due to electrostatic interaction, high SERS performance ascribed to the interparticle plasmonic coupling, and the stimulation of strong signal of 4-MBA, enabling the detection of MTA molecules at low concentrations. The fabricated capillaries allow rapid detection of MTA via a facile “aspiration-measure” detection mode. Based on the inherent SERS spectra of MTA and 4-MBA, the intensity ratio of MTA and 4-MBA versus the concentration of MTA shows a correlation coefficient of 0.990 in the range of 0.1 nM–110 nM. Given electrostatic force and capillary action, this developed portable SERS-enabled device holds great potential for practical applications in point-of-care (POC) diagnosis.

Using LC-MS/MS-based targeted proteomics to monitor the pattern of ABC transporters expression in the development of drug resistance.

PurposeThe overexpression of ATP-binding cassette transporters (ABC transporters), mainly including permeability glycoproteins (P-gp), multidrug resistance (MDR)-related protein 1 (MRP1), and breast cancer resistance proteins (BCRP), is one of the main reasons for the development of MDR which directly leads to chemotherapy failure. However, most of the currently used detection methods in MDR-related studies are qualitative or semiquantitative, but not quantitative. As a result, the measurement criteria of different experiments are not unified. Moreover, there are many contradictory results of the studies of the induction effect of drugs on ABC transporters. So, it is necessary to establish a quantitative assay for the quantification of P-gp, MRP1, and BCRP to study the mechanism of drug resistance.MethodsIn this paper, a novel and advanced liquid chromatography/mass spectrometry (MS)/MS-based targeted proteomics method for the quantification of P-gp, MRP1, and BCRP was developed and validated. Then, the cell lines MCF-7, HepG-2, and SMMC-7721 were, respectively, induced by different concentrations of doxorubicin (adriamycin [ADM]), mitoxantrone (MX), and methotrexate (MTX), to establish resistance cell lines. The method established was used to quantify the expression of P-gp, MRP1, and BCRP.ResultsThe result showed that the induction effects of drugs on protein were relatively stable and selective. ADM, MX, and MTX could induce overexpression of P-gp, MRP1, and BCRP. And, the induction effect of different drugs on proteins was selective. The pattern of overexpression of ABC transporters in the three types of resistance cell lines was different.ConclusionDuring the development of drug resistance, the cell type and patch, but not drug type, were the most important determinant factors of the overexpression level of ABC transporters in resistance cell lines. This study provides a good foundation for understanding the development of drug resistance in cell lines and can be used to explain the contradictory results in other published studies as described above.

In vitro- and in vivo Evaluation of Methotrexate-Loaded Hydrogel Nanoparticles Intended to Treat Primary CNS Lymphoma via Intranasal Administration.

Although it passes through blood-brain barrier (BBB) very poorly, methotrexate (MTX) is an important therapeutic in the treatment of many central nervous system malignancies. Accordingly, intranasal (IN) administration accompanied with a muco-adhesive chitosan-based nanoformulation is expected to overcome this problem. Nanogel containing MTX was prepared through an ionic gelation method and then characterized in terms of particle size, morphology, zeta potential, drug loading and drug release behavior. The drug release results were fitted on eight mathematical models to choose the model best describing the phenomenon. Then the nano-formulation and free drug solution in deionized water as control were administered in the nasal cavity for rats and after 15, 30, 60 and 240 minutes their brain and plasma were analyzed for MTX quantity. The nano-formulation demonstrated an average particle size near 100 nm with a zeta potential of 18.65±1.77 mv. Loading efficiency and loading capacity were calculated to be 65.46±7.66 and 3.02±0.34 respectively. The Weibull model was found to be best describing the release phenomenon as a combination of swelling and Fickian diffusion. Moreover in in vivo studies, drug targeting efficiency and direct transport percentage for nanogel (test) and free drug solution (control) were 424.88% and 76.46% and 34842.15% and 99.71% respectively. Conclusion: According to in vivo studies, nanogel produced significantly higher concentration of MTX in the brain but not in the plasma when compared to the free drug solution. Besides, in comparison to intravenous administration of the same nanogel it was indicated that intranasal administration significantly increases the brain concentration of MTX.

Application of microRNA Targeted 3'UTRs to Repress DHFR Selection Marker Expression for Development of Recombinant Antibody Expressing CHO Cell Pools.

The dihydrofolate reductase (DHFR) system is used for the selection of recombinant Chinese hamster ovary (CHO) cell lines using the inhibitor methotrexate (MTX). During clonal selection, endogenous DHFR expression, and resistance to MTX allows the selection of cells expressing sufficient DHFR to survive. Here, the authors describe a novel vector platform for the DHFR system, whereby addition of a synthetic 3'UTR destabilizes DHFR expression. miRs ability to negatively regulate gene expression by their near-complementary binding to the 3'UTR region of transcripts are harnessed. From the literature, the authors identified let-7f as a highly abundant, invariant miR in CHO cells. Three 3'UTR targets of the let-7f miR are then cloned in the DHFR host 3'UTR to determine the impact on gene expression (HMGA2 3'UTR sequence 1, 2, and 3). Using luciferase as a reporter, the authors show down-regulation of luciferase activity is mediated by the nature of the 3'UTR and its ability to bind let-7f. The same 3'UTRs downstream of the DHFR gene to show this also results in reduced transcript amounts are then applied. Finally, the authors applied this methodology to generate stable DG44-derived cell pools expressing a model monoclonal antibody (mAb), demonstrating this approach can be used for the selection of antibody-producing cells with low MTX concentrations.

Acute bone marrow suppression and gastrointestinal toxicity following acute oral methotrexate overdose

Objective: Acute methotrexate overdose rarely causes systemic toxicity due to saturable absorption and rapid renal elimination. We present a case of methotrexate toxicity following acute overdose.Case Report: A 56-year-old female presented soon after an overdose of 1250 mg of methotrexate, zopiclone and tramadol. The methotrexate was initially under-reported (500 mg) and folinic acid was not provided. Despite normal renal function, the patient developed toxicity. She represented 5 days following the overdose with mucositis, bone marrow suppression and prolonged febrile neutropenia. Treatment included folinic acid, broad-spectrum antibiotics, filgrastim, red cell and platelet transfusion. Her bone marrow began to recover 12 days following the overdose. She was discharged home on Day 17.Discussion: Severe toxicity following an acute ingestion of a large amount of methotrexate is rarely reported. The development of toxicity was unexpected in this case given methotrexate’s pharmacokinetics and the patient’s normal renal function. The serum methotrexate concentrations were below the treatment threshold of the folinic acid rescue therapy nomogram suggesting that the nomogram should not be relied on in acute ingestions. Large acute oral methotrexate poisoning can result in systemic toxicity and folinic acid therapy should be provided in ingestions >1000 mg.

Evaluation of Serum Methotrexate Level by Using Chemiluminescent Microparticle Immuno Assay (CMIA)

Methotrexate (MTX) plays important treatment modalities for several paediatric cancers. Several automated binding assays and chromatographic methods was developed for estimating Methotrexate levels in serum and these methods are time consuming & utility of these methods in hospital set up is minimum. The ARCHITECT Methotrexate Assay by Chemiluminescent Microparticle Immuno Assay (CMIA) technology offers a sensitive & rapid estimation of Methotrexate in serum samples. So aim of this study is to evaluate the performance of ARCHITECT Methotrexate Assay kit in Chemiluminescent Microparticle Immuno Assay (CMIA) technology and to determine the level of serum methotrexate after 48 hours of Methotrexate infusion. ARCHITECT Methotrexate Assay kit was validated as per U.S Food and Drug Administration (USFDA) Bio analytical guidelines. The linearity was found to be in the range of 0.02–1.49 μmol/L with R2 0.99. The ARCHITECT Methotrexate assay showed a maximal Limit Of Detection of 0.0060 μmol/L. The Limit of quantification was found to be 0.04 μmol/L. Recovery of the sample was found to be 98.9 %. The intraday precision study showed a C.V. of 4.48 % at the lowest level and 3.83 % at the highest level where as interday precision study showed a C.V. of 2.14 % at the lowest level and 2.89 % at the highest level. The mean concentration of serum MTX after 48 hours of High Dose Methotrexate Infusion was found to be 0.487±0.357 μMol/L. The ARCHITECT assay is suitable for monitoring serum Methotrexate and showed satisfactory performance in the analysis of low concentrated samples.

Simultaneous determination of plasma methotrexate and 7-hydroxy methotrexate by UHPLC–MS/MS in patients receiving high-dose methotrexate therapy

The plasma concentrations of methotrexate (MTX) and its major metabolite 7-hydroxy methotrexate (7-OH-MTX) are highly correlated with the toxicities in patients with high-dose MTX therapy. Routine monitoring of MTX and 7-OH-MTX plasma levels is useful for dose adjustment of rescue drugs and toxicity prevention. A UHPLC-MS/MS method for simultaneous determination of plasma MTX and 7-OH-MTX was developed, validated, and applied in 181 plasma samples. The ion transition was m/z 455.2 → 308.2 for MTX and m/z 471.2 → 324.1 for 7-OH-MTX. The flow rate was 0.4 mL/min with a run time of 2.6 min. The calibration range was 0.002–2 μM for MTX, and 0.01–10 μM for 7-OH-MTX. The intra-day and inter-day inaccuracy and imprecision were –5.50% to 10.93% and less than 9.20% for both analytes. The internal standard (MTX-D3) normalized recovery and matrix factor were consistent at four quality control levels. 14 h, 38 h, and 62 h after dosing, MTX and 7-OH-MTX plasma levels were significantly higher in patients with impaired renal function compared to those with normal renal function. 7-OH-MTX plasma levels were significantly higher in patients with impaired liver function compared to those with normal liver function.

Relationship of peak serum methotrexate concentration to prognosis and drug tolerance in non-metastatic extremity osteosarcomas.

This study aimed to explore whether peak serum methotrexate concentration (Cmax) correlated with adverse events, overall survival (OS) and event-free survival (EFS) in patients with primary extremity osteosarcoma. Patients with extremity osteosarcoma who were treated at our center between 2005 and 2015 were retrospectively studied. All the patients were Enneking stage II and had received standard perioperative chemotherapy composed of high-dose methotrexate, doxorubicin, cisplatin and ifosfamide. Cmax and treatment-associated toxicities of each cycle were recorded. OS and EFS were estimated and compared by Kaplan-Meier survival analysis, and Cox regression models were performed for univariate comparisons. In total, 567 patients were followed for an average of 53months (24-104months). The estimated 3- and 5-year EFS were 71.7 and 63.1%, and the 3- and 5-year OS were 78.2 and 72.9%, respectively. Cmax ranged from 527 to 2495µmol/L with a mean value of 931 ± 106µmol/L. No significant differences in EFS and OS (p = 0.18 and p = 0.28) were observed among patients with a mean Cmax > 1500, > 1000, > 700 and < 700µmol/L. However, patients with a mean Cmax > 1500µmol/L had significantly increased rates of grade 3-5 toxicity. In the univariate analysis, Cmax was not a prognostic factor for EFS (p = 0.08) or OS (p = 0.16). Cmax did not correlate significantly with the oncologic prognosis of non-metastatic extremity osteosarcoma patients treated by multi-agent chemotherapy; however, Cmax correlated closely with toxicities and complications. The persistent inclusion of methotrexate in classical multidisciplinary chemotherapy was questioned and should be examined in future trials.

Brain Activity Associated With Attention Deficits Following Chemotherapy for Childhood Acute Lymphoblastic Leukemia

The impact of contemporary chemotherapy treatment for childhood acute lymphoblastic leukemia on central nervous system activity is not fully appreciated. Neurocognitive testing and functional magnetic resonance imaging (fMRI) were obtained in 165 survivors five or more years postdiagnosis (average age = 14.4 years, 7.7 years from diagnosis, 51.5% males). Chemotherapy exposure was measured as serum concentration of methotrexate following high-dose intravenous injection. Neurocognitive testing included measures of attention and executive function. fMRI was obtained during completion of two tasks, the continuous performance task (CPT) and the attention network task (ANT). Image analysis was performed using Statistical Parametric Mapping software, with contrasts targeting sustained attention, alerting, orienting, and conflict. All statistical tests were two-sided. Compared with population norms, survivors demonstrated impairment on number-letter switching (P < .001, a measure of cognitive flexibility), which was associated with treatment intensity (P = .048). Task performance during fMRI was associated with neurocognitive dysfunction across multiple tasks. Regional brain activation was lower in survivors diagnosed at younger ages for the CPT (bilateral parietal and temporal lobes) and the ANT (left parietal and right hippocampus). With higher serum methotrexate exposure, CPT activation decreased in the right temporal and bilateral frontal and parietal lobes, but ANT alerting activation increased in the ventral frontal, insula, caudate, and anterior cingulate. Brain activation during attention and executive function tasks was associated with serum methotrexate exposure and age at diagnosis. These findings provide evidence for compromised and compensatory changes in regional brain function that may help clarify the neural substrates of cognitive deficits in acute lymphoblastic leukemia survivors.

Puerarin improves methotrexate-induced renal damage by up-regulating renal expression of Oat1 and Oat3 in vivo and in vitro

The regulation of renal transporters such as organic anion transporter (OATs) is a new target for treatment of acute renal failure. The purpose of this study was to investigate whether the effect of puerarin (Pur) on renal damage induced by methotrexate (MTX) is related to the expression of renal Oat1/3 in vivo and in vitro, and to explore the related mechanisms. Effect of Pur on the renal damage caused by MTX was evaluated by assessment of the changes of endogenous metabolites, toxins and H&E staining. Furthermore, Real-time PCR and western blot methods were taken to evaluate the modulation of Oat1/3 in rats. Then, the regulation of Oat1/3 by B-cell CLL/lymphoma (BCL)6 was explored by siRNA assay using NRK-52E cells in vitro. Pur reduced levels of endogenous metabolites and toxins, like creatinine, urea nitrogen and indoxyl sulfate in plasma in MTX-treated rats. Moreover, plasma concentration of MTX was significantly decreased, while the cumulative urinary excretion of MTX and the uptake of MTX by kidney slices were strongly increased after administration of multiple-dose of Pur via up-regulation of renal Oat1/3 expression. Knockdown of BCL6 by siRNA abrogated the Pur-induced Oat1/3 expression in NRK-52E cells. Pur improved MTX-induced renal toxicity through promotion of renal excretion of toxins by up-regulating renal Oat1/3 via BCL6. Pur was beneficial for the improvement of MTX-induced renal toxicity.

Ursodeoxycholate Restores Biliary Excretion of Methotrexate in Rats with Ethinyl Estradiol Induced-Cholestasis by Restoring Canalicular Mrp2 Expression.

The in vivo relevance of ursodeoxycholate (UDCA) treatment (100 mg/kg/day, per oral tid for 5 days before cholestasis induction followed by the same dosing for 5 days) on hepatic function was investigated in rats with 17α-ethinylestradiol (EE, 10 mg/kg, subcutaneous for 5 days)-induced experimental cholestasis. The bile flow rate and the expression level of hepatic multidrug resistance-associated protein 2 (Mrp 2) that were decreased in cholestasis were restored after UDCA treatment. Consistent with this, the biliary excretion clearance (CLexc,bile) of a representative Mrp2 substrate—methotrexate (MTX)—was decreased in cholestatic rats but was restored after UDCA treatment. Consequently, the plasma concentrations of MTX, which were increased by cholestasis, were decreased to control levels by UDCA treatment. Thus, the restoration of CLexc,bile appears to be associated with the increase in Mrp2 expression on the canalicular membrane by UDCA treatment followed by Mrp2-mediated biliary excretion of MTX. On the other hand, the hepatic uptake clearance (CLup,liver) of MTX was unchanged by cholestasis or UDCA treatment, suggestive of the absence of any association between the uptake process and the overall biliary excretion of MTX. Since UDCA has been known to induce the expression of canalicular MRP2 in humans, UDCA treatment might be effective in humans to maintain or accelerate the hepatobiliary elimination of xenobiotics or metabolic conjugates that are MRP2 substrates.

Efficient Enrichment of Gene-Modified Primary T Cells via CCR5-Targeted Integration of Mutant Dihydrofolate Reductase

Targeted gene therapy strategies utilizing homology-driven repair (HDR) allow for greater control over transgene integration site, copy number, and expression—significant advantages over traditional vector-mediated gene therapy with random genome integration. However, the relatively low efficiency of HDR-based strategies limits their clinical application. Here, we used HDR to knock in a mutant dihydrofolate reductase (mDHFR) selection gene at the gene-edited CCR5 locus in primary human CD4+ T cells and selected for mDHFR-modified cells in the presence of methotrexate (MTX). Cells were transfected with CCR5-megaTAL nuclease mRNA and transduced with adeno-associated virus containing an mDHFR donor template flanked by CCR5 homology arms, leading to up to 40% targeted gene insertion. Clinically relevant concentrations of MTX led to a greater than 5-fold enrichment for mDHFR-modified cells, which maintained a diverse TCR repertoire over the course of expansion and drug selection. Our results demonstrate that mDHFR/MTX-based selection can be used to enrich for gene-modified T cells ex vivo, paving the way for analogous approaches to increase the percentage of HIV-resistant, autologous CD4+ T cells infused into HIV+ patients, and/or for in vivo selection of gene-edited T cells for the treatment of cancer.

Leflunomide Increases Hepatic Exposure to Methotrexate and Its Metabolite by Differentially Regulating Multidrug Resistance-Associated Protein Mrp2/3/4 Transporters via Peroxisome Proliferator-Activated Receptor α Activation.

Methotrexate (MTX) is the gold standard drug for the treatment of rheumatoid arthritis (RA), and it is frequently combined with leflunomide (LEF) to enhance its clinical efficacy. However, this combination can exacerbate liver toxicity, and the underlying mechanism has not yet been clarified. We investigated whether LEF affects the pharmacokinetics of MTX and its primary toxic metabolite, 7-hydroxyl methotrexate (7OH MTX), in mice. LEF significantly increased the plasma concentration (area under the plasma concentration-time curve) of MTX and 7OH MTX (2.4 and 4.5 times, respectively), decreased their bile excretion, and increased their accumulation in the liver and kidneys. When we investigated the effect of LEF on the MTX absorption, distribution, metabolism, and excretion process, we found that LEF had little effect on liver aldehyde oxidase and 7OH MTX formation. However, LEF significantly decreased the expression of the apical efflux transporter multidrug resistance-associated protein 2 (Mrp2) and increased that of the basolateral efflux transporters Mrp3/4, except there was no significant change in Mrp4 protein expression. Mrp2/3/4 alteration changed the distribution of MTX and 7OH MTX in plasma and tissues. Further studies suggested that LEF indirectly activated peroxisome proliferator-activated receptor α (PPARα), which was likely responsible for the Mrp2/3/4 alteration in the liver. The MTX plasma concentration change induced by LEF was reversed by the PPARα-specific antagonist GW6471. These results may partially explain the exacerbated liver toxicity caused by combination treatment with MTX and LEF and may raise concerns regarding the risk of potential drug-drug interactions between PPARα agonists and Mrp substrates in the clinic.